Engineering the cell sheet tissue effect to fabricate high cytokine secretory mesenchymal stem cell constructs

Mesenchymal stem cell (MSC)-based therapies present several ideal attributes of a promising new drug candidate in their innate ability to secrete a dynamic pharmacology of bioactive cytokines and growth factors that can influence nearby cells via paracrine signaling. MSC signals in turn stimulate various endogenous biological processes desirable for tissue regeneration, including angiogenesis, immune modulation, and cell recruitment and proliferation. However, when injected as a suspension, these cells suffer from poor survival and localization, and suboptimal release of paracrine factors, limiting their clinical efficacy to date. Delivering MSCs within tissue-engineered threedimensional (3D) platforms not only resolves issues of cell localization, engraftment, and survival, but a growing body of evidence finds that 3D culture stimulates MSC paracrine activity in response to higher abundance physical cell contacts and biochemical cellular interactions, otherwise known as a tissue effect. The goal of this dissertation was to overcome historical limitations of MSC therapies by engineering engraftable 3D MSC tissue-like constructs optimized for therapeutic potency through a tailored tissue effect. To achieve this goal, we employ cell sheet tissue engineering, a foremost method for creating 3D cell constructs without the use of a biomaterial scaffold and comprising only cells and endogenous matrix, interconnected by cell-cell and cell-matrix interactions. The central hypotheses of this dissertation are that the desired tissue effect is fundamentally realized using cell sheet tissue engineering, and that unique 3D structure and physical and biochemical cellular interactions from cell sheet engineering can be enhanced within cell sheets to stimulate individual MSC cytokine production potency. These hypotheses are addressed by (1) demonstrating that 3D cell sheet structure and cellular interactions augment individual MSC pro-regenerative cytokine (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-10 (IL-10)) secretion capacity in vitro, (2) demonstrating that cell sheet centrifugation augments individual MSC proregenerative cytokine (VEGF, HGF, IL-10) secretion capacity by directly increasing 3D cellular interactions, and (3) demonstrating that cell sheet multilayering from centrifugation increases absolute MSC sheet construct secreted pro-regenerative cytokine (VEGF, HGF, IL-10) dose. Ultimately, this dissertation delivers a complete in vitro platform for engineering and multilayering MSC sheet tissue optimized for MSC cytokine production. This research culminates in the generation of highly functional MSC sheets as a 3D cell-delivery technology for cell therapy applications.

ENGINEERING THE CELL SHEET TISSUE EFFECT TO FABRICATE HIGH CYTOKINE SECRETORY MESENCHYMAL STEM CELL CONSTRUCTS by Sophia Bou-Ghannam A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biomedical Engineering The University of Utah December 2021 Copyright © Sophia Bou-Ghannam 2021 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Sophia Bou-Ghannam has been approved by the following supervisory committee members: David W. Grainger , Chair 08/28/2021 Date Approved Teruo Okano , Member Date Approved Michael Yu , Member Date Approved Vladimir Hlady , Member 08/28/2021 Date Approved Kyungsook Kim , Member 09/07/2021 Date Approved and by David W. Grainger the Department/College/School of , Chair/Dean of Biomedical Engineering and by David B. Kieda, Dean of The Graduate School. ABSTRACT Mesenchymal stem cell (MSC)-based therapies present several ideal attributes of a promising new drug candidate in their innate ability to secrete a dynamic pharmacology of bioactive cytokines and growth factors that can influence nearby cells via paracrine signaling. MSC signals in turn stimulate various endogenous biological processes desirable for tissue regeneration, including angiogenesis, immune modulation, and cell recruitment and proliferation. However, when injected as a suspension, these cells suffer from poor survival and localization, and suboptimal release of paracrine factors, limiting their clinical efficacy to date. Delivering MSCs within tissue-engineered threedimensional (3D) platforms not only resolves issues of cell localization, engraftment, and survival, but a growing body of evidence finds that 3D culture stimulates MSC paracrine activity in response to higher abundance physical cell contacts and biochemical cellular interactions, otherwise known as a tissue effect. The goal of this dissertation was to overcome historical limitations of MSC therapies by engineering engraftable 3D MSC tissue-like constructs optimized for therapeutic potency through a tailored tissue effect. To achieve this goal, we employ cell sheet tissue engineering, a foremost method for creating 3D cell constructs without the use of a biomaterial scaffold and comprising only cells and endogenous matrix, interconnected by cell-cell and cell-matrix interactions. The central hypotheses of this dissertation are that the desired tissue effect is fundamentally realized using cell sheet tissue engineering, and that unique 3D structure and physical and biochemical cellular interactions from cell sheet engineering can be enhanced within cell sheets to stimulate individual MSC cytokine production potency. These hypotheses are addressed by (1) demonstrating that 3D cell sheet structure and cellular interactions augment individual MSC pro-regenerative cytokine (vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-10 (IL-10)) secretion capacity in vitro, (2) demonstrating that cell sheet centrifugation augments individual MSC proregenerative cytokine (VEGF, HGF, IL-10) secretion capacity by directly increasing 3D cellular interactions, and (3) demonstrating that cell sheet multilayering from centrifugation increases absolute MSC sheet construct secreted pro-regenerative cytokine (VEGF, HGF, IL-10) dose. Ultimately, this dissertation delivers a complete in vitro platform for engineering and multilayering MSC sheet tissue optimized for MSC cytokine production. This research culminates in the generation of highly functional MSC sheets as a 3D cell-delivery technology for cell therapy applications. iv TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES .......................................................................................................... viii ABBREVIATIONS ............................................................................................................. x ACKNOWLEDGMENTS ................................................................................................. xii Chapters 1. INTRODUCTION ........................................................................................................... 1 1.1 Background .......................................................................................................... 1 1.2 Cell Sheet Tissue Engineering ............................................................................. 8 1.3 The Tissue Effect on Cell Functional Enhancement .......................................... 14 1.4 Human Umbilical Cord Mesenchymal Stem Cells ............................................ 18 1.5 Dissertation Overview: Motivation, Aims, and Scope ....................................... 20 1.6 References .......................................................................................................... 33 2. 3D CELL SHEET STRUCTURE AUGMENTS MESENCHYMAL STEM CELL CYTOKINE PRODUCTION ............................................................................................ 48 2.1 Abstract .............................................................................................................. 49 2.2 Introduction ........................................................................................................ 49 2.3 Results ................................................................................................................ 50 2.4 Discussion .......................................................................................................... 52 2.5 Conclusions ........................................................................................................ 54 2.6 Materials and Methods ....................................................................................... 54 2.7 References .......................................................................................................... 56 3. CELL SHEET CENTRIFUGATION AUGMENTS MESENCHYMAL STEM CELL CYTOKINE PRODUCTION ............................................................................................ 60 3.1 Abstract .............................................................................................................. 60 3.2 Introduction ........................................................................................................ 61 3.3 Results ................................................................................................................ 64 3.4 Discussion .......................................................................................................... 70 3.5 Conclusions ........................................................................................................ 80 3.6 Materials and Methods ....................................................................................... 81 3.7 References .......................................................................................................... 92 4. CELL SHEET MULTILAYERING FROM CENTRIFUGATION FUNCTIONALLY ENHANCES MESENCHYMAL STEM CELL TISSUE .............................................. 101 4.1 Abstract ............................................................................................................ 101 4.2 Introduction ...................................................................................................... 102 4.3 Results .............................................................................................................. 105 4.4 Discussion ........................................................................................................ 108 4.5 Conclusions ...................................................................................................... 119 4.6 Materials and Methods ..................................................................................... 120 4.7 References ........................................................................................................ 131 5. SUMMARY OF KEY FINDINGS ............................................................................. 136 5.1 References ........................................................................................................ 139 6. FUTURE WORK ........................................................................................................ 141 6.1 Evaluation of MSC Sheet Therapeutic Outcomes for Fibrotic Liver Disease . 141 6.2 Statistical Powering .......................................................................................... 146 6.3 Statistical Analysis ........................................................................................... 149 6.4 Quantitative Estimate of MSC Sheet Bioactive Factor Diffusion into Liver Tissue ..................................................................................................................... 151 6.5 MSC Sheet Exosomal Consideration as a Mediator of Therapeutic Paracrine Effect ...................................................................................................................... 156 6.6 References ........................................................................................................ 163 APPENDIX: PUBLISHED WORKS.............................................................................. 171 vi LIST OF TABLES 3.1 Single layer cell sheet attachment rate following medium addition and mechanical rotation test. ....................................................................................................................... 88 3.2 Cell sheet live cell numbers in static culture. .............................................................. 88 4.1 Cell sheet live cell numbers in static culture. ............................................................ 125 4.2 Summary of multicellular sheet layering. ................................................................. 130 LIST OF FIGURES 1.1 Poly(N-isopropylacrylamide) (PIPAAm). ................................................................... 29 1.2 Temperature-responsive cell culture dishes (TRCD). ................................................. 30 1.3 Conventional enzymatic cell harvest yielding a cell suspension. ............................... 30 1.4 Cell sheet harvest enhances MSC pro-regenerative cytokine production capacity relative to single cell harvest. ............................................................................................ 31 1.5 Concept for cell sheet tissue regeneration. .................................................................. 32 2.1 Microscopic cell morphology influences macroscopic tissue structure. ..................... 51 2.2 Spontaneous cell sheet contraction contributes a 3D tissue-like structure.................. 51 2.3 hUC-MSC actin structure changes in response to cell sheet contraction. ................... 52 2.4 Enhanced pro-regenerative cytokine gene expression related to 3D cell sheet tissuelike structure. ..................................................................................................................... 53 2.5 Cell sheet contraction increases actual cytokine production by hUC-MSCs. ............. 53 2.6 MSC sheet 3D structural and molecular transition. .................................................... 55 3.1 Single layer cell sheet adhesion optimization for stable centrifugation. ..................... 89 3.2 Centrifugation compacts cell sheet tissue structure. ................................................... 90 3.3 Cell sheet centrifugation enhances MSC pro-regenerative cytokine production related to cellular interactions. ...................................................................................................... 91 4.1 Flow chart illustrating the MSC sheet layering protocol with centrifugation (centrifugation method) and without centrifugation (conventional method). ................. 127 4.2 Centrifugation layering enables viable 2-layer cell sheet tissue fabrication. ............ 128 4.3 Centrifugation layering enhances MSC pro-regenerative cytokine production related to cellular interactions. .................................................................................................... 129 4.4 Cell sheet layering increases MSC sheet absolute pro-regenerative cytokine production........................................................................................................................ 130 6.1 Study design for generating a CCl4-induced disease model. .................................... 161 6.2 Study design for therapeutic evaluation of hUC-MSC transplantation..................... 161 6.3 The diffusion profile from MSC sheet secreted HGF into liver tissue. .................... 162 ix ABBREVIATIONS 2D Two-dimensional 3D Three-dimensional ANOVA Analysis of variance CD Cluster of differentiation DAPI 4’,6-diamidino-2-phenylindole DMEM Dulbecco’s modified eagle’s medium ECM Extracellular matrix EDTA Disodium ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay EV Extracellular vesicle FBS Fetal bovine serum FGF Fibroblast growth factor GAPDH Glyceraldehyde 3-phosphate dehydrogenase H&E Hematoxylin & eosin HGF Hepatocyte growth factor IHC Immunohistochemical IL-10 Interleukin-10 IV Intravenous IP Intraperitoneal LCST Lower critical solution temperature MHC Major histocompatibility complex MI Myocardial infarction MSC Mesenchymal stem cell NEAA Nonessential amino acids PBS Phosphate buffered saline PFA Paraformaldehyde PIPAAm poly(N-isopropylacrylamide) PS Penicillin streptomycin qRT-PCR Quantitative real time polymerase chain reaction RT Room temperature (i.e., ~20oC) SD Standard deviation SE Standard error TCP Tissue culture plastic dish TRCD Temperature-responsive culture dish VEGF Vascular endothelial growth factor xi ACKNOWLEDGMENTS Throughout the writing of this dissertation, I have received a great deal of support and assistance. I would first like to thank Professor D.W. Grainger, whose expertise was invaluable in guiding the research questions and composing manuscripts as practical documents to propagate the technical edge beyond the work described here. Thank you for always challenging me to think and speak as an expert. Thank you for your friendship, mentorship, and caring advisory spanning the professional and the personal. I feel lucky to have you in my life. I would like to thank Professor T. Okano, whose scientific expertise was the foundation for the research questions pursued. Your expertise guided the motivation and formulated the methodology. You provided me with life opportunities that few doctoral students would ever receive by making available to me a network of people and resources, both in Japan and in the United States, that supported my professional development and my personal growth. I would like to thank Professor K.S. Kim, whose scientific expertise was invaluable in formulating the research questions and methodology, and in shaping the scientific impact of the results. Thank you for your time and dedication devoted to teaching me. Thank you for always motivating and guiding me to deliver my absolute best. As a mentor, you provided me with tools that make me a stronger scientist and a better person. In addition, I thank my committee members, Professor M. Yu, and Professor V. Hlady, for their wise counsel and critical lens that helped distill the research questions into a poignant thesis and an impactful scientific document. xiii CHAPTER 1 INTRODUCTION 1.1 Background Drug discovery has evolved to treat some of the most debilitating diseases. Small molecular compounds have been powerful in their ability to address single-target disease pathways to successfully manage symptoms and control disease stasis [1, 2]. Drug development has evolved for disease treatment, incorporating biological drugs with target specificity that can block or even reverse diseased pathways [2, 3]. However, most complex diseases that implicate multiple biological pathways still lack adequate treatments. In the case of liver fibrosis, for example, disease onset begins with hepatocyte damage and cascades into the recruitment of inflammatory cells and activation of collagen-producing cells (hepatic stellate cells); chronic inflammation and continued cell death facilitates fibrotic scarring that propagates to disrupt tissue vasculature, ultimately contributing to whole organ failure [4, 5]. In this case, a poly-pharmacological approach that can act on the underlying cell condition, mitigate inflammatory responses, and repair vasculature is required for adequate disease treatment. Notably none are available. This is the case for many complex human pathologies. Recently, as knowledge of cell processes increases, cell-based therapies have increasingly emerged as a new treatment option in medicine and pharmacy, spurring a 2 new field of medicine known as regenerative medicine. Regenerative medicine aims to provide therapy by replacing or repairing impaired cells, tissues, or organs, a promising approach for conditions that lack adequate or existing treatments, such as chronic, autoimmune, and metabolic diseases, cancers, and tissue degeneration [6]. Cell therapies offer the promise of tailored, dynamic pharmacology for diseases that cannot be adequately treated by existing pharmaceuticals. Live cells can respond to environmental stimuli by adapting their lineage phenotype, proliferating, and secreting various responsive cytokines and trophic factors that can simultaneously elicit immunemodulating and regeneration-mediating effects. Cell therapy, by means of cell replacement or paracrine/trophic mechanisms, centers around the idea that cell communication pathways can be harnessed for their regenerative functions to treat diverse diseases [7]. Toward this end, mesenchymal stem cells (MSCs) have become the most contemporary clinically studied experimental cell therapy platform worldwide for their clinically useful intrinsic features of high-capacity self-renewal, multipotent differentiation potential, migration ability to damaged or diseased tissue sites (homing), regeneration-mediating bioactive factor secretion, low immunogenicity, and high antiinflammatory potential [8]. First identified as stromal cells that could be isolated from whole bone marrow aspirates by Friedenstein in 1960 [9], the existence of an adult MSC that could be derived from stromal cell populations was later separately proposed by Owen and Caplan [10, 11]. Originating from the mesoderm germ layer, MSCs are defined by their progenitor nature capable of giving rise to at least three mesodermal lineages in vitro: chondrocytes, osteoblasts, and adipocytes [12]. Additionally, in vitro 3 studies have made it possible to derive endodermal lineage cells by MSC transdifferentiation into hepatocytes [13] and β-cells of pancreatic islets [14, 15], as well as cells derived ontogenetically from ectoderm by stimulating MSC differentiation into neurons [16-18]. Much of the early preclinical and clinical efforts using MSCs focused predominantly on this differentiation potential to mediate therapy by direct cell replacement to restore structure and function that had been lost due to tissue injury or disease [19]. However, as new knowledge of MSC developmental plasticity is understood, and as more animal and human in vivo data are revealed across various tissue sites, evidence that MSCs are capable of differentiating, or maintaining a differentiated phenotype in vivo is sparse and overwhelmingly points to the contrary [20-23]. Rather, there is substantial evidence that MSCs exert therapeutic benefits largely by direct cell contact and local secretion of numerous growth factors, angiogenic factors, and immunomodulatory proteins that stimulate endogenous tissue repair, known as the paracrine effect [24, 25]. This attribution of paracrine function to MSC-mediated therapy is based on in vivo observations that MSC delivery without differentiation and engraftment into injured tissues still yields regeneration via alternative therapeutic mechanisms [26-29]. One of the initial reports identifying the paracrine mechanism of MSCs by Gnecchi and colleagues (2005) characterized the secretion of cytoprotective factors by MSCs, reporting that modified MSCs could prevent ventricular remodeling and reestablish heart function in less than 72 hours following surgical myocardial infarction (MI) and cell transplantation [30, 31]. This rapid ischemic amelioration raised the possibility of an action other than a myogenic pathway that would not be evident in such a brief period. Thus, the mechanism by which injected MSCs might release trophic 4 factors that contribute to host-tissue protection and immune modulation, or the paracrine effect, was proposed and has since become a functional focal point of MSCs that researchers, clinicians, and cell therapy companies (e.g., Pluristem Therapeutics Inc, Athersys Inc, Mesoblast Ltd) alike have worked to harness, enhance, and implement for regenerative therapies. The most clinically used method for delivering MSCs for therapy has been by systemic (i.e., by intravenous infusion, IV) or direct-to-tissue injection of dissociated cells suspended in saline or a dilute collagen solution [32, 33]. Anticipating an MSC trophic response and disease-site homing that does not require direct tissue localization and engraftment, intravenous injections or infusions are the most clinically expedient methods for MSC delivery. Although over 1000 clinical trials (see clinicaltrials.gov) utilizing MSC therapies in the form of cell injections have been reported to date, the reliance on direct injections for clinical administration is a substantial limitation to significant therapeutic efficacy. This is perhaps a primary reason that, despite much clinical research, so few MSC therapies are clinically approved for human therapy to date. Irrespective of tissue source, MSC engraftment following direct injection is generally low and transient in nature [24, 34]. In the application of MSCs for ischemic MI, individual reports recognize that 1% of injected MSCs remain 24 hours after transplantation in a rat heart with experimental MI [35] and <3% are retained in the infarct region within 50–75 minutes of transplantation in human patients with ischemic heart disease [36]. Part of this low engraftment efficiency is owed to systemic delivery, which subjects the cells to a first-pass effect, entrapping a large proportion of cells within the microvasculature of certain organs (primarily the lungs) [24, 37, 38]. Clinical 5 administration using MSC therapies compensate with doses of higher absolute numbers of cells and repeat injections to ensure that a minimum necessary number of cells reach the injury site. In a review that considered 276 clinical trials administering MSCs as systemic injections, a mean dose per injection of 2.16x108 cells/person, and a mean dose per kilogram (kg) of 7.24x106 cells/kg was reported [24]. Generating cell numbers of this magnitude and the necessity for repeat infusions pose a technical and practical hurdle for the translation of MSC therapies to broader patient populations. Additionally, IV delivery has been found to facilitate rapid removal of administered MSCs by innate host immune cells [39, 40], and cell suspensions delivered locally to the target tissue (e.g., intramuscularly, intraperitoneally) appear to leave the site of administration by immunologic or lymphatic clearing mechanisms [34, 41, 42]. Many studies suggest that MSCs delivered by injection have a short lifespan, owing to the loss of ECM attachment (anoikis) as well as metabolic stress in response to the ischemic, low-serum in vivo condition and the presence of reactive oxygen species and hostile inflammatory cytokines in the disease tissue environment [43, 44]. Should transplanted MSCs survive and engraft at the target tissue location, low exposure to instructive cues results in suboptimal and unpredictable activity of the cells, undercutting the expected therapeutic paracrine outcomes [41, 45]. To overcome the problems of dissociated cell injections as a delivery method for MSC therapies, tissue engineering approaches have become second-generation cell therapies. Tissue engineering is based on the concept that 3D biomaterial scaffolds can be used as an alternative to the 3D tissue network in which nearly all cells naturally reside, with cells seeded into scaffolds to replicate native tissue-like structures in vitro [46]. In 6 addition to providing an environmental niche that drives cell attachment and function, the use of a 3D biomaterial scaffold provides an engraftable matrix that can localize seeded cells directly to the target tissue. The cell-seeded scaffold can be injected into or sutured onto the tissue of interest to circumvent first-pass elimination of cell doses. By default, a scaffold-seeded formulation of therapeutic cells mitigates cell death from loss of attachment. Because the cells are introduced within an instructive microenvironment, cell activity is stimulated as directed at the site of scaffold engraftment. However, use of biomaterial scaffold-based designs has had only limited success in complete tissue reconstruction in humans due to several complications. First, when biomaterial scaffolds degrade, large amounts of ECM are deposited in their place [47-49]. In the re-creation of structures with a large proportion of ECM to a sparse cell population, such as cartilage or bone, fabricated structures may resemble native tissues. However, toward the reconstruction of cell-dense and perfused structures, such as the heart or liver, constructs engineered with degradable scaffolds fail in their recapitulation of native tissue structure and function. Second, in cases involving larger constructs, the distribution of cells throughout scaffolds is not incidentally homogenous: due to the regression of oxygen diffusion toward the center of the scaffold, the highest concentration of seeded cells is present on the periphery of the scaffold, while the hypoxic center is cell-sparse and often necrotic [46, 50]; the result is often unsatisfactory long-term tissue regeneration. Third, most biomaterial choices for tissue scaffolds are not readily adhesive to biologic surfaces. This requires that the biomaterial seeded with cells must be sutured or glued into an already injured tissue environment, which can cause further tissue damage. Although localized, the biomaterial encasement disrupts cell interactions and 7 direct contact crosstalk with the native tissue; cells are tasked with migrating through the biomaterial scaffold to engage with the native tissue. Signals from tissue are hindered in the scaffold from natural presentation to seeded cells. Therefore, although biomaterial scaffolds are touted as a solution to the engraftment and localization limitations of systemic cell suspension injections, cells within a biomaterial can only support paracrine mediated signaling, and this is likely compromised from non-native scaffold effects. Finally, fourth, the human response to the scaffold material must be considered. No material, whether natural or synthetic, is entirely bioinert. The long-term presence of a biomaterial activates a foreign body response that drives further inflammatory activation in addition to further inflammation of the disease state. Degradation of the material scaffold is necessary to prevent end-stage fibrous encapsulation of the engrafted material. This host inflammatory response can damage the transplanted cells, resulting in failure of the engineered tissues [51]. Clearly, there is a need for a next-generation advancement from scaffold-based tissue engineering that better addresses clinical requirements with reliable, viable solutions. Cell sheet tissue engineering is the foremost approach to creating 3D cell constructs without the use of a biomaterial scaffold. By utilizing a temperature responsive polymer engrafted to a cell culture surface, cells can adhere to and proliferate across the cell culture surface at 37°C, and then detach from the culture surface at room temperature without the use of enzymatic degradation [52]. The temperature-responsive culture surface presents dual surface properties, which are cell adhesive at 37°C and cell nonadhesive at room temperature (~20°C). Cells cultured to confluence across 2D surfaces form cell-cell interactions and junctions between cells, creating an interconnected cell 8 network that is further unified by endogenously deposited ECM with which the cells form cell-matrix interactions. A reduction in temperature allows the cells to detach from the culture surface as a contiguous sheet without the need for enzymatic degradation, maintaining the cell-cell interactions and deposited ECM [53, 54]. Cell suspensions typical of conventional culture harvest on plastic can therefore be avoided. By detaching cells at confluence, cultured cells are homogenously incorporated throughout the cell sheet. The maintenance of deposited, endogenous ECM enables protein-mediated adhesion to biologic surfaces, allowing cell sheets to spontaneously engraft to a tissue surface without the need for sutures or glues. Cell sheets can be layered, mediated by sheet-to-sheet biologic adhesion, to produce 3D structures of a desired thickness and cell loading capacity. Because cell sheets are composed entirely of cells, direct contact is possible between cells and the target tissue. Cell sheets therefore allow cell therapy to be mediated by paracrine signaling, as well as by direct signaling via cell-to-cell crosstalk. This direct crosstalk with the diseased tissue provides instructive signaling to the cell sheet so that the cellular response is tailored to the therapeutic needs of the injury. 1.2 Cell Sheet Tissue Engineering This section introduces cell sheet tissue engineering for the generation of scaffold-free 3D tissue constructs. Details regarding the temperature-responsive culture dish technology, cell sheet characteristics and application feasibility, and prior MSC sheet designs, and implementations are discussed in this section. 9 1.2.1 Temperature-responsive cell culture surfaces Poly(N-isopropylacrylamide) (PIPAAm) is a temperature-responsive polymer that experiences competing forces from the interactions of water with the hydrophilic amide groups and the hydrophobic isopropyl and polyacrylamide backbone groups. PIPAAm exhibits a lower critical solution temperature (LCST) of about 32°C in water. Amide groups of PIPAAm undergo hydration with water molecules below the LCST, and hydrophobicity is increased above the LCST due to displacement of weakly hydrating waters and resulting strong associations between pendant isopropyl groups [55, 56]. Therefore, PIPAAm networks swell below the LCST but shrink above the LCST because of the reversible hydration and dehydration of the polymer chains (Figure 1.1). Considering its many advantages, including real-time monitoring, operational simplicity, high sensitivity, and fast response, PIPAAm has been widely studied in temperatureresponsive medical applications [55, 56]. Okano et al. invented temperature-responsive cell culture dishes by grafting PIPAAm onto the surface of tissue culture plastics (TCP) [53, 57]. IPAAm monomer is polymerized and covalently immobilized on the surface of TCP dishes at nanometer-scale thicknesses using several different methods, commercialized using e-beam grafting. PIPAAm-grafted temperature-responsive TCP exhibits temperature-dependent changes in wettability consistent with the LCST transitions of the non-grafted polymer form, attachment/detachment as a function of temperature. facilitating reversible cell 10 1.2.2 Harvest and manipulation of cell sheets Generally, cells can attach and grow on the hydrophobic surface of TCP, as well as on the surface of PIPAAm-grafted temperature-responsive TCP (temperatureresponsive cell culture dish, TRCD) at 37°C. When the temperature is reduced to 20°C, PIPAAm grafted on TCP spontaneously undergoes a hydration transition, with the surface wettability changing from hydrophobic to hydrophilic and the hydrating PIPAAm chains expanding away from the TCP surface. Using this approach, cells are cultured on the TRCD surface at 37 °C. When cells reach confluence, they form a sheet-like structure comprising endogenous intercellular junctions and ECMs [52]. At temperatures below 25°C (room temperature), aqueous media spontaneously penetrates the PIPAAm polymer interface between adherent cells and the TRCD, swelling and expanding PIPAAm chains under hydration and physically separating cells from TRCD surfaces without any damage to the intercellular junctions, ECMs, or cells (Figure 1.2) [54, 57]. These preserved endogenous intercellular junctions and ECMs influence cellular function and transplantation efficacy to target tissues. 1.2.3 Structural and functional features of cell sheets In conventional cell therapies, cultured cells are ultimately harvested from the cell culture dishes (TCPs) for transplantation. Since cultured cells generally attach to cell culture dishes strongly with intrinsic adhesion proteins (e.g., deposited ECM and cell membrane receptors), these adhesive proteins or receptors must be released to harvest cells from culture surfaces for experimental and clinical use [58]. To separate adherent cultured cells from TCPs, chemical disruption methods are most used. In the chemical 11 disruption method, proteolytic enzymes (e.g., trypsin EDTA or dispase) are added to cell culture media and general enzymatic digestion nonspecifically cleaves endogenous myriad proteins [59]. This uncontrolled proteolytic disruption compromises various important cell functions such as cell proliferation, adhesion, survival, and migration [60]. Significantly, the resulting harvested cell product is a single cell suspension where endogenous cell-cell associations common to tissue formation and engraftment are destroyed (Figure 1.3). Although much remains unclear, major in vivo mechanisms related to cellmediated tissue regeneration include: (1) differentiation to the desired tissue lineage to replace damaged tissue and cells and (2) paracrine factor secretion to regenerate damaged tissue and cells [53]. In a previous study, MSC sheet fabrication preserved stem cellspecific surface markers associated with differentiation potential [61]. Additionally, cell sheets prepared with human bone marrow stem cells (hBMSCs) expressed significantly higher gene expression levels of pro-regenerative cytokines (hepatocyte growth factor: HGF; vascular endothelial growth factor: VEGF; Interleukin 10: IL-10) related to antiinflammation, tissue regeneration, and vascularization, respectively, compared to a single cell suspension of hBMSCs (Figure 1.4). Also, trypsin-treated hepatocytes decreased their albumin production, while hepatocytes harvested from PIPAAm-grafted surfaces, by means of temperature reduction, preserved their albumin secretion capacity [54]. The enhanced pro-regenerative cytokine and albumin protein secretion capabilities by cells in sheet form compared to those enzymatically dissociated is likely due to the preservation of endogenous intercellular and extracellular proteins (i.e., cell adhesion proteins). The amount of cell adhesion proteins was compared between cells as cell sheets and as single 12 cell suspensions. Yamato, Okano, et al (2004) reported that cell sheets maintained significantly higher intercellular and extracellular proteins (i.e., laminin, E-cadherin, and desmosomes) compared to single cell suspensions in an immunoblotting assay [62]. Furthermore, the structure of intercellular and extracellular proteins was considerably different. Cell sheet had an aligned and connected structure of cell adhesion proteins throughout the cell sheet (i.e., F-actin, vinculin, fibronectin, laminin, integrin β-1, and connexin 43) in immunohistochemistry (IHC) analysis, while cells in suspension had cleaved cell adhesion protein structures that aggregated on the cell surface without any organized patterns [63]. These findings indicate that retaining cell adhesion proteins with structures typical of native tissue-like interconnected cells is crucial to the cells’ functionality [62, 63]. These results support 15 years of clinical findings using cell sheet technology, demonstrating enhanced therapeutic effects in heart, cornea, esophagus, periodontal ligament, middle ear, knee cartilage, and lung regeneration [62, 64-70]. 1.2.4 Review of mesenchymal stem cells in cell sheet use MSCs have been used for cell sheet fabrication to regenerate diverse tissues and support tissue repair across various disease indications, primarily mediated by the following modes of action: first, MSC sheet fabrication and directed differentiation, or in vivo transplantation for signal-induced differentiation, toward the tissue of interest to replaced damaged tissues or cells directly in the case of bone, cartilage, periodontal ligament, and hepatic tissue engineering (Figure 1.5a) [71-79]. Second, MSC sheets, transplanted in an undifferentiated state, secreted pro-regenerative cytokines to regenerate damaged tissue or cells and ameliorate disease propagation indirectly in the case of heart, 13 liver, pancreas, kidney, and brain regeneration (Figure 1.5b) [73, 80-86]. In the second case, the intrinsic paracrine capacity of MSCs was combined with the superior localized delivery and engraftment capability of cell sheet technology to increase therapeutic effects in ischemic and fibrotic diseases compared against MSC suspension injection controls. In one MSC sheet application for heart failure in a rat model, Narita and colleagues (2013) compared cardiac functional recovery following MI and myocardium retention of syngeneic bone marrow derived MSCs (BMSC) grafted either by epicardial placement as MSC sheets or by intramyocardial (IM) injection as a single cell suspension [80]. Their findings validated that epicardial placement of BMSC sheets is more effective in treating heart failure compared with IM MSC injection, primarily due to the superior localization and retention of donor cells by cell sheet transplantation, as well as establishment of communication between the sheet and the epicardium that allowed unhindered permeation of MSC-secreted molecules into the myocardium to facilitate a paracrine effect [80]. Similarly, Imafuku and colleagues (2019) compared MSC sheets transplanted onto the kidney surface and suspended MSCs administered intravenously (IV) in a rat renal ischemia-reperfusion-injury model and found that donor-cell retention and survival was superior in the cell sheet transplantation group relative to the IV administered group [85]. Additionally, cell sheet transplantation secreted VEGF and HGF for 14 days, increased the expression of activated VEGF/HGF receptors in the kidney, and strongly suppressed renal fibrosis via microvascular injury protection mediated by prolonged MSC secretion of these vasoprotective and cytoprotective cytokines [85]. These studies evidence the advantage of MSC sheets over injected MSCs in localizing and retaining grafted cells, as well as enabling local and sustained pro-regenerative 14 cytokine secretion driving therapeutic paracrine effects for the treatment of wide-ranging ischemic and fibrotic diseases. However, these studies of MSCs fabricated as cell sheets to date have only demonstrated the advantage over single MSCs in terms of in vivo localization and engraftment. No investigation has yet probed whether the 3D tissue-like structure and cell-dense, interconnected organization of MSCs in cell sheets enhance the intrinsic cytokine secretory capacity; that is, to address if individual MSCs are more paracrinepotent fabricated as a cell sheet or as a single cell. The following section explains this concept, known as the tissue effect, provides context for how the tissue effect could increase MSC potency, and introduces how cell sheet fabrication and associated in vitro engineering strategies could modulate this tissue effect. 1.3 The Tissue Effect on Cell Functional Enhancement The last 20 years have seen extensive investigation and reporting in the literature of a phenomena whereby signaling and other cellular functions differ in threedimensional (3D) compared with two-dimensional (2D) mammalian cell culture systems, notably characterized by in vitro cell geometry and adhesion structures evolving toward in vivo-like tissue organization in 3D [87-91]. Culturing cells within 3D matrices provides environments that mimic in vivo microenvironments, promoting physiologic morphology, polarity, and functions such as differentiation or complex cell organization [92-95]. This type of 3D culture has long been used as a modeling system to understand tumor biology and cell behaviors such as motility, migration, and matrix remodeling [96, 97]. 15 More recently, these advantages of 3D culture have intersected with the therapeutic use of MSCs, recognizing that MSC capacity for differentiation is enhanced by, and in many cases requires, 3D culture [98-101]. Similarly, MSC trophic factor secretion in vitro and paracrine activity in vivo is notably enhanced in 3D MSC cultures compared to single, suspended MSCs or MSCs cultured under plastic-adherence as 2D monolayers [95, 102-107]. Although MSCs are characterized by their plastic adherence, investigations into MSC immunomodulatory potential have widely established that 3D culture upregulates MSC production of anti-inflammatory cytokines compared to 2D culture [108-112], more effectively suppressing immune cell activation [113, 114] and stimulating macrophage anti-inflammatory polarization [115-117]. While the mechanisms controlling MSC signaling stimulation in 3D are not clearly defined, many groups have sought answers in the most striking differences observed between cells in 2D and 3D: morphology, and, related, the organization, composition, and abundance of cell interactions. Cells grown in a 2D monolayer are flat and can adhere and spread freely in the horizontal plane but have no support for spreading in the vertical dimension. This creates a forced apical-basal polarity and limits cell interactions to the basal plane (with the substrate) and to the cell perimeter (with neighboring cells). In one study investigating MSC therapeutic potency as 3D spheroids compared to single cells generated from monolayer culture, Lee and colleagues (2012) found that 3D MSC spheroids showed significantly higher improvement in left ventricular contractility and ejection fraction in a rat MI model [118]. To explain the enhanced therapeutic efficacy, this study determined that cell-cell interactions were essential for 3D spheroid formation, and that expression of key cell interaction proteins 16 enhanced VEGF secretion pathways essential for MSC paracrine activity [118]. In a similar study utilizing biomaterials, Qazi and colleagues (2017) demonstrated that 3D cell-cell interactions between MSCs within porous scaffolds were responsible for increased in vitro MSC cytokine secretion and enhanced paracrine-mediated myoblast migration and proliferation compared to both 2D plastic-adherent MSCs and hydrogel encapsulated MSCs [119]. These studies, among others, illustrate a well-established correlation between 3D tissue-mimetic culture upregulation of cell-cell and cell-matrix interactions and increased MSC paracrine benefit compared to single cell and 2D monolayer culture. In the context of improving cell function by introducing tissue structures and tissue-like organization, we have defined the tissue effect: physical interactions between cells and matrices, as well as biochemical interactions facilitated by adhesions interactions and cross-membrane-communication junctions, promote improved (or closer to physiologic) cell functions. This tissue effect was verified in a study within our group comparing enzyme treated single MSCs that have had all physical and biochemical interactions cleaved, rendering morphologically spheroidal cells, and MSCs cultured to high-density confluence as an adherent 2D monolayer [63]. Nakao and colleagues (2019) identified the importance of the structure of cell interactions: while single MSCs expressed cell-cell adhesion proteins (vinculin), gap junction proteins (connexin 43), cellECM adhesion proteins (integrin ß1) and well as extracellular proteins related to cell adhesion (fibronectin, laminin) cleaved on their surface, the confluent 2D MSC monolayers demonstrated a tissue-like interconnected structure of these cell adhesion proteins that correlated with significantly higher gene expression of paracrine factors 17 VEGF, HGF, and IL-10 per cell relative to the single MSC condition [63]. Cell shape was also evaluated, showing the actin cytoskeleton aligned as stress fibers between cell-cell attachment interfaces, whereas actin within the single MSCs had not been actively polymerized and showed no signs of stress fiber formation [63]. This study highlighted the importance of physical interactions between cells that impart biochemical adhesion and communication interactions to enhance MSC function. Cell sheet tissue engineering provides a unique opportunity to exploit the tissue effect for MSC therapy. Building from Nakao’s (2019) findings that 2D monolayer culture, characterized by cell-cell and cell-matrix binding interactions, enhances MSC cytokine secretion capacity relative to single MSCs, this dissertation fabricates MSCs as scaffold-free, 3D cell sheets. In transition from confluent culture, temperature-responsive cell detachment induces spontaneous contraction into a 3D cell sheet, structurally comprising cell-dense interactions and culture-derived matrix with morphologically changed constituent cells. Given current understanding that 3D culture enhances clinically relevant MSC paracrine function, this dissertation describes for the first time using scaffold-free 3D cell sheet culture to achieve this MSC functional improvement. While cell sheet centrifugation has been extensively employed to construct multilayered functional tissues in vitro to replace diseased tissue in vivo [120-124], centrifugation as a tool to manipulate 3D cellular interactions and stimulate intrinsic MSC cytokine production in cell sheets has never been previously investigated. This dissertation explores centrifugation as a tool to form biochemical interactions and binding protein formation within 3D cell sheets, as well as a means to generate viable, multilayered MSC sheets with controlled thicknesses and cell doses to increase absolute 18 MSC sheet construct secreted cytokine dose. This dissertation provides a novel framework for engineering the tissue effect within cell sheets, as well as a clear measure of the value of the tissue effect in the context of MSC cytokine production function. 1.4 Human Umbilical Cord Mesenchymal Stem Cells Human umbilical cord (hUC) provides a rich source of MSCs that can be derived from the cord blood [125], Wharton’s Jelly [126], as well as the subepithelial layer of the cord lining [127]. hUC-MSCs are being increasingly researched and evaluated for clinical use considering their ease of tissue sourcing from patient birthing discards and cell isolation relative to other MSC sources that require patient biopsies [128, 129]. Additionally, in contrast to adult stem cells, UC-derived MSCs demonstrate a faster rate and higher capacity of self-renewal owing to their fetal annex origin [130]. The original experiments and data reported in this manuscript use an MSC population isolated from the subepithelial layer of umbilical cord tissue. This hUC-MSC population exhibits the minimal criteria for definition as a multipotent mesenchymal stromal cell by The International Society for Cellular Therapy, demonstrating adherence to plastic maintained in standard culture conditions, surface antigen expression (≥95%) of CD73 (cluster of differentiation 73), CD90, and CD105, and multipotent differentiation to osteoblasts, adipocytes, and chondroblasts in vitro [131]. In a previous study, we characterized these subepithelial-derived hUC-MSCs across subculture passage numbers 4 through 12 [61]. Cell growth rates showed 16- to 20-fold increases from initial cell seeding numbers through passages 4-8, declining to 14fold in passage 9 to 3.1-fold in passage 12 [61]. Regarding multipotency, hUC-MSCs 19 showed a strong capacity for directed differentiation to osteogenic and adipogenic cell lineages in passages 6 through 10, indicated by Alizarin Red and Oil Red O staining, respectively, but showed minimal differentiation in passage 12 [61]. Similarly, hUCMSCs expressed a surface phenotype consistent with mesenchymal stem cells from passage 6 to 10, showing positive expression of CD73, CD105, and CD90; passage 12 cells showed lower positive expression of these surface antigens by flow cytometry [61]. To achieve cell numbers useful for fabricating high cell density cell sheets that maintained a defined MSC phenotype, hUC-MSCs in passage 6 were selected for cell sheet fabrication and experimentation reported in the body chapters of this dissertation. Considering potential future translational implications for ischemic and fibrotic diseases, hUC-MSC are a promising candidate for therapeutic cell sheet development because of their high paracrine potency and low immunogenicity relative to other wellinvestigated MSC sources. hUC-MSCs secrete proteins important for tissue regeneration following ischemic and fibrotic injury, namely vascular endothelial growth factor (VEGF; angiogenic [132]), hepatocyte growth factor (HGF; anti-apoptotic [133]), and interleukin-10 (IL-10; anti-inflammatory [134]) [63, 135]. Additionally, hUC-MSCs showed significantly lower relative gene expression of major histocompatibility complex (MHC) class II antigens (known to activate host immune systems against allogenic agents), HLA-DR, DP, and DQ, per cell compared to bone marrow-derived and adipose tissue-derived MSCs [61]. Having established this baseline phenotype, function, and antigenicity profile for hUC-MSCs as single cells, the work described in this dissertation investigates how the tissue effect contributed by 3D cell sheet fabrication and culture, modulated by 20 centrifugation, alters, enhances, or preserves hUC-MSC clinically relevant character and behavior. 1.5 Dissertation Overview: Motivation, Aims, and Scope Though MSC therapies are currently reported in over 900 clinical trials globally (see clinicaltrials.gov), few convincing pivotal trial results and virtually no approved cell therapy products have yet been realized. Despite growing expectations and research familiarity, current human MSC therapies administered primarily as dissociated cell suspension injections are underwhelming in human translation. These poor clinical outcomes are attributed in large part to the low in situ engraftment and retention (i.e., poor dosing), and low potency (i.e., survival and concentrated paracrine factor delivery) of MSCs administered by suspension injection into circulation [23, 39, 40]. There is a clear need for a new method of MSC administration that delivers the MSC dose directly to the target tissue with high-fidelity localization and engraftment, simultaneously enabling cell survival, paracrine signaling and the intended pharmacological function. Regarding MSC’s mechanism of action, contemporary studies and clinical applications focus on paracrine growth factors and immunomodulatory factors as key mediators of MSC’s therapeutic effect, identified to protect injured tissues and encourage endogenous repair mechanisms [136]. Arnold Caplan has recently made clear his position that MSCs should be viewed as “medicinal signaling cells” that provide a unique pharmacology delivered by paracrine actions [137]. Many investigators and industrydriven studies have hence focused on MSC product optimization using conditioning strategies to boost MSC pro-regenerative cytokine production capacity [138-140]. 21 Cytokine and growth factor priming has been widely implemented to increase MSC potency [141], but these tactics do not address the accompanying issue limiting MSC paracrine efficacy, that is the mode of cell delivery. Interestingly, tissue engineering provides a solution for both considerations: first, by engineering cells into 3D tissues, an engraftable, dose-controlled structure that promotes cell adhesion and survival is available for localized administration. Additionally, 3D culture is a well-established method for MSC conditioning to increase paracrine potency, particularly when compared to 2D adherent culture that precedes cell suspension harvest for injection [95, 142, 143]. Engineering 3D MSC tissues, therefore, simultaneously generates highly functional and locally transplantable MSC therapy products. Over 20 years of clinical data assert the clinical advantages of cell sheet tissue engineering in overcoming the therapeutic limitations of suspension-based cell therapy [64, 65, 67-69, 144-149]. Cell sheets retain endogenous cell matrix, receptors, and adhesive proteins that permit spontaneous, protein-mediated adhesion to biological surfaces without suturing, enabling high-fidelity cell dose administration, localization, and engraftment at the target tissue [150]. Furthermore, cell sheets preserve myriad proteins responsible for key cell functions of proliferation, adhesion, survival, and migration that are disrupted in the preparation of cell suspensions [54]. Seven first-inhuman clinical studies/trials have been initiated, applying cell sheets for multiple disease indications based on superior cell dose administration and engraftment to support therapeutic outcomes [62, 64-70]. The idea that these intrinsic advantages of cell sheet technology could be applied to resolve issues of MSC therapy administration laid the foundation for this dissertation’s work, engineering MSC sheets as a platform technology 22 that could be applied for various disease indications. Additionally, our group reported significantly greater pro-regenerative cytokine secretion capacity in MSCs fabricated as cell sheets relative to MSCs dissociated into single cell suspensions (Figure 1.4), indicating that not only could cell sheets provide the advantage of concentrating MSC paracrine factor secretion to the target tissue through high-fidelity localization, acting as therapeutic factor delivery depots, but that the intrinsic 3D tissue-like structure that cell sheet fabrication provides contributes to enhanced individual MSC function. This finding provided the impetus to investigate the tissue effect contributed by 3D cell sheet fabrication, and how this correlates with MSC functional augmentation, defined as the stimulation of intrinsic MSC cytokine production. Further, understanding of the cell sheet tissue effect prompted investigation into centrifugation strategies to further modulate tissue interactions within the cell sheet that could reciprocally augment MSC function. Therefore, the motivation driving this dissertation was to overcome the limitations of clinical gold standard MSC suspension injections by engineering engraftable, dosecontrolled, scaffold-free MSC sheet tissue, as well as to maximize MSC therapeutic potency for tissue regeneration by enhancing individual MSC cytokine secretory capacity through a tailored tissue effect. Ultimately, this dissertation delivers a complete in vitro platform for engineering, centrifuging, and multilayering MSC sheet tissue optimized for MSC cytokine production, with adaptable parameters available for sheets of other cell types. Further, this research generates highly functional MSC sheets as a 3D cell-delivery technology immediately available for therapeutic efficacy studies as a bioactive factor delivery depot supporting tissue regeneration. 23 The central hypotheses of this dissertation are that the tissue effect is fundamentally realized using cell sheet tissue engineering, and that 3D structure and instances of physical and biochemical cell-cell and cell-matrix interactions can be enhanced within cell sheets to stimulate individual MSC cytokine production potency. Specifically, we hypothesize that (1) cell sheet spontaneous 3D structural and morphological transition, characterized by cell shape change and increased instances of cellular interactions, augments individual MSC cytokine production relative to the 2D monolayer counterpart, that (2) cell sheet centrifugation increases cell-experienced 3D cellular interactions, augmenting individual MSC cytokine production relative to noncentrifuged sheets, and that (3) cell sheet multilayering from centrifugation generates viable 2-layered constructs with controlled thicknesses and cell doses, increasing absolute MSC sheet construct secreted cytokine dose. To test these hypotheses, three specific aims were pursued as outlined in Sections 1.4.1 through 1.4.3. 1.5.1 Aim 1: Demonstrate that 3D cell sheet structure and cellular interactions augment individual MSC pro-regenerative cytokine (VEGF, HGF, IL-10) secretion capacity in vitro Adherent cells grow to confluence with endogenous matrix deposition, forming adhesion interactions and junction with the matrix and with adjacent cells [54, 151]. Cell sheets’ temperature-mediated detachment from adherent monolayer culture induces spontaneous contraction into a continuous, 3D tissue-like structure with upregulated cellular interactions [79, 152]. Relative to 2D adherent culture, 3D culture is known to 24 upregulate MSC paracrine mechanisms [95, 143]. Therefore, the goal of this aim is to combine cell sheet engineering with MSCs to fabricate a 3D cell delivery platform that boosts intrinsic MSC pro-regenerative cytokine secretion potential. The hypothesis of aim 1 is that spontaneous post-detachment cell sheet contraction contributes a 3D structural and morphological transition with higher abundance cellular interactions necessary to augment individual MSC cytokine secretory capacity. To address this hypothesis, studies described in detail in Chapter 2 of this dissertation evaluated: (i) cell sheet post-detachment tissue structural differences following the 2D-to-3D transition from adherent monolayer culture; (ii) cytoskeletal, morphological, and cellular interaction differences in 3D contracted sheet and 2D adherent monolayer culture; (iii) differences in pro-regenerative cytokine production per MSC in 3D cell sheets and 2D monolayers [152]. Specifically, human umbilical cord MSCs (hUC-MSCs) were prepared as confluent 2D adherent cultures and as standard cell sheets using temperature-responsive culture dishes (TRCDs). Cell sheets were allowed to spontaneously contact upon temperature-mediated harvest and were compared to 2D adherent monolayers for changes in tissue thickness, diameter, and volume. Analysis of cell shape 3D transition in response to cell sheet contraction included staining for cytoskeletal organization (f-actin) and nuclei shape (DAPI) with nuclei circularity quantified. Differences in cell-experienced cellular and tissue interactions in 2D monolayers and 3D cell sheets were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). Soluble pro-regenerative cytokine production by 2D monolayers and 3D cell sheets was quantified using an enzyme-linked immunosorbent assay (ELISA) and normalized per cell in each construct to measure the impact of 3D 25 culture on individual MSC secretory function. This aim introduced cell sheet technology as a 3D culture platform that enhances MSC paracrine capacities attributed to improved MSC clinical utility. 1.5.2 Aim 2: Demonstrate that cell sheet centrifugation augments individual MSC pro-regenerative cytokine (VEGF, HGF, IL-10) secretion capacity by directly increasing 3D cellular interactions Therapeutic factors secreted by MSCs promote tissue regeneration and immunomodulation in vivo [153-156]. However, delivery of MSCs in the absence of an engraftable environment offers limited efficacy due to low cell retention, poor graft survival, and the non-maintenance of a physiologically relevant dose of growth factors at the injury site [34, 35]. While the cell-delivery platform was previously thought to be inert, there is an increasing body of evidence that the delivery of MSCs on 3D platforms that support physical and cellular interactions increases MSC paracrine activity and resultant therapeutic outcomes [95, 103, 105, 107, 143]. Cell sheet engineered tissue is scaffold-free with no biomaterial interruption between cellular and tissue interactions. Therefore, the goal of aim 2 to was to exploit centrifugation as a means of compacting cell sheets and introducing higher abundance 3D cellular interactions. The hypothesis is that centrifuging 3D MSC sheets enhances cell-experienced 3D cellular interactions, augmenting individual MSC cytokine production relative to non-centrifuged sheets. To address this hypothesis, studies described in detail in Chapter 3 of this dissertation determined: (i) optimal parameters for stable and consistent cell sheet centrifugation and sustainable subsequent aqueous culture; (ii) effects of MSC sheet 26 centrifugation on cell packing arrangement, construct thickness, cytoskeletal structure, and paracrine-related cellular interactions; (iii) effects of sheet centrifugation on MSC pro-regenerative cytokine production over time. Specifically, culture surface conditions were optimized to support stable and consistent cell sheet adhesion under centrifugal force. Centrifugation speed and duration were selected for their ability to support stable and reliable cell sheet adhesion to the culture surface under aqueous culture conditions. Once a centrifugation method was established, centrifuged hUC-MSC sheets were evaluated for cell packing arrangement, tissue thickness, cell viability, and cytoskeletal changes relative to non-centrifuged sheets. Cellular interaction gene expressions were quantified with qRT-PCR. To determine the sustained impact of centrifugation on MSC cytokine production function, soluble cytokines were quantified over 4 days in static culture and normalized per cell in centrifuged and non-centrifuged constructs. This aim delivers a platform for highly generating cytokine secretory MSCs by exploiting sheet centrifugation’s ability to enhance 3D cellular interactions. 1.5.3 Aim 3: Demonstrate that cell sheet multilayering from centrifugation increases absolute MSC sheet construct secreted pro-regenerative cytokine (VEGF, HGF, IL-10) dose MSC paracrine function is arguably one of their most clinically beneficial attributes, mediated by MSC secretion of myriad pro-regenerative cytokines and subsequent host-mediated tissue regeneration [19, 98, 153, 157, 158]. The inability of injection delivered MSCs to localize at the site of injury dilutes potential paracrine benefit, with clinical applications compensating by administering higher absolute cell 27 doses [24, 36-38]. Cell sheet tissue engineering fundamentally resolves the issues of cell delivery by localizing and engrafting the full cell dose directly to the site of tissue injury [68, 159-161]. Still, the exact cell dose required to achieve physiologically relevant paracrine benefit is unknown, and larger cell doses within 3D cell-delivery platforms could provide higher concentrations and sustained therapeutic factor release. The number of cells in a single cell sheet can be tailored with initial cell seeding density, but maximal density is limited by the surface attachment area. Cell sheet layering is a well-studied approach for doubling the absolute cell dose within engraftable cell sheet constructs, rendering larger functional benefits in vivo [83, 120-123, 144, 162]. Therefore, toward the generation of an optimal 3D MSC delivery platform, the goal of aim 3 is to generate multilayered MSC sheets that provide incremental control over MSC-secreted growth factor dose from the 3D tissue construct. The hypothesis of this aim is that cell sheet multilayering from centrifugation generates viable 2-layered constructs with controlled thicknesses and cell doses, increasing the absolute MSC sheet construct-secreted cytokine dose. To address this hypothesis, studies described in detail in Chapter 4 of this dissertation determined: (i) the incremental thickness, cell number, and cell viability change from 1-layer to 2-layer centrifuged and non-centrifuged sheets; (ii) the effects of multilayering from centrifugation on 3D cellular interactions and MSC pro-regenerative cytokine production compared to statically multilayered sheets; (iii) the sustained increase in absolute secreted cytokine dose contributed by MSC sheet multilayering. To do so, 1-layer and 2-layer MSC sheets fabricated by conventional, static methods or by centrifugation methods were histologically analyzed for tissue thickness. Conventional 28 layering did not produce a 2-to-1 increase in tissue thickness relative to the single layer, prompting investigation into the hypoxic conditions and cell viability of statically multilayered sheets. Differences in cellular interaction gene expressions between 2-layer conventional and centrifuged cell sheets were quantified with qRT-PCR. MSC cytokine production was quantified by ELISA to measure the impact of centrifugation multilayered on MSC function, as well as to determine the incremental increase in secreted cytokine dose contributed by sheet multilayering. This aim delivers a method for increasing the absolute therapeutic factor dose secreted by 2-layer cell sheets of controlled thicknesses and cell numbers by exploiting cell sheet multilayering from centrifugation. 29 Figure 1.1 Poly(N-isopropylacrylamide) (PIPAAm). Poly(N-isopropylacrylamide) (PIPAAm) swells below the lower critical solution temperature (LCST: 32°C) and shrinks above 32°C. The image was created using BioRender.com. The figure is reprinted from Kim, K., Bou-Ghannam, S., Okano, T. Cell sheet tissue engineering for scaffold-free three-dimensional (3D) tissue reconstruction, Methods in Cell Biology, 157 (2020), 143-167, with permission from Elsevier. 30 Figure 1.2 Temperature-responsive cell culture dishes (TRCD). Cells are recovered from a temperature-responsive cell culture dish (TRCD) as a sheet with intact endogenous cell adhesion proteins by simple temperature reduction. Hydration of grafted PIPAAm polymer below its LCST swells the surface, pushing away adherent cells from the TRCD. The figure is reprinted from Kim, K., Bou-Ghannam, S., Okano, T. Cell sheet tissue engineering for scaffold-free three-dimensional (3D) tissue reconstruction, Methods in Cell Biology, 157 (2020), 143-167, with permission from Elsevier. Figure 1.3 Conventional enzymatic cell harvest yielding a cell suspension. Conventional cell therapy depends on enzymatic harvest to collect cells from the cell culture surface. Enzyme treatment disrupts endogenous cell adhesion proteins, yielding cells in media suspension. TCP: tissue culture plastic. The figure is reprinted from Kim, K., Bou-Ghannam, S., Okano, T. Cell sheet tissue engineering for scaffoldfree three-dimensional (3D) tissue reconstruction, Methods in Cell Biology, 157 (2020), 143-167, with permission from Elsevier. 31 Figure 1.4 Cell sheet harvest enhances MSC pro-regenerative cytokine production capacity relative to single cell harvest. Pro-regenerative cytokine secretion is increased when cells are harvested as a sheet compared to an enzymatically collected single cell suspension. hBMSCs were used for this experiment. The single cell group was harvested from TCP using enzyme treatment. The cell sheet group was detached from TRCD by temperature reduction from 37°C to room temperature. HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; IL-10, Interleukin 10. *p < 0.05 (n=3) and NS, no statistically significant difference. The figure is reprinted from Kim, K., Bou-Ghannam, S., Okano, T. Cell sheet tissue engineering for scaffold-free three-dimensional (3D) tissue reconstruction, Methods in Cell Biology, 157 (2020), 143-167, with permission from Elsevier. 32 Figure 1.5 Concept for cell sheet tissue regeneration. Conceptual illustration of tissue regeneration using cell sheet technology. Cell sheet (A) directly replaces damaged tissue and cells, and (B) secretes pro-regenerative cytokines to stimulate host-mediated regeneration of damaged tissue and cells (paracrine stimulus). The image was created using BioRender.com. The figure is reprinted from Kim, K., Bou-Ghannam, S., Okano, T. 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Gimble, Adipose tissue derived stem cells secretome: soluble factors and their roles in regenerative medicine, Curr Stem Cell Res Ther 5(2) (2010) 103-10. [156] A. Trounson, C. McDonald, Stem Cell Therapies in Clinical Trials: Progress and Challenges, Cell Stem Cell 17(1) (2015) 11-22. [157] A.I. Caplan, Why are MSCs therapeutic? New data: new insight, J Pathol 217(2) (2009) 318-24. [158] T. Squillaro, G. Peluso, U. Galderisi, Clinical Trials With Mesenchymal Stem Cells: An Update, Cell Transplant 25(5) (2016) 829-48. [159] T. Iwata, M. Yamato, H. Tsuchioka, R. Takagi, S. Mukobata, K. Washio, T. Okano, I. Ishikawa, Periodontal regeneration with multi-layered periodontal ligamentderived cell sheets in a canine model, Biomaterials 30(14) (2009) 2716-23. [160] T. Ohki, M. Yamato, M. Ota, R. Takagi, D. Murakami, M. Kondo, R. Sasaki, H. Namiki, T. Okano, M. Yamamoto, Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets, Gastroenterology 143(3) (2012) 582-588 e2. 47 [161] N. Yamaguchi, H. Isomoto, S. Kobayashi, N. Kanai, K. Kanetaka, Y. Sakai, Y. Kasai, R. Takagi, T. Ohki, H. Fukuda, T. Kanda, K. Nagai, I. Asahina, K. Nakao, M. Yamato, T. Okano, S. Eguchi, Oral epithelial cell sheets engraftment for esophageal strictures after endoscopic submucosal dissection of squamous cell carcinoma and airplane transportation, Sci Rep 7(1) (2017) 17460. [162] T. Shimizu, M. Yamato, T. Akutsu, T. Shibata, Y. Isoi, A. Kikuchi, M. Umezu, T. Okano, Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets, J Biomed Mater Res 60(1) (2002) 110-7. CHAPTER 2 3D CELL SHEET STRUCTURE AUGMENTS MESENCHYMAL STEM CELL CYTOKINE PRODUCTION Bou-Ghannam, S., Kim, K., Grainger, D. W. & Okano, T. 3D cell sheet structure augments mesenchymal stem cell cytokine production. Sci Rep 11, 8170, doi:10.1038/s41598-021-87571-7 (2021). https://doi.org/10.1038/s41598-021-87571-7. Copyright © 2021, The Author(s). Reprinted with permission from Springer Nature. 49 2.1 Abstract 2.2 Introduction 50 2.3 Results 51 Figure 2.1 Microscopic cell morphology influences macroscopic tissue structure. Figure 2.2 Spontaneous cell sheet contraction contributes a 3D tissue-like structure. 52 Figure 2.3 hUC-MSC actin structure changes in response to cell sheet contraction. 2.4 Discussion 53 Figure 2.4 Enhanced pro-regenerative cytokine gene expression related to 3D cell sheet tissue-like structure. Figure 2.5 Cell sheet contraction increases actual cytokine production by hUC-MSCs. 54 2.5 Conclusions 2.6 Materials and Methods 55 Figure 2.6 MSC sheet 3D structural and molecular transition. 56 2.7 References 57 58 59 CHAPTER 3 CELL SHEET CENTRIFUGATION AUGMENTS MESENCHYMAL STEM CELL CYTOKINE PRODUCTION Bou-Ghannam, S., Kim, K., Grainger, D. W. & Okano, T. Cell sheet multilayering from centrifugation generates high cytokine secretory mesenchymal stem cell tissue. In submission (2021). 3.1 Abstract The focal advantage of cell sheet technology has been as a scaffold-free 3D celldelivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence now also demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment conditions mesenchymal stem cells (MSCs), stimulating their proregenerative cytokine production. In this capacity, cell sheets not only function as 3D cell-delivery platforms, but also prime MSC therapeutic paracrine capacity. Toward optimizing 3D MSC sheets for cell therapy, this study expands on our previous assessment of cell-cell and cell-matrix interactions in 3D cell sheets and their role in boosting MSC cytokine production potency by introducing centrifugation as a noninvasive tool to augment MSC-experienced cellular interactions. To achieve this goal, cell sheets fabricated by temperature-mediated harvest were centrifuged using optimized 61 conditions of rotational speed and time to support tight adhesion to the culture surface. Centrifugation distinctly rendered a dense cell-packing arrangement within the sheet and compacted the overall sheet thickness 1.8-fold. Enhanced cell physical interactions due to dense packing is supported by measured upregulation of genes related to intercellular communication and matrix interactions, including connexin 43, integrin β1, and laminin. Cell sheet centrifugation triggered functional enhancement of individual MSCs, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), notably regulated by 3D culture and increased cellular interactions. Together, these data validate cell sheet centrifugation as a non-invasive tool for enhancing MSC therapeutic potency. 3.2 Introduction Mesenchymal stem cell (MSC)-based therapy is a promising alternative to the administration of growth factors and genes to treat complex tissue degenerative diseases that implicate multiple biological pathways [1]. Clinical efforts have increasingly shifted toward exploiting MSCs for their paracrine-centric therapeutic mechanism, whereby MSCs secrete a broad range of bioactive cytokines and growth factors that can stimulate nearby cells via paracrine signaling [2-4]. This MSC paracrine effect guides tissue regeneration by exerting immunomodulation and stimulating progenitor cell angiogenesis, proliferation, and differentiation in the target tissue [5-7], and has been therapeutically utilized for applications in wound healing [8], myocardial repair [9], and liver fibrosis mitigation [10]. However, MSC delivery in the absence of a transplantable graft or scaffold offers limited clinical efficacy due to low cell retention, poor graft 62 survival, and non-maintenance of a physiologically relevant dose of growth factors at the injury site [11, 12]. Engineering MSCs as 3D constructs inherently resolves critical issues of cell survival and localization by structuring cells within scaffold or hydrogel microenvironments, or as self-aggregated spheroids, that can be directly engrafted to the injured tissue [13, 14]. 3D MSC-delivery platforms therefore operate as local bioactive drug delivery depots that concentrate and sustain therapeutic factor delivery [15]. Historically, tissue engineered delivery systems were considered inert structural platforms for cellular attachment and survival [16]. With rapid advancements in understanding how the tissue microenvironment instructs MSC behavior and function, 3D culture is now well understood to provide MSCs with critical morphological and biochemical cues that directly impact MSC paracrine potency [17, 18]. Particularly, 3D environments that promote cell-cell and cell-matrix interactions boost MSC cytokine production in vitro corresponding with enhanced paracrine benefit in vivo. For instance, Thomas et al (2014) demonstrated that higher abundance cell-matrix interactions within extracellular matrix (ECM) microgels mediated via integrin ligand signaling promoted angiogenic paracrine human MSC phenotypes [19, 20]. Qazi et al (2017) isolated cell-cell interaction protein formation responsibility in augmenting MSC paracrine function, demonstrating that MSC-seeded porous scaffolds that enabled cell-cell contact significantly increased paracrine-mediated myoblast migration and proliferation compared to hydrogel encapsulated MSCs with inhibited intercellular contact [21]. Consistently, MSC spheroids that enable intercellular adhesion protein [22] and gap junction protein [23] formation demonstrated superior paracrine potency compared to 63 dissociated MSCs and to 2D cultured MSCs that are respectively deficient in or void of cellular interactions. Additionally, fundamental advances in understanding of the MSC secretome has shown that secreted extracellular vesicles (EV) may be dominating the observed paracrine response by trafficking these regenerative proteins and RNAs directly into the target cells (see Section 6.2); 3D culture has similarly been recognized to improve MSC-EV yield and therapeutic efficacy [24-27]. Cell sheet tissue engineering, a method for generating scaffold-free 3D tissues, intrinsically comprises cell-cell and cell-matrix interactions. Utilizing commercial temperature-responsive polymer-grafted cell culture dishes (TRCD), cells grown to confluence deposit ECM and form adhesion interactions and junctions with the ECM and with adjacent cells. Aqueous temperature reduction from 37°C to 20°C prompts a surface property change from hydrophobic to hydrophilic that releases the confluent layer from the surface [28, 29], inducing spontaneous contraction that reorganizes the interconnected sheet into a 3D tissue comprised entirely of cells and endogenous matrix [30, 31] (see Figures 1.1-1.5 and description, Chapter 1). The focal advantage of cell sheet technology has been superiority as a 3D cell-delivery platform: stably biologically adhesive without requiring suturing [32-36], absent any interruption of transplanted cell and host tissue communication commonly caused by biomaterial scaffolds or encapsulations [37-39], and supportive of sustained cell engraftment, survival, and reparative function [40]. For example, MSC sheet transplantation studies attribute improved outcomes in ischemic heart failure to superior engraftment, survival, and localized paracrine function compared to systemically administered MSCs [40, 41]. Recently, though, it has been demonstrated that the intrinsic cell sheet 3D tissue-like microenvironment conditions the constituent 64 MSCs, stimulating pro-regenerative cytokine gene expression and production in response to 3D organization that increases cell-cell and cell-matrix interactions relative to MSCs cultured as 2D adherent monolayers [30]. Cell sheets not only function as 3D celldelivery platforms, but also prime MSC therapeutic paracrine capacity. Toward optimizing 3D MSC sheets as bioactive factor delivery depots, this study expands on our previous assessment of cell-cell and cell-matrix interactions in 3D cell sheets and their role in boosting MSC cytokine production potency [30]. Because cell sheets contain no interruptive biomaterial scaffolding, comprising entirely interacting cells and endogenous matrix, we expected centrifugation could be used as a tool to noninvasively increase instances of physical and biochemical cellular interactions related to cytokine secretion within MSC sheets. This study optimizes conditions for stable and reliable cell sheet centrifugation, and evaluates centrifuged tissue structure, viability, cellular interaction variations, and pro-regenerative cytokine production over time relative to conventionally prepared cell sheets. 3.3 Results 3.3.1 Cell sheet adhesion optimization for stable centrifugation Pre-coating of the cell sheet culture surface was optimized to prevent cell sheet sliding or deformation during centrifugation. The insert membranes were coated with FBS to promote cellular adhesion. In a test of 4 cell sheets per group, insert membranes pre-coated for 1 (Figure 3.1a) or 16 (Figure 3.1b) hours were compared for rate of cell sheet deformation following centrifugation at 1030 relative centrifugal force (g force, RCF) for 30 seconds (Figure 3.1e). On 1-hour FBS coated insert membranes, 25% 65 experienced cell sheet deformation during centrifugation (Figure 3.1c), while 16-hour coating demonstrated no deformation (Figure 3.1d). To prevent cell sheet sliding under centrifugation, insert membranes were FBS pre-coated for 16 hours. Additionally, excess medium remaining at the cell sheet-insert membrane interface could inhibit stable cell sheet adhesion to the culture surface following centrifugation. To remove excess interfacial medium without dehydrating the cells, adherent cell sheet tilting time on the culture surface was optimized. In the case of 1-minute tilting, the blue-dyed solution seeped past the cell sheet perimeter, indicating medium remained at the cell sheet-insert membrane interface (Figure 3.1g). For 10-minute tilting, the blue-dyed solution concentrated on the cell sheet surface, indicating the cell sheet had drained this excess medium from the cultureware and was visually dehydrated (Figure 3.1i). After 5-minute tilting, the blue-dyed solution spread to the perimeter of the cell sheet, indicating that excess interfacial medium had been cleared without drying the cell sheet (Figure 3.1h). From these findings, 5-minute tilting was implemented for the sheet centrifugation protocol. 3.3.2 Quantification of cell sheet adherence to the culture surface Cell sheet adherence to the culture surface is necessary for further culture and analysis of 1-layer centrifuged sheets, as well as for consistent and stable subsequent sheet layering. Adherence under a range of centrifugation speeds and durations was evaluated quantitatively by a mechanical rotation test. Cell sheets centrifuged for 2 minutes at 29 RCF maintained adherence to the insert membrane surface in all three trials; however, cell sheets centrifuged at 29 RCF for 1 minute detached partially from the surface in one of three trials, and when centrifugation duration was decreased to 30, 66 15, and 5 seconds, cell sheets detached from the surface under mechanical rotational shear testing in all three trials, indicating weak adhesion (Table 3.1). Centrifugation at 114 RCF for 2 minutes adhered cell sheets in all 3 trials but demonstrated weaker adhesion with only 1 minute (66% adherence) or 30 seconds (33% adherence) of centrifugation, and consistently failed to adhere the cell sheet with only 15 and 5 seconds of centrifugation (Table 3.1). Increasing centrifugation speed to 458 RCF consistently maintained cell sheet adherence to the surface in 1 minute and in 30 seconds, although partial cell sheet detachment was observed with only 15 seconds (66% adherence) or 5 seconds (33% adherence) of centrifugation (Table 3.1). Cell sheets centrifuged at 1030 RCF for 30, 15, or 5 seconds were adhered in all three trials following mechanical rotation testing, indicating that this speed forced tight adherence between the cell sheet and the culture surface (Table 3.1). Compared to the conventional incubation method that requires 1 hour to tightly adhere the cell sheet to the culture surface, centrifugation shortened adherence time to 2 minutes at a maximum, and 5 seconds at a minimum. Cell sheets centrifuged at 1832 RCF for 5 seconds displayed visible deformation due to sliding in all three trials, therefore considered a failure for stable cell sheet centrifugation and not tested for adherence under mechanical rotational shear (Table 3.1). 3.3.3 Centrifugation compacts 1-layer cell sheet tissue structure Histological images of conventional and centrifuged (114 RCF, 2 minutes) 1layer cell sheet cross-sections stained with H&E are shown in Figure 3.2. Within 1-hour after fabrication, conventional cell sheets show a loose cell packing arrangement within the tissue structure and an inconsistent apical tissue surface (Figure 3.2a). Conversely, 67 centrifuged cell sheets 1-hour after fabrication have a visibly tight-packed physical arrangement; the apical tissue surface is level and flat, rendering a consistent thickness across the tissue (Figure 3.2c). The cross-sectional tissue structure of centrifuged cell sheets 24 hours after fabrication (Figure 3.2d) was 1.8-fold decreased in thickness (Figure 3.2e) compared to conventional cell sheets (Figure 3.2b) (22 ± 3.2 μm and 41 ± 7.3 μm, respectively (p < 0.0001)). To confirm that this tight-packed physical structure due to centrifugation does not contribute to hypoxic tissue conditions, the relative gene expression of HIF-1α per MSC was compared against the conventional cell sheet at 24 hours. No significant upregulation of HIF-1α in centrifuged cell sheets was measured (Figure 3.2f). Additionally, the population percentage of dead cells was not significantly different between conventional and centrifuged cell sheets at 24 hours, suggesting that the near halving in tissue thickness was not contributed by cell loss due to cell death (Figure 3.2g). The cytoskeletal structure, a determinant of cell shape, was investigated as a potential contributor to the tight-packed tissue structure in centrifuged cell sheets. While decreased slightly on average, cytoskeleton related β-actin gene expression was not significantly different between conventional and centrifuged cell sheets at 24 hours (Figure 3.2h). 3.3.4 Centrifugation enhances tissue interaction gene expression in 1-layer cell sheets Centrifugation clearly increases physical interactions between cells within 1-layer cell sheets, evidenced by the visible tight-packed tissue structure 1 hour after fabrication. To understand how centrifugation impacts cell biochemical interactions, relative gene 68 expression of cell-cell and cell-matrix interaction proteins was compared between conventional (i.e., uncentrifuged) and centrifuged 1-layer sheets 24 hours after fabrication. Gene expression for connexin 43, a cross-membranal gap junction protein that forms between physically adjacent cells and facilitates direct intracellular molecular signaling exchange, is significantly upregulated in centrifuged cell sheets relative to conventional cell sheets (p = 0.020) (Figure 3.3b). Gene expression for integrin β1, a cell surface receptor that extracellularly binds ECM ligands, and, concomitantly, laminin, a cell-adhering ECM glycoprotein, was significantly upregulated due to cell sheet centrifugation (p = 0.0011 and p = 0.011, respectively) (Figure 3.3c and 3.3d, respectively). Gene expression for β-catenin, that intracellularly binds extracellular cadherin to mediate cell adhesion between adjacent cells, was significantly decreased due to centrifugation relative to conventional cell sheets (p = 0.023) (Figure 3.3a). These data suggest that 1-layer cell sheet centrifugation promotes biochemical interactions related to cell-cell and cell-matrix adhesion per MSC, likely corresponding with increased physical interactions due to the observed tight-packed tissue structure. 3.3.5 Cell sheet centrifugation increases MSC pro-regenerative cytokine production To evaluate the impact of increased physical and biochemical interactions between MSCs due to cell sheet centrifugation on MSC function, cytokine production by centrifuged and conventional 1-layer cell sheets was compared. Vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) gene expression per MSC are significantly upregulated in 1-layer centrifuged cell sheets 69 relative to conventional cell sheets 24 hours after fabrication (p = 0.018, p = 0.036, and p = 0.0047, respectively) (Figure 3.3e-g, respectively). In the analysis of culture supernatants, VEGF secretions per sheet were approximately equal at 1 day but increased 1.9-fold on average at 2 days (p = 0.036) and 4.0-fold on average at 3 days (p = 0.033) in centrifuged sheets relative to conventional sheets, returning to approximately equal at 4 days (Figure 3.3h). HGF secretions remained consistently higher on average by centrifuged cell sheets compared to conventional cell sheets over 4 days, showing average increases of 1.1-fold, 1.2-fold, 1.3-fold, and 1.4-fold at 1, 2, 3, and 4 days, respectively (Figure 3.3i). IL-10 secretions increased 2.0-fold at 1 day (p = 0.033) and 2.3-fold at 4 days (p = 0.0030) and were slightly higher on average at 2 and 3 days by centrifuged cell sheets compared to conventional cell sheets (Figure 3.3j). To account for cell number in the total production of cytokines, live cell numbers were counted per sheet (Table 3.2). The live cell numbers at 4 days were not significantly changed from 1 day in conventional or centrifuged 1-layer cell sheets, reflected in the proliferation p value (Table 3.2). Additionally, live cell numbers in conventional and centrifuged cell sheets were not significantly changed from 1 day to 4 days in culture (Table 3.2). Normalizing for cell number per sheet, MSCs in 1-layer centrifuged cell sheets secreted significantly more VEGF, HGF, and IL-10 per cell at each time point in culture (IL-10 increased on average at 3 days) (Figure 3.3k-m, respectively) compared to MSCs in 1-layer conventional cell sheets. These data show that individual MSC cytokine production function is augmented due to 1-layer cell sheet centrifugation. 70 3.4 Discussion The aim of this study was to stimulate intrinsic MSC cytokine production potency through cell sheet engineering that would feasibly enhance clinically useful paracrine effects for applications in regenerative medicine. A previous study demonstrated the importance of physical and biochemical cellular interactions in a 2D cell sheet monolayer on upregulating MSC paracrine capacity relative a dissociated MSC formulation [42], and, further, an additional study found that upon spontaneous cell sheet contraction into a 3D tissue with higher abundance cellular interactions, MSC paracrine capacity was similarly increased, measured as in vitro pro-regenerative cytokine production per cell [30]. The present study explores for the first time the feasibility of centrifugation as a tool to non-invasively increase instances of physical and biochemical cellular interactions related to cytokine secretion within MSC sheets. To accomplish this, conditional optimizations of the culture surface on which MSC sheets would be centrifuged were designed to prevent sheet slipping or structural deformation due to centrifugation. Serum containing cell attachment factors, predominantly matricellular glycoproteins fibronectin and laminin, was used to pre-coat insert membranes and support cell sheet adhesion by binding intact matrix receptors [43, 44]. Pre-coating times of 1 hour or 16 hours (overnight) were selected for research utility. Unlike 16 hours pre-coating (Figure 3.1b, d), 1 hour pre-coating (Figure 3.1a, c) could not consistently prevent cell sheet deformation under centrifugation forces (Figure 3.1e), suggesting that the concentration of surface-adsorbed attachment factors was too low to strongly bind cell sheet matrix-adhesion proteins and overcome tissue deformation under force. Cell sheet adhesion to the culture surface requires direct interaction between the 71 sheet and the protein-adsorbed surface. Because the cell sheet was cultured, harvested, and manipulated onto the culture surface in aqueous conditions, a residual volume of medium trapped between the two surfaces prevents their direct contact. Although the force of centrifugation could be enough to force the interfacial medium out, higher gforces risked the cell sheet slipping during centrifugation and would require longer centrifugation durations to achieve adherence. Therefore, before centrifugation, cell sheets on dishes were tilted upright at an approximate 60° angle to passively drain medium from the cell-cultureware interface (Figure 3.1f). Tilting time was an important consideration: 1 minute was not long to clear interfacial medium and 10 minutes was too long so that the cell sheets became dry, evidenced by blue-dyed solution applied to the center of the cell sheet leaking beyond the sheet perimeter (Figure 3.1g) or concentrating on the sheet surface (Figure 3.1i), respectively. Tilting for 5 minutes was enough to passively expel interfacial medium while keeping the cell sheet hydrated (Figure 3.1h). This way, applied centrifugal forces would not be buffered by the residual interfacial medium, but rather act directly on protein interactions between the cell-cultureware surfaces. Optimal cell sheet centrifugation conditions require that the sheet be stably adhered to the culture surface in medium. Therefore, conditions of centrifuge rotational speed and time were investigated for complete cell sheet attachment in medium under orbital rotation immediately after centrifugation (Table 3.1). Centrifugation generates a radial force (RCF), or “g force”, that is exponentially related to the rotational speed defined as revolutions per minute (RPM) [45]. Higher rotational speeds accelerated cell sheet adhesion to the culture surface, withstanding displacement mechanical rotation 72 testing (Table 3.1). These results imply that higher g force can increase cell-to-surface adhesion, either through physical protein interactions or biochemical binding. As applied g force was relatively decreased, longer durations of this applied force were required to achieve a similar level of adherence (Table 3.1). Similar outcomes were seen with myoblast sheets adhered by centrifugation [46], as well as by weighted manipulators[47], where increased centrifuge speeds and heavier manipulators, respectively, accelerated complete cell sheet-to-surface adherence. Under the conditions described in this manuscript, centrifugation at 1832 RCF resulted in cell sheet deformation (Table 3.1). However, conditions of centrifuge acceleration or adjustment of the applied force vector on the cell sheet could potentially be altered to withstand high rotational speed [48]. Histological analysis of 1-layer cell sheets revealed visually obvious structural contrasts 1 hour after fabrication (Figure 3.2a, c). Conventional cell sheets displayed a loose cell packing arrangement and an inconsistent apical tissue surface (Figure 3.2a), contrasted by the visibly tight-packed physical arrangement, level and flat apical tissue surface, and consistent thickness across the tissue in centrifuged cell sheets (Figure 3.2c). The conventional cell sheet approximately represents the centrifuged cell sheet structure without any centrifugation, suggesting that the force of centrifugation imparted the observed organized cell packing arrangement within the cell sheet. After 24 hours in culture, the centrifuged sheet was significantly 1.8-fold decreased in thickness compared to the conventional sheet 24 hours after fabrication (Figure 3.2b, d, e). No differences in the gene expression of HIF-1α, a transcriptional regulator of the adaptive response to hypoxia [49], or in cell death at 24 hours confirmed that this near halving in centrifuged tissue thickness was not due to hypoxic thickness limitations or cell loss due to cell death 73 (Figure 3.2f, g). Importantly, gene expression for connexin 43, a cross-membranal gap junction protein [50], and integrin β1[51], an extracellular ECM binding protein, and laminin, an integrin-receptor ligand[52], were significantly upregulated due to cell sheet centrifugation (Figure 3.3b-d). Conceivably, centrifugation triggered increased physical cell interactions within the sheet that led to higher abundance biochemical cell-cell and cell-matrix interactions. Physical and biochemical binding interactions would impart adhesive tension between the cells[53], potentially contributing the 1.8-fold more compacted tissue thickness. Cell shape change, regulated by the actin cytoskeletal structure [54], did not appear to be a major contributing factor to the tight-packed tissue structure or compacted tissue thickness, indicated by only a slight change in cytoskeleton related β-actin gene expression in centrifuged sheets relative to conventional sheets (Figure 3.2h). It is unclear whether this cytoskeletal structure change occurred immediately in response to centrifugation, or if the cytoskeleton compacted in response to adhesive tension by abundant cell binding interactions, or in direct response to integrin protein formations that are intracellularly bound by β-actin filaments[55]. β-catenin regulates cadherin-mediated cell adhesion and the migratory ability of cells[56, 57]; the measured decreased β-catenin gene expression in centrifuged sheets could be attributed to an inhibited cell migratory potential in the tight-packed tissue structure with high intercellular adhesive tension (Figure 3.3a). The MSC ability to secrete myriad pro-regenerative cytokines and stimulate subsequent paracrine activity in host tissue is one of their most clinically relevant attributes [58, 59]. This study sought to enhance MSC production of cytokines that regulate tissue repair and regeneration using cell sheet centrifugation. Therefore, MSC 74 production of VEGF, HGF, and IL-10, cytokines with specific implications in vascularization [60], fibrosis mitigation [10], and inflammation mediation [61], respectively, by conventional and centrifuged cell sheets was assessed. Centrifugation significantly increased gene expression of VEGF, HGF, and IL-10 per cell relative to conventional sheets in 24-hour samples (Figure 3.3e-g). Concomitantly, centrifuged sheets secreted higher concentrations of VEGF, HGF, and IL-10 in static culture over 4 days following fabrication compared to conventional sheets (Figure 3.3h-j). Live cell numbers were not significantly different between groups at 1 or 4 days culture, and each group showed no significant change in cell number from 1 to 4 days culture (Table 3.2). Normalizing for cell number, MSCs in 1-layer centrifuged cell sheets secreted significantly higher concentrations of VEGF, HGF, and IL-10 at each time point in static culture compared to MSCs in 1-layer conventional cell sheets (Figure 3.3k-m). This augmented MSC cytokine production potency is a feature of cell sheet centrifugation, likely linked to enhanced cellular interactions. For instance, MSC upregulation of gap junction protein formation has previously been attributed to boosting MSC VEGF production, promoting angiogenesis[62]. MSC HGF production is significantly increased with facilitated cell-cell interactions[21]. Also, tissue-like 3D culture that enabled cellECM interactions upregulated MSC IL-10 production relative to non-matrix-interacting MSCs[63]. Centrifugation, therefore, provides an external tool to promote and upregulate physical and biochemical interactions related to MSC cytokine production capacity within a cell sheet. We recognize that this manuscript’s investigation of only three cellular interaction proteins represents a limited selection for asserting the broader claim that cell sheet 75 centrifugation upregulates biochemical cellular interactions. Indeed, we demonstrated histologically that centrifugation notably impacts cell sheet structure, compacting the tissue without any significant changes to cell number, viability, or cell shape. These results can be taken together to conclude that centrifugation confers a tighter cell packing arrangement within cell sheets, characterized by increased physical cell-cell contacts, leading us to hypothesize that biochemical cell-cell interactions would be similarly upregulated. β-catenin, connexin 43, and integrin β1 (with laminin) were selected specifically for their previously identified and mechanistically isolated roles in modulating MSC in vitro cytokine production and in vivo paracrine activity within 3D culture systems [17, 21, 22, 64]. However, to ameliorate possible selection bias, broader transcriptomic evaluation of related cell interaction genes could be evaluated using RNA sequencing (RNA-seq) rather than our described method of RT-qPCR that compares one gene of interest between samples at a time. Multiplex RNA-seq would enable comparison of replicates in both groups (1-layer conventional and 1-layer centrifuged sheets) and read and quantify expression across the complete cell transcriptome [65, 66]. RNA-seq data analysis would still require transcriptome grouping to address a clear comparative point about cell interactions, for instance analyzing all genes surrounding integrin-related binding [65, 66]. This type of analysis still narrows investigation to pre-determined proteins of interest but would illustrate much more broadly the trends of interactionrelated genes to make a more definitive claim regarding the impact of centrifugation [65, 66]. To isolate these interaction proteins as mechanistically underpinning the observed change in MSC function, further experiments using a genetic inhibitor of the target proteins would be needed [21, 65-67]. For example, 1-layer centrifuged cell sheets with 76 or without connexin 43 inhibitor treatment could be compared for cytokine production quantity to determine if connexin 43 formation directly regulates secreted cytokine quantities [21, 65-67]. Of course, careful optimization would be needed to ensure that the protein inhibitor does not disrupt cell sheet structural integrity, which is dependent on cell-cell interaction protein formation [28, 68]. If the inhibitor compromises cell sheet structure or cell viability, further evaluation of secreted cytokines could be biased. These same considerations apply to our selection of cytokines investigated in this study, which were limited to those that are most documented to contribute to in vivo paracrine actions by MSC sheets, notably VEGF and HGF (angiogenesis), and IL-10 (immune modulation) [41, 69-75]. RNA-seq could again be employed here to investigate a much broader range of cytokines, of which some are likely upregulated and downregulated by cell sheet centrifugation. To determine if the cytokine profile importantly contributes to our desired function, that is paracrine-mediated regeneration, a functional assay (in vitro or in vivo) would need to be employed. An in vitro functional assay could involve indirect co-culture of 1-layer conventional or centrifuged cell sheets with human vascular endothelial cells (HUVECs), and quantifying endothelial cell sprouting over time as function of paracrinemediated angiogenesis [76]. Chapter 6 details a therapeutic efficacy evaluation of MSC sheets in the liver that can similarly give context to functional enhancement attributed to cell sheet centrifugation. In summary, cell sheet centrifugation allows non-invasive manipulation of cell packing within cell sheet tissue, contributing to more physical interactions between cells and matrix which were hypothesized to influence biochemical interactions related to MSC cytokine production potential. Confirming this hypothesis, this study demonstrates 77 that (1) MSC sheets are compacted by centrifugal force, contributing a tight cell packing arrangement within the resulting layer that increases physical cellular interactions, leading to (2) higher abundance cell-cell and cell-matrix interactions that enhance the pro-regenerative cytokine secretory capacity per MSC in centrifuged cell sheet tissues. For these reasons, centrifuged cell sheets represent a novel 3D MSC-delivery platform that intrinsically boosts therapeutic MSC paracrine capacities attributed to clinical utility. This study also details the cell sheet centrifugation strategy with modular conditions for cell sheet adhesion and stability under centrifugation, enabling future testing using centrifugation conditions to control various therapeutic cell functions. 3.4.1 Hydrodynamic compression and mechanotransduction in MSCs In this study, we ascribe MSC functional enhancement to the upregulation of cellular interactions, responsible for intercellular communication and direct binding adhesion with the extracellular matrix, facilitated by applied centrifugal force. This identified mechanism of action may be overbroad but is tangentially supported by a body of research employing hydrodynamic compressive forces on MSCs within 3D culture systems for the modulation of cell function via mechanotransduction [77-85]. Mechanotransduction, the conversion of external, environmental forces acting on a cell into internal activation of biochemical signaling cascades that control cell functions, has been extensively studied for directed differentiation of MSCs toward osteogenic or chondrogenic lineages, but is increasingly being explored for the impact on stem cell pharmacodynamics [82, 84, 86-88]. Research in this area largely implicates the force transduction pathway regulated by extracellular matrix-integrin-actin cytoskeleton 78 deformation. Integrins transduce force from the matrix to the actin cytoskeleton, activating downstream effector protein signaling cascades that influence long-term gene expression regulating cell fate and function [80, 89, 90]. For instance, Bandaru et al (2020) found that dynamic mechanical compression of hydrogel-seeded MSCs promoted angiogenesis within human dermal endothelial cells, attributed to increased production of growth factors by mechanically loaded cells [77]. Yes associated protein (YAP), a key molecular component regulating gene expression in response to matrix-integrin-actin deformation, was inhibited to demonstrate that YAP-mediated mechanosensing regulated the secretion of pro-angiogenic molecules from MSCs [77]. Apart from external compressive force, matrix stiffness and resultant cell adhesive tension are well known to transduce mechanosignals related to cell function [84, 86, 87, 91, 92]. For instance, Engler et all (2006) first described tuning hydrogel mechanical stiffness on 2D materials, finding that matrix stiffness directs MSC differentiation to osteolineages through focal adhesion formation [93]; Huebsch et al (2010) reproduced this outcome using variable matrix stiffness in a 3D culture environment, finding that differentiation was guided by altering integrin clustering [94]. Toward enhancing MSC paracrine function, Wan et al (2018) demonstrated that the 3D culture of adipose-derived MSCs on aligned electrospun fibers upregulated production of immunomodulatory factors that promoted macrophage M2 polarization [95]. Activation of focal adhesion kinase (FAK), YAP, and transcriptional coactivator with PDZ binding motif (TAZ), molecular components regulating gene expression responses to mechanical stimulation, tightly regulated this MSC immunomodulatory function, finding MSCmediated macrophage suppression was inhibited by a YAP inhibitor [95]. 79 Centrifugation as a compressive force has not been directly studied in the context of stimulating mechanical transduction in 3D MSC tissues for analogous comparison to our reported findings with cell sheet centrifugation. However, cell pelleting by centrifugation for the generation of 3D MSC spheroids may provide context. The process of spheroid formation begins with loose cell aggregation; in response to increased cellcell interactions, cadherin gene expression levels upregulate, and cadherin protein accumulates on the cell membrane [96]. Ultimately, cadherin-to-cadherin binding induces dynamic compaction into cell spheroids from the cell aggregates [96]. To our knowledge, there are no studies specifically investigating centrifugation speed or duration on cell aggregation and resultant cell interaction protein abundance in self-assembled spheroids, likely because interaction proteins are formed via the subsequent condensation process in culture [96]. However, versus static methods for cell aggregation (or pelleting), such as hanging drop or non-adhesive surface culture, centrifugation pelleting is most commonly used for MSC scaffold-free chondrogenesis, imitating the cell condensation process by densely packing cells and enhancing 3D cellcell and cell-matrix interactions [97-102]. This approach and collective results are mirrored in centrifuged sheets, histologically visualized as a tight-packed cell arrangement compared to non-centrifuged sheets and supports our findings that centrifugation induced cell interaction protein gene expression related to MSC function. Conceivably, mechanotransduction in response to cell sheet centrifugation could be regulating the observed MSC functional enhancement. High cell density physical packing was observed in centrifuged sheets, coupled with upregulation of integrin β1 and laminin gene expression within 24 hours compared to non-centrifuged tissues. 80 Concomitantly, β-actin gene expression was changed in MSCs in centrifuged sheets, potentially induced by adhesive tension related to increased cellular adhesive interactions. Matrix-integrin-cytoskeletal deformation in response to centrifugal compression could activate downstream signaling pathways that regulate cytokine gene expression, stimulating the measured protein production enhancement. Using the same hUC-MSC, Nakao et al (2019) found that trypsin treatment cleaved integrin β1, inhibiting YAP via phosphorylation of Ser127 (phosphor-Yap, pYAP) and, related, showed that trypsin treatment inhibited F-actin polymerization, similarly known to induce pYAP. In contrast, 2D hUC-MSC sheets preserved integrin β1, actively polymerized F-actin fibers, and reduced pYAP expression in MSCs [42]. hUC-MSCs in these 2D sheets showed significantly upregulated cytokine production capacity relative to the trypsin treated single cells, suggesting a possible correlation between matrix-integrincytoskeletal mechanotransductive activation of the YAP signaling pathway and enhanced MSC paracrine factor secretory capacity [42]. Closer investigation of proteins specifically linked to MSC mechanotransduction would need to be investigated to confirm this phenomenon in centrifuged cell sheets; cytokine production per MSC in centrifuged cell sheets could be evaluated following treatment with a YAP-inhibitor to confirm whether mechanosignaling regulates MSC paracrine factor stimulation [95]. 3.5 Conclusions This study demonstrated that cell sheet centrifugation increases cell-experienced 3D cellular interactions, augmenting individual MSC cytokine production relative to noncentrifuged sheets. Importantly, these data highlight relationships between intercellular 81 and cell-matrix interactions and MSC paracrine-related function in the absence of any biomaterial, eliminating material considerations that could impact cell function. Based on these findings, and with a range of centrifugation conditions optimized for stable cell sheet centrifugation, future work will consider the impact of higher rotational speeds and centrifugation durations on further increasing biochemical interactions within cell sheets and the effect on MSC gene expression and protein production related to paracrine function. Overall, this cell sheet centrifugation platform can be readily implemented to generate scaffold-free, 3D MSC tissue-like constructs with enhanced paracrine-relevant secretory functions for applications in regenerative medicine. 3.6 Materials and Methods 3.6.1 Human umbilical cord mesenchymal stem cell (hUC-MSC) culture Banked hUC-MSCs provided by Jadi Cell (LLC, FL, USA) were initiated at 4,500 cells/cm2 and expanded in growth medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 1.0% penicillin streptomycin (PS) (Gibco, NY, USA), 1.0% Glutamax (Life Technologies), and 1.0% non-essential amino acids (Life Technologies) and incubated in a humidified environment (37°C, 5.0% CO2). Growth medium was exchanged after 24 hours of initiating culture and every 2 days subsequently. hUC-MSCs were passaged upon reaching 85% confluence. 82 3.6.2 hUC-MSC sheet fabrication Passage 5 hUC-MSCs were sub-cultured using 0.05% Trypsin-EDTA (Gibco) and the cell suspensions were counted using a hemocytometer. The resultant passage 6 hUCMSCs were aliquoted in the growth medium supplemented with 20% FBS and 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, MO, USA). P6 hUC-MSCs were seeded at 41,580 cells/cm2 onto 35 mm diameter UpCell™ temperature-responsive culture dishes (TRCDs) (CellSeed, Tokyo, Japan) and cultured for 4 days in a humidified environment without exchanging medium. At 4 days, confluent hUC-MSCs on TRCDs were moved to 20°C and spontaneously detached within 30 minutes, generating cell sheets. 3.6.3 Serum coating optimization Serum coating conditions were optimized for cell sheet stability under centrifugation. Briefly, 1.0 μm-diameter pore 6-well cell culture insert membranes (Falcon, NE, USA) were pre-coated with 2 mL of 100% FBS for either 1 hour or 16 hours under standard incubation conditions (37°C, 5.0% CO2). Immediately following detachment, cell sheets were transferred onto pre-coated insert membranes (FBS aspirated and rinsed twice with phosphate buffered saline (PBS) (Gibco)). The insert membranes with centrally placed cell sheets were then excised from their insert well and placed onto the centers of 35-mm tissue culture plastic (TCP) dishes (CELLTREAT, MA, USA). Cell sheets on 1-hour or 16-hour FBS pre-coated insert membranes/TCP were then centrifuged (Eppendorf 5810R, 16 cm rotor radius; Eppendorf, Hamburg, Germany) at 1030 RCF for 30 seconds for 4 trials per condition (n=4 sheets). Afterward, the gross cell sheet was assessed with optical images. 83 3.6.4 Cell sheet tilting optimization Cell sheet tilting was optimized for excess medium removal at the cell sheet and culture surface interface without cell drying. Briefly, cell sheets on the insert membrane/TCP dish were tilted upright at an approximate 60° angle for 1, 5, or 10 minutes to remove excess medium. Afterward, PBS dyed with 0.4% trypan blue solution (Cell Culture Tested Trypan Blue Solution, Sigma Aldrich) was added to the center of the cell sheet and assessed for excess media presence (blue solution beyond the cell sheet perimeter) or cell sheet dehydration (blue solution concentrated and localized on the cell sheet surface) (n=3 sheets per condition). 3.6.5 Mechanical rotation test Adherence between a MSC sheet and the insert membrane culture surface following centrifugation was quantified as the rate of attachment following rotational agitation in medium. First, cell sheets detached from TRCD by temperature reduction were transferred to 16-hour FBS-coated insert membranes on TCPs and tilted for 5 minutes. Cell sheets were then centrifuged at 29, 114, 458, 1030, 1832 RCF using a swing-type plate centrifuge with 16 cm rotor radius (Eppendorf) set to 37°C internal temperature. Immediately afterward, 2 mL warmed growth medium was added to the dish containing the cell sheet and set on a horizontal orbital shaker (VEVOR, Shanghai, China) for agitation at 90 RPM for 10 minutes. To determine adherence, the cell sheet was observed for partial or whole detachment from the insert membrane culture surface (n=3 sheets for each condition). 84 3.6.6 Conventional cell sheet fabrication Immediately following detachment from TRCDs by temperature reduction, single cell sheets were transferred to 16-hour FBS-coated insert membranes on TCPs and tilted for 5 minutes. The cell sheets were then incubated for 1 hour (37°C, 5.0% CO2) to achieve adherence with the culture surface. For further analysis, 1-layer cell sheets were cultured in 10% FBS medium for 1 hour or 24 hours after fabrication. 3.6.7 Cell sheet centrifugation Single cell sheets detached from TRCD by temperature reduction were transferred to 16-hour FBS-coated insert membranes on TCPs, tilted for 5 minutes, and centrifuged (37°C) at 114 RCF for 2 minutes. For further analysis, 1-layer cell sheets were cultured in 10% FBS medium for 1 hour or 24 hours after fabrication. 3.6.8 1-layer cell sheet structural analysis 1-layer cell sheets fabricated by conventional or centrifugation methods were collected 1 hour or 24 hours after layering and fixed with 4.0% paraformaldehyde (PFA) (Thermo Fisher Scientific) for 30 minutes and paraffin embedded. Embedded samples were sectioned at 4.0 μm and stained with Mayer’s Hematoxylin (Sigma-Aldrich) and Eosin (Thermo Scientific) (H&E) to visualize the cell sheet dimensions in cross section. Stained cell sheet sections were dried overnight and imaged with a Bx41 widefield microscope (Olympus, Tokyo, Japan) using AmScope Software (AmScope, CA, USA). To calculate 1-layer cell sheet thicknesses, 5 H&E pictures were taken along the length of 24 hour cultured samples (n=3 per group), and 5 linear measurements from the apical to 85 basal plane were made per image using AmScope Software (AmScope), these 75 linear measurements of thickness were averaged per group. 3.6.9 Cell number counting The absolute cell number in 1-layer cell sheet samples was quantified 24 hours after layering using a tissue destructive method. Briefly, culture medium was removed, and each sample was rinsed twice with PBS. Trypsin-EDTA (0.25%, 2 mL) (Gibco) was added directly on the samples to be incubated at 37°C first for 10 minutes in a humidified incubator, then for 15 minutes in a 37°C water bath. Afterwards, trypsin was removed from the cells by centrifugation (1200 RPM, 5 minutes) and supernatant aspiration. Cell pellets were dispersed with 0.5 mL collagenase P (0.05%, Sigma Aldrich) and incubated for 10 minutes in a 37°C water bath. At this point, cell sheets were fully digested into single cell suspensions. An additional 0.5 mL of cell growth medium was added to each cell suspension to total 1.0 mL, and exact cell numbers and population viability were measured using a trypan blue exclusion assay (n=3 sheets per group). Table 3.2 reports live cell numbers as 104 cells. 3.6.10 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated from 1-layer cell sheet samples (n=3 sheets per group) 24 hours after fabrication in TRIzol (Ambion, Life Technologies, CA, USA) with the PureLink 18 RNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to manufacturer instructions. Isolated RNA was quantified with a NanoDrop Spectrophotometer (Thermo Scientific) and all cDNA samples were prepared from 1.0 μg 86 of RNA/sample using a high-capacity cDNA reverse transcription kit (Life Technologies). Genes were quantified using quantitative PCR with Applied Biosystems primers (glyceraldehyde 3-phosphate dehydrogenase [GAPDH, Hs99999905_m1] as a housekeeping gene, β-catenin [Hs00355049_m1], integrin β1 [Hs01127536_m1], connexin 43 [Hs04259536_g1], laminin [Hs00966585_m1], VEGF [Hs99999070], HGF [Hs00379140_m1], IL-10 [Hs00961622_m1], β-actin [Hs99999903_m1], and hypoxia inducible factor 1 alpha [HIF-1α, Hs00153153_m1]), and was performed on Applied Biosystems Step One Plus (Applied Biosystems, CA, USA). Relative gene expression was determined using the comparative threshold cycle (CT) change algorithm. 3.6.11 Soluble cytokine secretion quantification 1-layer cell sheets fabricated by conventional or centrifugation methods were transferred to 6-well cell culture inserts and cultured with 5 mL of fresh growth medium for 4 days (n=3 sheets per group). During the culture, the media were changed every 24 hours and the supernatants were collected at each medium change. At the end of culture, cell sheets were digested, and live cell numbers were counted in the same manner as previously described. The concentration of soluble VEGF, HGF, and IL-10 was quantified in the collected supernatants using human VEGF, human HGF, and human IL10 Quantikine ELISA kits (R&D Systems, MN, USA), respectively, according to manufacturer’s instructions. To determine the concentration of cytokines secreted per cell, concentration values at each time point were normalized by the average live cell number counted at 1 day. Table 3.2 reports live cell numbers as 104 cells. 87 3.6.12 Statistical analysis All statistical analysis was conducted on data sets of n ≥ 3 biological replicates via at least 2 experimental repetitions and incorporating technical replicates to ensure consistency of results [103]. All quantitative values expressed as a mean ± standard error (SE). All data sets were evaluated for normality using a Shapiro-Wilk test. Comparisons of two groups were tested using a two-tailed, unpaired Student’s t-test. Statistical significance between three or more groups was tested using a one-way analysis of variance (ANOVA) with either a Tukey or Bonferroni test correction for multiple comparisons depending on normality results. Statistical significance between live cell numbers (n ≥ 3 biological replicates) counted after 1 day and 4 days in static culture was tested using a two-tailed, unpaired Student’s t-test assuming a 95% confidence level, reported as the “proliferation p value” in Table 3.2. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. No statistical significance (NS) was defined as p > 0.05. Statistical analysis was conducted using GraphPad Prism 9 software (Prism 9.0.0, https://www.graphpad.com/scientific-software/prism/). 88 Table 3.1 Single layer cell sheet attachment rate following medium addition and mechanical rotation test. Table 3.2 Cell sheet live cell numbers in static culture. 89 Figure 3.1 Single layer cell sheet adhesion optimization for stable centrifugation. Single cell sheets were placed on cell culture-insert membranes coated with 100% FBS for (a) 1 hour or (b) 16 hours and centrifuged. The rate of centrifuge-induced cell sheet deformation was (c, d) observed and (e) quantified. To remove excess interfacial medium without dehydrating cell sheets, (f) cell sheets on insert membranes/TCP dishes were tilted upright at an approximate 60° angle for (g) 1 minute, (h) 5 minutes, or (i) 10 minutes. 90 Figure 3.2 Centrifugation compacts cell sheet tissue structure. Histological cross-sections of conventional and centrifuged 1-layer cell sheets stained with H&E at (a, c) 1 hour and (b, d) 24 hours after fabrication, with quantified comparisons for 1-layer conventional and 1-layer centrifuged cell sheet (e) tissue thickness, (f) HIF-1α gene expression, (g) cell viability, and (h) β-actin gene expression in 24-hour samples. All gene expression normalized to GAPDH and compared to the 1layer conventional cell sheet. Scale bars = 200 μm. Values are means ± SE (***p < 0.001). NS = not significant (p > 0.05). 91 Figure 3.3 Cell sheet centrifugation enhances MSC pro-regenerative cytokine production related to cellular interactions. Quantitative gene expression of proteins related to cellular interactions, including (a) β-catenin (cell-cell interaction), (b) connexin 43 (gap junction), (c) integrin β1 (cellECM interaction), and (d) laminin (ECM), and to cytokine production, including (e) VEGF, (f) HGF, and (g) IL-10, in conventional and centrifuged cell sheets at 24 hours. Analysis of cell sheet supernatants in static culture quantified (h) VEGF, (i) HGF, and (j) IL-10 secretion per sheet over 4 days and normalized (k) VEGF, (l) HGF, and (m) IL-10 concentration per average live cell numbers in 1-layer conventional and centrifuged sheets counted at 1 day in static culture. All gene expression normalized to GAPDH and compared to the 1-layer conventional cell sheet. Values are means ± SE (*p < 0.05 and **p < 0.01). NS = not significant (p > 0.05). 92 3.7 References [1] A. Trounson, C. McDonald, Stem Cell Therapies in Clinical Trials: Progress and Challenges, Cell Stem Cell 17(1) (2015) 11-22. [2] P.R. Baraniak, T.C. 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Gentili, R. Cancedda, E. Boux, A cell-free scaffoldbased cartilage repair provides improved function hyaline-like repair at one year, Clin Orthop Relat Res 470(3) (2012) 910-9. [102] M. Ullah, H. Hamouda, S. Stich, M. Sittinger, J. Ringe, A reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures, Biores Open Access 1(6) (2012) 297-305. [103] T.D. Schmittgen, K.J. Livak, Analyzing real-time PCR data by the comparative C(T) method, Nat Protoc 3(6) (2008) 1101-8. CHAPTER 4 CELL SHEET MULTILAYERING FROM CENTRIFUGATION FUNCTIONALLY ENHANCES MESENCHYMAL STEM CELL TISSUE Bou-Ghannam, S., Kim, K., Grainger, D. W. & Okano, T. Cell sheet multilayering from centrifugation generates high cytokine secretory mesenchymal stem cell tissue. In submission (2021). 4.1 Abstract Three dimensional (3D) MSC-delivery platforms operate as local bioactive drug delivery depots that concentrate and sustain therapeutic factor delivery. Tailored control of the cell dose, potency and corresponding secreted factor dose, therefore, is desirable for engineering cell delivery systems of clinical relevance. Having previously demonstrated that cell sheet centrifugation boosts MSC cytokine production potency via higher abundance cellular interactions (Chapter 3), this study pursues multilayered MSC sheet fabrication from centrifugation to achieve tailored cell dose control. Cell sheet multilayering from centrifugation incrementally increased 2-layered tissue thickness and live cell number, exactly twice that measured in the 1-layer centrifuged counterpart. By contrast, non-centrifuged, statically fabricated (conventional) 2-layer sheets experienced 102 a loss in tissue thickness and cell number relative to the anticipated incremental increase, only 32% thicker and with only 68% more cells, possibly contributed by a hypoxic thickness threshold. 2-layer centrifuged cell sheets demonstrated significantly upregulated MSC gene expression of tissue interaction proteins, connexin 43, integrin β1, and laminin, and increased MSC production of pro-regenerative cytokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10), by 1.2-fold, 1.7-fold, and 2.0-fold, respectively, relative to 2-layer conventional cell sheets. Comparing MSC protein secretions in centrifuged cell sheets revealed that layering indeed doubled secreted HGF and IL-10 concentrations, and non-linearly increased secreted VEGF concentrations, attributed to additional intercellular interactions at the layered sheet interface. Toward the generation of an optimal 3D cell-delivery platform, the present study details a strategy for cell sheet multilayering using centrifugation that generates 3D functionally enhanced 2-layer MSC tissues. 4.2 Introduction Clinical applications of mesenchymal stem cells (MSCs) for therapy have largely shifted away from the hypothesis of MSC differentiation in situ to replace or regenerate host cells toward exploiting MSCs as a generous source of bioactive factors that, via paracrine-centric mechanisms, orchestrate various biological processes desirable for tissue regeneration [1-3]. These goals include therapeutic angiogenesis, immune modulation, cell proliferation and differentiation, and fibrotic mitigation [4-9]. To harness and maximize MSC bioactive drug delivery, transplantation and engraftment fidelity and cell viability are critical. However, most clinical applications of MSC 103 therapies rely on systemic (e.g., intravenous infusions) or direct injection of dissociated cell suspended to deliver cells to target tissue [10-12]. Significantly, long-term outcomes of these MSC therapies show largely marginal clinical benefits with high inter-patient variability [13]. These therapeutic limitations are attributed generally to poor engraftment of the transplanted MSCs to target tissue from injected cell suspension delivery, which subjects the cells to a first-pass effect entrapping a large proportion of cells within the microvasculature of non-target organs (primarily the lungs) [13]. The necessary MSC dose required to stimulate physiologically relevant paracrine outcomes is unknown, as clinical studies compensate for low localization and survival and paracrine effect dilution by administering higher absolute numbers of MSCs [14]. Nonetheless, increasing cell dosing has not impacted clinical success to date, indicating a more complex cell delivery issue in producing therapeutic goals. To overcome the problems of dissociated cell injection delivery, methods that localize, engraft, and concentrate MSCs are necessary to impart therapeutic efficacy and clinical benefit. Cell sheet technology is a unique cell delivery method capable of high cell-engraftment efficacy and targeted delivery [15-17]. Cell sheet technology enables cultured cell harvest without enzymatic treatment or cell or protein disruption by using cell culture-ware grafted with temperature-responsive polymer chains exhibiting an aqueous lower critical solution temperature of 32 ◦C [15-17]. Temperature-responsive cell culture dishes (TRCD) transition from hydrophobic to hydrophilic as aqueous temperature is reduced below 32 ◦C [15-17]. Aqueous media penetrates the grafted polymer interface between adherent cells and TRCD at temperatures below 32 ◦C, expanding polymer chains under hydration and physically separating cell surfaces from 104 culture surfaces [15-17]. Cell sheets retain endogenous cell junctions, extracellular matrix, receptors, and adhesive proteins; enhance cell viability and communication; and retain natural cellular environments that promote sustained cellular activity [18]. These key features of cell sheet technology have been used over several human disease indications to fabricate a unique cell sheet vehicle for therapeutic cell delivery and engraftment [19-27]. Cell dose is a key parameter for the clinical success of cell therapies. Cell sheet engineering generally controls the absolute cell dose in two ways: first, through the initial seeding density in a single cell sheet culture that reaches a maximum based on culture surface attachment area. To go beyond the single sheet cell dose, the second method is by cell sheet layering that effectively doubles the total cell dose within the harvested transplantable tissue, an important consideration for achieving a clinically relevant pharmacologic effect from bioactive factor secreting MSCs. Strategies for cell sheet layering have been extensively employed to increase the absolute cell dose and create tissues with thicknesses and functional outputs of physiological significance for tissue regeneration and repair [28-33]. Having previously demonstrated that cell sheet centrifugation boosts MSC cytokine production potency via higher abundance cellular interactions (Chapter 3), this study pursues a strategy for generating multilayered MSC sheets from centrifugation that conserves the centrifugation-induced cell functional enhancement relative to noncentrifuged, statically multilayered sheets, and with the advantage of incremental control over MSC-secreted growth factor dose from the 3D tissue construct. Toward the generation of an optimal 3D cell-delivery platform, the present study provides an 105 adaptable protocol for cell sheet multilayering using centrifugation that generates functionally enhanced 2-layer MSC tissues. 4.3 Results 4.3.1 Comparative assessment of 2-layer cell sheet structure and viability fabricated by conventional and centrifugation layering methods Layering protocols using centrifugation (centrifugation method) or using passive incubation (conventional method) were developed to support sheet-to-sheet attachment (Figure 4.1). 1-layer conventional and 1-layer centrifuged cell sheets 1 hour after layering (Chapter 3) were visualized in cross-section (Figure 4.2a and 4.2e, respectively) for reference against the 2-layer counterpart following layering (Figure 4.2c and 4.2g, respectively). Histological assessment of 2-layer cell sheets fabricated by the conventional method (H&E stained, visualized as cross-sections) revealed that, although adhered in medium, layered sheets did not interface within 1 hour following layering (Figure 4.2c), but sheet-sheet interfacing was achieved in 24 hours of culture (Figure 4.2d). Analysis of tissue structure in 24-hour samples showed that 2-layer centrifuged cell sheets (Figure 4.2h) were exactly double the thickness of 1-layer centrifuged cell sheets (Figure 4.2f) on average (44 ± 4.8 μm and 22 ± 3.2 μm, respectively (p < 0.0001)) (Figure 4.2i). Conversely, 2-layer conventional cell sheets (Figure 4.2d) were only 32% increased in thickness relative to 1-layer conventional cell sheets (Figure 4.2b) (54 ± 3.1 μm and 41 ± 7.3 μm, respectively (p < 0.0001)) (Figure 4.2i). The population percentage of dead cells in 2-layer conventional cell sheets was significantly increased compared to 1-layer conventional cell sheets (p = 0.0007), while no significant change in 2-layer 106 centrifuged cell sheets was measured relative to 1-layer centrifuged cell sheets, suggesting that the loss in tissue thickness could be contributed in part by cell loss due to cell death (Figure 4.2j). Also, the percentage of dead cells in 1-layer and 2-layer centrifuged cell sheets was not significant compared to 1-layer conventional cell sheets (Chapter 3) (Figure 4.2j). To investigate whether hypoxia due to tissue thickness could be implicated in the cell death and limited tissue thickness in 2-layer conventional cell sheets, the relative gene expression of HIF-1α per MSC was compared against 1-layer conventional cell sheets at 24 hours. HIF-1α was significantly upregulated in 2-layer conventional cell sheets relative to 1-layer conventional sheets (p < 0.0001), while no significant change between 1-layer and 2-layer centrifuged cell sheets was measured (Figure 4.2k). Additionally, 1-layer and 2-layer centrifuged cell sheets did not significantly impact HIF-1α gene expression relative to 1-layer conventional cell sheets (Figure 4.2k). These data suggest that conventional cell sheet layering under the conditions reported does not immediately render consistent, tight, layered sheet-sheet interfacing, despite requiring 1 hour to achieve physical adherence between the two sheet layers. Further, conventional layering appears to compromise tissue structure and negatively impact cell viability within the 2-layer cell sheet tissue, potentially due to hypoxic conditions related to thickness. 4.3.2 Cell sheet centrifugation layering enhances MSC pro-regenerative cytokine production related to cellular interactions Consistent with the results observed in 1-layer cell sheets, 2-layer sheets fabricated by centrifugation layering demonstrated significantly increased MSC gene 107 expression for cell-cell interaction-related protein, connexin 43 (p = 0.0067), as well as for cell-ECM interaction-related proteins, integrin β1 (p = 0.0048) and laminin (p = 0.0003) (Figure 4.3b-d, respectively) relative to MSCs in 2-layer conventional cell sheets. MSC gene expression for β-catenin, though, was significantly upregulated in 2-layer conventional cell sheets relative to 2-layer centrifuged cell sheets (p = 0.018) (Figure 4.3a). These data implicate centrifugation effects observed with 1-layer sheets, whereby MSC experienced cell-cell and cell-matrix interactions are promoted (Chapter 3), is conserved in 2-layer cell sheets using the centrifugation layering method. VEGF, HGF, and IL-10 gene expression per MSC are significantly upregulated in 2-layer centrifuged cell sheets relative to conventional cell sheets 24 hours after fabrication (p = 0.037, p = 0.015, and p = 0.021, respectively) (Figure 4.3e-g, respectively). Analysis of culture supernatants collected 24 hours after layering showed that VEGF, HGF, and IL-10 secretions were 1.2-fold, 1.7-fold, and 2.0-fold increased from 2-layer centrifuged cell sheets relative to 2-layer conventional cell sheets (p = 0.017, p = 0.0002, and p = 0.0003, respectively) (Figure 4.3h-j, respectively). Live cell numbers were similar in 2-layer conventional sheets and 2-layer centrifuged sheets at 24 hours (Table 4.1). Normalizing for cell number, 2-layer centrifuged cell sheet MSC secretions of VEGF, HGF, and IL-10 were 1.3-fold, 1.8-fold, and 2.0-fold increased per cell relative to 2-layer conventional cell sheet MSCs (p = 0.011, p = 0.0001, and p = 0.0003, respectively) (Figures 4.3k-m, respectively). 108 4.3.3 Cell sheet layering increases MSC sheet absolute secreted cytokine dose To measure the impact of cell sheet layering on total cytokine production, culture supernatants from 1-layer and 2-layer centrifuged cell sheets were analyzed over 4 days. 2-layer centrifuged cell sheets secreted 3.2-fold, 8.7-fold, 10-fold, and 4.8-fold more VEGF than 1-layer centrifuged cell sheets at 1, 2, 3, and 4 days, respectively (1 day: p < 0.0001, 2 days: p = 0.0001, 3 days: p = 0.0096, 4 days: p = 0.0020) (Figure 4.4a). HGF secretions remained significantly higher on average by 2-layer centrifuged cell sheets compared to 1-layer centrifuged cell sheets over 4 days, producing 1.2-fold, 1.4-fold, 2.1fold, and 2.0-fold more HGF at 1, 2, 3, and 4 days, respectively (1 day: p = 0.015, 2 days: p = 0.0046, 3 days: p = 0.0021, 4 days: p = 0.0032) (Figure 4.4b). IL-10 secretions by 1layer centrifuged sheets and 2-layer centrifuged sheets were similar at 1 day, but 2-layer centrifuged sheet IL-10 secretion increased 2.4-fold on day 2, 2.1-fold on day 3, and 1.9fold on day 4 in culture (2 days: p = 0.0093, 3 days: p = 0.024, 4 days: p = 0.033) (Figure 4.4c). 2-layer centrifuged cell sheets comprised 2.0-fold more cells at 1 day (p = 0.0002), and 1.8-fold more cells at 4 days (p = 0.0061) than 1-layer centrifuged cell sheets (Table 4.1). Live cell numbers in 1-layer and in 2-layer centrifuged cell sheets were not significantly changed from 1 day to 4 days in culture (Table 4.1). Overall, centrifugation layering is an effective method for incrementally increasing the MSC dose within cell sheet tissue, thereby increasing the overall secreted dose of therapeutic cytokines. 4.4 Discussion The aim of this study was to develop a strategy for controlling the MSC-secreted cytokine dose from 3D MSC sheets for future clinical utility in providing physiologically 109 relevant paracrine outcomes. Toward the development of 3D MSC sheets as bioactive factor delivery depots for cell-based therapy, cell sheet layering is an invaluable tool for maximizing the cell densities realized within engineered cell sheets [28-33]. Building from conditions for first-layer cell sheet adhesion (Chapter 3), layering protocols using centrifugation (centrifugation method) or using passive incubation (conventional method) were developed to support sheet-to-sheet attachment (Figure 4.1). Data in Figure 4.2 show that centrifugation rendered a tight-packed cell structural arrangement that resulted in a more compacted tissue structure relative to conventional cell sheets, and, consistently, layering from centrifugation generated 2-layer centrifuged cell sheets with exactly double the thickness and cell number as 1-layer centrifuged cell sheets (Figure 4.2i). By contrast, 2-layer cell sheets fabricated by the conventional method experienced a loss in tissue thickness and cell number relative to the anticipated incremental increase, only 32% thicker and with only 68% more cells (Table 4.1) than 1-layer conventional cell sheets (Figure 4.2i). The significant upregulation of HIF-1α gene expression in 2-layer conventional sheet MSCs within 24 hours of fabrication suggests an oxygen diffusion threshold had been exceeded by layering, inducing hypoxia within the tissue, and limiting the 2-layer construct thickness to approximately 55 µm (Figure 4.2k). Thickness limitations have previously been reported in studies using multilayered cell sheets: human endometrial-derived mesenchymal cell sheets cultured on a dish experienced a thickness limitation around 40 µm [34]. Five-layer human skeletal muscle myoblast sheets approximately 70-80 µm thick showed signs of hypoxia that three-layer HSMM sheets (approximately 40-50 µm thick) did not [33]. A thickness limitation in 2-layer conventional MSC sheets reported here is conceivable, but the non-maintenance of tissue 110 thickness could also be attributed in part to cell loss due to cell death evidenced by 10% population cell death within 24 hours of conventional layering (Figure 4.2j). Low oxygen conditions are likely causing cell death within the tissue, as this magnitude of cell death was significantly higher than the percentage of dead cells within 1-layer conventional and 1- and 2-layer centrifuged cell sheets that demonstrated significantly lower relative HIF1α gene expression levels and diffusive transport distances (Figure 4.2j-k). Although similar hypoxic thresholds related to tissue thickness have been reported in layered cell sheet constructs, the classic study by Krogh et al (1922) defines the oxygen diffusion limit in tissues to be 100-200 µm [35], a thickness threshold our 2-layer MSC sheets are well below (~55 µm). Of course, diffusion limitations must take into consideration the metabolic demand of the constituent cell type, as well as the cell packing density within the tissue. Accounting for cell type for the sake of comparison with our results, a study comparing umbilical cord tissue-derived MSC spheroids found gradient oxygen diffusion contributing to reduced cellar metabolism in the core of UCMSC spheroids with 30,000 cells per spheroid and a 251 µm radius [36]. By contrast, the hUC-MSC sheets described here contain approximately 1,260,000 total cells. Cell density would have to be normalized per unit tissue volume to make a direct comparison with UC-MSC spheroid studies, but the cell-dense nature of cell sheets could impose unique oxygen transport limitations not experienced in 3D spheroid cultures. To definitively conclude whether an oxygen diffusion threshold is contributing to cell-experienced hypoxia in 2-layer conventional cell sheets, an oxygen transport study is necessary. Oxygen tension could be measured using an oxygen microsensor that could be placed near the apical plane of the cell sheet and measurements taken every 10 µm through the 111 center and to the basal plane of the cell sheet [37]. Consistent with the centrifugation effect observed in 1-layer cell sheets, whereby centrifugation increased MSC-experienced biochemical cell-cell and cell-matrix interactions related to their cytokine production capacity, MSCs in 2-layer cell sheets fabricated by centrifugation layering demonstrated significantly higher gene expression for gap junction protein, cell-matrix interaction-related proteins, and pro-regenerative cytokines relative to MSCs in 2-layer conventional cell sheets (Figure 4.3b-g). Also, 2layer centrifuged cell sheets secreted significantly higher concentrations of therapeutic factors, VEGF, HGF, and IL-10, within 24 hours of fabrication than did 2-layer conventional cell sheets (Figure 4.3h-j). Live cell numbers in 2-layer conventional and centrifuged cell sheets were similar at 24 hours (Table 4.1), contributed by cell losses possibly related to hypoxic cell death in 2-layer conventional sheets, resulting in similar relative fold changes between the two groups in cytokines secreted per sheet and cytokines secreted per cell (Figure 4.3k-m). Certainly, cell interaction protein and cytokine gene expression and production could be impaired due to hypoxic conditions impacting cell viability in 2-layer conventional cell sheets. However, given these findings of a centrifugation effect in 1-layer cell sheets (also Chapter 3) that showed no signs of hypoxia or reduced viability, and the exact incremental increase in thickness and cell dose due to 2-sheet centrifugation layering, we expect that the centrifugation effect observed in 1-layer cell sheets is conserved in 2-layer cell sheets, contributing to the higher reported cellular interactions and secretory function compared to 2-layer conventional sheets. Lastly, incrementally increased total cytokine production due to sheet layering 112 was confirmed in centrifuged cell sheets over 4 days in culture. The concentration of HGF secreted by 2-layer centrifuged cell sheets was twice that of 1-layer centrifuged cell sheets by days 3 and 4 in culture, and concentration of secreted IL-10 was twice that of the 1-layer from days 2 to 4 in culture (Figure 4.4b-c). VEGF secretions by 2-layer centrifuged sheets were on the order of 3.2-fold, 8.7-fold, 10-fold, and 4.8-fold higher than the 1-layer at 1, 2, 3, and 4 days, respectively (Figure 4.4a), although live cell number within the construct was only 2.0-fold greater in 2-layer sheets on average (Table 4.1). This result could be attributed to additional cellular interactions formed at the interface of the two cell sheet layers. Previous cell sheet multilayering studies have used centrifugation as an effective method to introduce cellular communication between stratified cell sheets: centrifugation of stacked cardiomyocytes derived from induced pluripotent stem cells formed immediate cell-cell interfacing between sheet layers, and within 30 minutes, electrical coupling of inter-sheet cardiomyocytes occurred via gap junction formation, indicated by synchronous beating throughout the multilayered tissue [28]. That cytokine concentrations were at least double that of the 1-layer supports that the centrifuge effect is conserved in 2-layer centrifuged cell sheets. Taken together, these results highlight several key features of cell sheet centrifugation layering that collectively generate highly cytokine secretory MSC tissue: (1) tissue compaction due to centrifugation enables MSC sheet layering without exceeding oxygen diffusion thresholds, supporting (2) increment thickness and cell dose control with quality tissue structure; (3) centrifugation layering increases conserves the centrifuge effect of higher abundance cell-cell and cell-matrix interactions that enhance MSC pro-regenerative cytokine secretory capacity, thereby (2) doubling the secreted 113 factor dose of engraftable MSC tissue compared to the single-layer counterpart. Based on these findings, centrifugation-based cell sheet layering represents a valuable platform for tailoring the thickness, cell dose, and secreted bioactive factor dose within MSC tissues to further develop optimized 3D MSC-delivery depots for future paracrine-mediated tissue regeneration. 4.4.1 Centrifugation layering for multicellular sheet engineering In this chapter, cell sheet multilayering from centrifugation was utilized as a tool to increase the absolute cell number within engraftable MSC tissue for the purpose of higher concentration and sustained secreted factor release that would, hypothetically, support a larger therapeutic paracrine response in vivo. Multicellular sheet fabrication from centrifugation multilayering, though, is of considerable interest for increasing overall graft function or for engineering complex tissue function that can replace diseased or injured tissues. Cell sheet layering represents a unique co-culture platform that cannot be easily replicated by seeded-scaffold, cell encapsulation, or spheroid tissue engineering strategies. While these strategies generally mix cell types in suspension, resulting in a randomized and heterogeneous instance of “A-type” and “B-type” cell interactions within the 3D tissue (e.g., A-B, A-A, B-B interaction possibilities), cell sheet layering allows precise control over cell interactions in 3D tissues. B-type cell sheets can be precisely stacked on A-type cell sheets, controlling the instances of A-B cell interactions at the layered sheet interface. Many complex human tissues exhibit this defined stratum that regulates physiologic function (e.g., corneal epithelium, liver parenchymal tissue, intestinal tissue, bone tissues). 114 A large and continuously growing body of cell sheet research, therefore, has been devoted to investigating cell sheet layering methods with novel multicellular combinations and layered organizations for the creation of complex tissues in vitro that can restore disease biological functions in vivo [28-33, 38-40]. To do so, a range of cell sheet multilayering techniques have been developed. A non-assisted form of cell sheet layering involves manipulating one cell sheet atop the other in medium, removing the medium by aspiration, and culturing the layered sheets under incubated conditions to promote their attachment to each other. This is described as the conventional layering method in this chapter. Centrifugation layering is an assisted layering technique, whereby the applied centrifugal force promotes rapid attachment between layered cell sheets and helps eliminate gaps or delamination within 2-layered tissues. Another assisted layering method utilizes a weighted plunger coated with gelatin: this plunger is placed on a cell sheet and incubated so that the sheet can attach to the gelatin. The cell sheet on the plunger is then manipulated onto another cell sheet (constituting the basal layer) and cultured under incubated conditions to promote sheet-to-sheet attachment, then the gelatin is dissolved, and the weighted plunger is removed. The pressure from the plunger creates a tightly laminated and homogenous layered sheet interface without gaps. The plunger method can be applied to confluent cell monolayers prior to temperaturemediated detachment to prevent contraction, allowing monolayer sheets to be harvested upon temperature reduction and subsequently layered. Table 4.2 summarizes the use of these various cell sheet manipulation techniques for the generation of layered, multicellular tissues. The main purpose of employing these manipulator techniques is to achieve a cohesive, consistent interface of interaction and cell communication between 115 the layered sheets, eliminating any gaps between sheet strata to ensure homogenous function throughout the tissue. For clinical translation, the assisted manipulator techniques allow precise control over applied force on the cell sheets so that multicellular tissue fabrication is reliable and reproducible. Conceivably, the strategy for cell sheet multilayering from centrifugation described in this dissertation could be adapted for multicellular sheet layering. Regardless of the cell type, the initial step is to optimize conditions for stable and consistent firstlayer cell sheet adhesion to the culture surface in medium to enable subsequent sheet layering. Described in detail in Chapter 3, conditions of dish pre-coating with serum for 16 hours and cell sheet tilting for 5 minutes to remove excess interfacial medium can likely be applied to cell sheets of different cell types, potentially pre-coating for longer durations if cell attachment under centrifugation is weak or tilting for longer if the cell sheet is thicker and holds a larger volume of residual medium. Chapter 3 outlines a range of centrifuge rotational forces and applied force durations and corresponding ability to strongly adhere the first layer cell sheet to the culture surface, quantified as the rate of complete cell sheet attachment to the dish following medium addition and mechanical agitation testing. Rotational speed and time contributing to applied centrifugal force will need to be optimized to adhere a cell sheet of a different cell type than the hUC-MSC used in this dissertation, but, importantly, our finding that higher relative centrifugal force (g force) can consistently adhere cell sheets in a shorter time than lower g forces can help guide optimization strategies. Of course, cell viability should be evaluated in response to centrifugation, immediately and within 1-3 days after centrifugation; this can be done using tissue destructive methods to quantify population dead cell percentages, 116 and to histologically visualize cell and tissue quality and integrity in sheet cross-sections. Once the first-layer cell sheet is stably adhered to the culture surface in medium, the second-layer sheet of a different cell type than the first can be positioned atop the first layer by manipulation in medium and subsequently stacked by medium aspiration. Cell sheets fabricated from different cell types are generally not identical in sheet diameter, thickness, and cell density. Overlaid sheets of different diameters can be resized with a biopsy punch to remove non-interacting sheet excess. Conditions of second-layer cell sheet centrifugation must be optimized not only for tight adhesion to the first-layer cell sheet, but also for immediate, cohesive interfacing between the two sheets without any gaps or delamination. Structural sheet interfacing can be manipulated by applied g force in the same manner as with the first-layer sheet, where higher g forces can likely interface the sheets more rapidly and can be evaluated with histologic visualization of the layered sheets in cross-section immediately following centrifugation layering. Cell viability impacted by centrifugation layering should be assessed as previously described for the first layer. If the resultant multicellular tissue will be cultivated in vitro for further analysis, a mixed media formulation must be optimized to support survival, phenotypic stability, and desired functionality of each cell type. In the case of multilayered sheets, the tissue effect experienced by cells is not only due to the 3D tissue-like structure of cell sheets, but layering is an effective method to introduce physical interaction, molecular adhesion, and intercellular communication between stratified cell sheets that enables homogenous function throughout the tissue. For instance, Haraguchi et al (2012) stacked cardiomyocytes derived from induced pluripotent stem cells (iPSCs) using centrifugation and conducted electrophysiological 117 analysis to measure electrical coupling between sheet layers and throughout the multilayered tissue [28]. Centrifugation enabled immediate interfacing between sheet layers, and, within 30 minutes after this physical cell-cell contact was formed, electrical coupling of neighboring cardiomyocytes occurred, indicated by synchronous beating throughout the multilayered tissue. This group found that gap junction formation (connexin 43) was rapidly established following cell-cell contact between layers and mediated the synchronous electrical coupling [28]. For multicellular sheet fabrication, this interface of cellular interactions is imperative to achieve complex, multicellular tissue functions in vitro. To construct vascularized cardiac tissue, Sasagawa et al (2010) used a weighted plunger manipulator system to sandwich human umbilical vein endothelial cells (HUVECs) between two myoblast sheets out to five-layered constructs [39]. Following co-culture, the interfaced structural support allowed HUVECs to form capillary-like networks with lumen-like structures within the five-layer myoblast sheet constructs [39]. This cell sheet layering strategy to achieve stratified multicellular interactions has also been implemented to construct functional hepatic tissue [40] as well as periodontal tissue in vitro [41]. Mono- or multicellular layered cell sheet interfacing, therefore, is a key parameter for which assisted manipulation techniques are optimized. Achieving this trans-sheet tissue effect that influences multicellular tissue function depends on cohesive sheet interfacing. In the case of 2-layer conventional hUCMSC sheets, evident interfacing was achieved within 24 hours culture in growth medium despite a distinct delamination within 1 hour of layering by the conventional method (Figure 4.2). Relative to other MSC types, hUC-MSCs are highly motile [42] and likely migrated and formed cell interactions contributing to homogenous layered sheet 118 interfacing under optimal normoxic culture in serum-supplemented medium. However, different cell types without this migratory capacity may never interface in culture. In fact, this was the case with multilayered human bone marrow derived MSC (hBMSC) sheets that were fabricated by the conventional method described in this dissertation (Thorp et al (2021), in submission). 2-layer conventional hBMSC sheets demonstrated clear sheet delamination at 3 days in culture (Thorp et al (2021), in submission). Additionally, multilayered cell sheets for pre-clinical and clinical engraftment would be transplanted immediately following fabrication. The low serum, hypoxic, and potentially inflammatory in vivo environment would not necessarily be hospitable for sheet interfacing that could otherwise occur under in vitro growth culture conditions. Apart from structural tissue interfacing that can be observed histologically in cross-section, molecular interfacing indicating trans-sheet communication can also be confirmed. As described above, Haraguchi et al (2012) confirmed this layered tissue effect through synchronous electrical coupling propagated by the formation of gap junction proteins [31]. Without electrically conductive cells, the presence of gap junction proteins can be observed immunohistochemically by injecting a fluorescent dye into the apical sheet layer that is only transferred via gap junctions [43]. Dye present in the bottom strata of the multilayered sheet would evidence the formation of multicellular communication across the layered sheet interface. Fabrication of multicellular tissue using the cell sheet multilayering from centrifugation strategy described in this dissertation is feasible. Appropriate functional evaluation would be dependent on the component cell types and the multicellular tissue attempting to be fabricated. Table 4.2 provides examples of complex tissue functions that 119 have previously been evaluated in multicellular sheets fabricated by layering manipulation techniques, including vessel formation by endothelial cells or successful drug metabolism by functional hepatocytes, and could provide relevant context to guide future multicellular layering studies. 4.5 Conclusions This study demonstrated that cell sheet multilayering from centrifugation generates viable 2-layered constructs with controlled, incremental thicknesses and cell doses, increasing absolute MSC sheet construct secreted cytokine dose. Based on these findings, future work will likely investigate the impact of additional layers on MSCexperienced cellular interactions and resultant function. The addition of layers must consider oxygen diffusion thresholds that limit tissue thickness in static in vitro culture. To navigate this limitation, future investigation could implement cell sheets with smaller initial seeding densities (thinner tissues) to maximize the number of layers achieved before a hypoxic threshold is reached, or co-culture with microvasculature-producing endothelial cells could provide an avenue for oxygen distribution throughout thick tissues. Overall, this cell sheet centrifugation layering platform can be readily implemented to generate highly cytokine secretory, 3D MSC bioactive factor delivery depots for applications in tissue regeneration. 120 4.6 Materials and Methods 4.6.1 Human umbilical cord mesenchymal stem cell (hUC-MSC) culture Banked hUC-MSCs provided by Jadi Cell (LLC, FL, USA) were initiated at 4,500 cells/cm2 and expanded in growth medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 1.0% penicillin streptomycin (PS) (Gibco, NY, USA), 1.0% Glutamax (Life Technologies), and 1.0% non-essential amino acids (Life Technologies) and incubated in a humidified environment (37°C, 5.0% CO2). Growth medium was exchanged after 24 hours of initiating culture and every 2 days subsequently. hUC-MSCs were passaged upon reaching 85% confluence. 4.6.2 hUC-MSC sheet fabrication Passage 5 hUC-MSCs were sub-cultured using 0.05% Trypsin-EDTA (Gibco) and the cell suspensions were counted using a hemocytometer. The resultant passage 6 hUCMSCs were aliquoted in the growth medium supplemented with 20% FBS and 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, MO, USA). P6 hUC-MSCs were seeded at 41,580 cells/cm2 onto 35 mm diameter UpCell™ temperature-responsive culture dishes (TRCDs) (CellSeed, Tokyo, Japan) and cultured for 4 days in a humidified environment without exchanging medium. At 4 days, confluent hUC-MSCs on TRCDs were moved to 20°C and spontaneously detached within 30 minutes, generating cell sheets. 121 4.6.3 Conventional cell sheet layering Immediately following detachment from TRCDs by temperature reduction, first layer cell sheets were transferred to 16-hour FBS-coated insert membranes on tissue culture plastic dishes (TCPs) and tilted for 5 minutes. The cell sheets were then incubated for 1 hour (37°C, 5.0% CO2) to achieve adherence with the culture surface. A second detached cell sheet was moved onto the first layer cell sheet using warmed growth medium, spread over the surface of the first layer cell sheet by medium aspiration, and tilted for 5 minutes. The 2-layer cell sheet was incubated at 37°C for 1 hour to achieve adherence between the layered cell sheets. For further analysis, 2-layer cell sheets were cultured in 10% FBS medium for 1 hour or 24 hours after layering. 4.6.4 Centrifugation cell sheet layering First layer cell sheets detached from TRCD by temperature reduction were transferred to 16-hour FBS-coated insert membranes on TCPs, tilted for 5 minutes, and centrifuged (37°C) at 114 RCF for 2 minutes. Afterward, the second layer cell sheet was spread over the surface of the first layer cell sheet by medium aspiration, tilted for 5 minutes, and incubated at 37°C for 30 minutes before centrifuging the 2-layer sheets at 114 RCF for 2 minutes at 37°C. For further analysis, 2-layer cell sheets were cultured in 10% FBS medium for 1 hour or 24 hours after layering. 4.6.5 2-layer cell sheet structural analysis 2-layer cell sheets fabricated by conventional or centrifugation methods were collected 1 hour or 24 hours after layering and fixed with 4.0% paraformaldehyde (PFA) 122 (Thermo Fisher Scientific) for 30 minutes and paraffin embedded. Embedded samples were sectioned at 4.0 μm and stained with Mayer’s Hematoxylin (Sigma-Aldrich) and Eosin (Thermo Scientific) (H&E) to visualize the cell sheet dimensions in cross section. Stained cell sheet sections were dried overnight and imaged with a Bx41 widefield microscope (Olympus, Tokyo, Japan) using AmScope Software (AmScope, CA, USA). To calculate 2-layer cell sheet thicknesses, 5 H&E pictures were taken along the length of 24 hour cultured samples (n=3 per group), and 5 linear measurements from the apical to basal plane were made per picture using AmScope Software (AmScope) and averaged per group. 4.6.6 Cell number counting The absolute cell number in 2-layer cell sheet samples was quantified 24 hours after layering using a tissue destructive method. Briefly, culture medium was removed, and each sample was rinsed twice with PBS. Trypsin-EDTA (0.25%, 2 mL) (Gibco) was added directly on the samples to be incubated at 37°C first for 10 minutes in a humidified incubator, then for 15 minutes in a 37°C water bath. Afterwards, trypsin was removed from the cells by centrifugation (1200 RPM, 5 minutes) and supernatant aspiration. Cell pellets were dispersed with 0.5 mL collagenase P (0.05%, Sigma Aldrich) and incubated for 10 minutes in a 37°C water bath. At this point, cell sheets were fully digested into single cell suspensions. An additional 0.5 mL of cell growth medium was added to each cell suspension to total 1.0 mL, and exact cell numbers and population viability were measured using a trypan blue exclusion assay (n=3 sheets per group). Table 4.1 reports live cell numbers as 104 cells. 123 4.6.7 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated from 2-layer cell sheet samples (n=3 sheets per group) 24 hours after layering in TRIzol (Ambion, Life Technologies, CA, USA) with the PureLink 18 RNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to manufacturer instructions. Isolated RNA was quantified with a NanoDrop Spectrophotometer (Thermo Scientific) and all cDNA samples were prepared from 1.0 μg of RNA/sample using a high-capacity cDNA reverse transcription kit (Life Technologies). Genes were quantified using quantitative PCR with Applied Biosystems primers (glyceraldehyde 3-phosphate dehydrogenase [GAPDH, Hs99999905_m1] as a housekeeping gene, β-catenin [Hs00355049_m1], integrin β1 [Hs01127536_m1], connexin 43 [Hs04259536_g1], laminin [Hs00966585_m1], VEGF [Hs99999070], HGF [Hs00379140_m1], IL-10 [Hs00961622_m1], β-actin [Hs99999903_m1], and hypoxia inducible factor 1 alpha [HIF-1α, Hs00153153_m1]), and was performed on Applied Biosystems Step One Plus (Applied Biosystems, CA, USA). Relative gene expression was determined using the comparative threshold cycle (CT) change algorithm. 4.6.8 Soluble cytokine secretion quantification 2-layer cell sheets fabricated by conventional or centrifugation methods were transferred to 6-well cell culture inserts and cultured with 5 mL of fresh growth medium for 4 days (n=3 sheets per group). During the culture, the media were changed every 24 hours and the supernatants were collected at each medium change. At the end of culture, the cell sheets were digested, and live cell numbers were counted in the same manner as previously described. The concentration of soluble VEGF, HGF, and IL-10 was 124 quantified in the collected supernatants using human VEGF, human HGF, and human IL10 Quantikine ELISA kits (R&D Systems, MN, USA), respectively, according to manufacturer’s instructions. To determine the concentration of cytokines secreted per cell, concentration values at each time point were normalized by the average live cell number counted at 1 day. Table 4.1 reports live cell numbers as 104 cells. 4.6.9 Statistical analysis All statistical analysis was conducted on data sets of n ≥ 3 biological replicates via at least 2 experimental repetitions and incorporating technical replicates to ensure consistency of results [44]. All quantitative values expressed as a mean ± standard error (SE). All data sets were evaluated for normality using a Shapiro-Wilk test. Comparisons of two groups were tested using a two-tailed, unpaired Student’s t-test. Statistical significance between three or more groups was tested using a one-way analysis of variance (ANOVA) with either a Tukey or Bonferroni test correction for multiple comparisons depending on normality results. Statistical significance between live cell numbers (n ≥ 3 biological replicates) counted after 1 day and 4 days in static culture was tested using a two-tailed, unpaired Student’s t-test assuming a 95% confidence level, reported as the “proliferation p value” in Table 3.2. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. No statistical significance (NS) was defined as p > 0.05. Statistical analysis was conducted using GraphPad Prism 9 software (Prism 9.0.0, https://www.graphpad.com/scientific-software/prism/). 125 Table 4.1 Cell sheet live cell numbers in static culture. Table 4.2 Summary of multicellular sheet layering. Multicellular tissue of interest Component cell types Layering method Complex tissue function Human umbilical vein endothelial cells (HUVECs) & human skeletal muscle myoblasts [39] Weighted plunger manipulator [39] Capillary-like network formation [39] Vascularized cardiac tissue Rat neonatal cardiomyocytes & rat endothelial cells (ECs) [45, 46] Non-assisted manipulation with incubation (conventional method) [46] EC sprouting and network formation; increased angiogenic factor secretion in vitro; in vivo neovascularization and vessel fusion with host [45, 46] Vascularized tissue Human umbilical vein endothelial cells (HUVECs) & neonatal normal human dermal fibroblasts (NHDFs) [47] Weighted plunger manipulator [47] Capillary-like network formation [47] 126 Table 4.2 Continued. Multicellular tissue of interest Component cell types Human hepatoma cells (HepG2) & bovine pulmonary artery endothelial cells (BPAEC) [48, 49] Layering method Non-assisted manipulation with incubation (conventional method) [48-50] Rat hepatocytes & human aortic endothelial cells (HAECs) [50] Maintenance of hepatocyte-specific cuboidal morphology and albumin production in culture [50] Hepatic tissue Periodontal tissue Complex tissue function Upregulated gene expression of liverspecific cytochrome P450 (CYP) enzymes [48, 49] Rat hepatocytes & bovine aortic endothelial cells (ECs) [40, 51] Weighted plunger manipulator [40, 51] Periodontal ligament (PDL) cells & osteoblast-like cells (MC3T3-E1) [41] Non-assisted manipulation with incubation (conventional method) [41] Maintenance of hepatocyte-specific cuboidal morphology, albumin production, and urea synthesis in culture; upregulated hepatocyte-specific gene expression; chemical uptake and bile efflux function [40, 51] Bone-ligament regeneration following ectopic and orthotopic transplantation in mice [41] 127 Figure 4.1 Flow chart illustrating the MSC sheet layering protocol with centrifugation (centrifugation method) and without centrifugation (conventional method). 128 Figure 4.2 Centrifugation layering enables viable 2-layer cell sheet tissue fabrication. Histological assessment of 1-layer conventional and centrifuged cell sheets (a, e, respectively) 1 hour and (b, f, respectively) 24 hours after fabrication, and the corresponding 2-layer cell sheets fabricated by conventional or centrifugation layering methods at (c, g, respectively) 1 hour and (d, h, respectively) 24 hours after layering, visualized as cross-sections stained with H&E. 1-layer and 2-layer cell sheets fabricated by conventional or centrifuge methods were compared for (i) tissue thickness, (j) cell viability, and (k) HIF-1α gene expression in 24 hour samples. All gene expression normalized to GAPDH and compared to the 1-layer conventional cell sheet. Scale bars = 200 μm. Values are means ± SE (***p < 0.001). NS = not significant (p > 0.05). 129 Figure 4.3 Centrifugation layering enhances MSC pro-regenerative cytokine production related to cellular interactions. Quantitative gene expression of cell interaction proteins (a) β-catenin, (b) connexin 43, (c) integrin β1, and (d) laminin (ECM), and cytokines (e) VEGF, (f) HGF, and (g) IL-10 in 2-layer conventional and centrifuged cell sheets at 24 hours. Analysis of cell sheet supernatants in static culture quantified (h) VEGF, (i) HGF, and (j) IL-10 secretion per 2-layer sheet after 1 day in static culture, and normalized (k) VEGF, (l) HGF, and (m) IL-10 concentration per average live cell numbers in 2-layer conventional and centrifuged sheets counted at 1 day in static culture. All gene expression normalized to GAPDH and compared to the 2-layer conventional cell sheet. Values are means ± SE (*p < 0.05, **p < 0.01, and ***p < 0.001). 130 Figure 4.4 Cell sheet layering increases MSC sheet absolute pro-regenerative cytokine production. Analysis of 1-layer and 2-layer centrifuged cell sheet supernatants in static culture quantified (a) VEGF, (b) HGF, and (c) IL-10 secretion per sheet over 4 days. 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Shimizu, M. Yamato, A. Kikuchi, T. Sasagawa, S. Sekiya, J. Kobayashi, G. Chen, T. Okano, Cellular control of tissue architectures using a threedimensional tissue fabrication technique, Biomaterials 28(33) (2007) 4939-46. [48] M. Ohno, K. Motojima, T. Okano, A. Taniguchi, Up-regulation of drugmetabolizing enzyme genes in layered co-culture of a human liver cell line and endothelial cells, Tissue Eng Part A 14(11) (2008) 1861-9. [49] M. Ohno, K. Motojima, T. Okano, A. Taniguchi, Induction of drug-metabolizing enzymes by phenobarbital in layered co-culture of a human liver cell line and endothelial cells, Biol Pharm Bull 32(5) (2009) 813-7. [50] M. Harimoto, M. Yamato, M. Hirose, C. Takahashi, Y. Isoi, A. Kikuchi, T. Okano, Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes, J Biomed Mater Res 62(3) (2002) 464-70. [51] K. Kim, K. Ohashi, R. Utoh, K. Kano, T. Okano, Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets, Biomaterials 33(5) (2012) 1406-13. CHAPTER 5 SUMMARY OF KEY FINDINGS In this dissertation, cell sheet tissue engineering was investigated for purposes of modulating the tissue effect experienced by mesenchymal stem cells (MSC) toward stimulating clinically relevant intrinsic MSC cytokine production potency. The operative hypotheses guiding this research were that the MSC tissue effect is fundamentally realized using cell sheet tissue engineering, and that 3D structure and instances of physical and biochemical cell-cell and cell-matrix interactions can be enhanced within cell sheets to stimulate individual MSC cytokine production potency. To address this goal, we assessed 3D structural and chemical transitions that impact MSC secretory potency as 2D and 3D cell sheets, determined how cell sheet manipulation techniques could enhance tissue interactions related to paracrine potency, and determined whether this paracrine-related secreted factor dose could be modulated using cell sheet layering strategies. Chapter 2 investigated how cell sheet fabrication affected MSC cytokine production potency in vitro [1]. MSC cultivation in three-dimensional (3D) tissue like microenvironments is known to enhance MSC paracrine function [2-4]. Cell sheet technology, a scaffold-free 3D culture platform, had not previously been investigated for 3D-related augmentation of MSC paracrine-related cytokine production function. 137 Therefore, this study assessed cell sheets following their temperature-induced 2D-to-3D structural transition following spontaneous detachment contraction from adherent monolayer culture. Comparisons of 2D monolayer cultures to contracted 3D cell sheets showed notable structural and cytoskeletal reorganization, resulting in significantly greater MSC gene expression of cellular interaction and ECM proteins in 3D cell sheets. Pro-regenerative cytokine gene expression was significantly greater in 3D cell sheet MSCs, and, consistently, concentration of secreted angiogenic factor, VEGF, was significantly higher by 3D cell sheets accounting for differences in cell number and proliferation rate. These studies demonstrated that cell sheet spontaneous 3D structural and morphological transition, characterized by cell shape change and increased instances of cellular interactions, augments individual MSC cytokine production relative to the 2D monolayer counterpart. Collectively, this research informed key findings about the relationship between MSC 3D culture and scaffold-free cellular interactions on the regulation of MSC paracrine-relevant function. Chapter 3 expands on our previous assessment of cell-cell and cell-matrix interactions in 3D cell sheets and their role in boosting MSC cytokine production potency [1, 5]. Because cell sheets contain no interruptive biomaterial scaffolding and are comprised entirely of interacting cells and endogenous matrix, we expected centrifugation could be used as a tool to non-invasively increase instances of physical and biochemical cellular interactions related to cytokine secretion within MSC sheets. To test this, conditions for stable and reliable cell sheet centrifugation were first optimized. Comparisons of conventional (non-centrifuged) and centrifuged cell sheets showed notable tissue compaction and tight-packed cellular arrangement within centrifuged cell 138 sheets, as well as significantly greater MSC gene expressions of cellular interaction proteins, ECM proteins, and pro-regenerative cytokine proteins in centrifuged cell sheets. Centrifugation indeed augmented individual MSC cytokine production compared to MSCs in conventional cell sheets, quantified by ELISA, and this functional enhancement subsisted over 4 days in static in vitro culture. These studies demonstrated that cell sheet centrifugation increases cell-experienced 3D cellular interactions, augmenting individual MSC cytokine production relative to non-centrifuged sheets. Overall, this cell sheet centrifugation platform can be readily implemented to generate scaffold-free, 3D MSC tissue with enhanced paracrine-relevant secretory function for applications in regenerative medicine. Having demonstrated that cell sheet centrifugation boosts MSC cytokine production potency via higher abundance cellular interactions within a sheet (Chapter 3), Chapter 4 pursues a strategy for generating multilayered MSC sheets from centrifugation that conserves the centrifugation-induced functional enhancement relative to noncentrifuged, statically multilayered sheets, and with the advantage of incremental control over MSC-secreted growth factor dose from the 3D tissue construct. Building from conditions for first-layer cell sheet adhesion, sheet layering protocols using centrifugation (centrifugation method) or using passive incubation (conventional method) were developed to support sheet-to-sheet interfacing and attachment. Comparisons of 2-layer centrifuged and 2-layer conventional cell sheets revealed a compacted tissue structure with a controlled, incremental increase in thickness and cell dose in centrifuged 2-layers but not in conventional 2-layers. Consistent with the centrifugation effect observed in 1layer cell sheets, MSCs in 2-layer cell sheets fabricated by centrifugation layering 139 demonstrated significantly higher gene expression for gap junction protein, cell-matrix interaction-related proteins, and pro-regenerative cytokines, as well as secreted cytokine concentrations per sheet and per cell. Centrifugation layering indeed incrementally increased MSC-secreted cytokine dose, quantified by ELISA, that subsisted over 4 days in static in vitro culture. Significant upregulation of VEGF secretion per cell in 2-layer centrifuged cell sheets could indicate increased cellular interactions contributed by the layered interface. Overall, this study demonstrated that cell sheet multilayering from centrifugation generates viable 2-layered constructs with controlled thicknesses and cell doses, increasing absolute MSC sheet construct secreted cytokine dose. Toward the generation of an optimal 3D cell-delivery platform, the present study provides an adaptable protocol for cell sheet multilayering using centrifugation that generates functionally enhanced 2-layer MSC tissues. Overall, these findings support assertions that 3D MSC sheets represent a novel platform for enhancing paracrine-related MSC function that is. These studies lay the foundation for developing cell sheets as 3D MSC-delivery systems with tailored bioactive factor delivery function for future in vivo cell therapy in regenerative medicine applications. 5.1 References [1] S. Bou-Ghannam, K. Kim, D.W. Grainger, T. Okano, 3D cell sheet structure augments mesenchymal stem cell cytokine production, Sci Rep 11(1) (2021) 8170. [2] B. Follin, M. Juhl, S. Cohen, A.E. Pedersen, J. Kastrup, A. Ekblond, Increased Paracrine Immunomodulatory Potential of Mesenchymal Stromal Cells in ThreeDimensional Culture, Tissue Eng Part B Rev 22(4) (2016) 322-9. 140 [3] T.H. Qazi, D.J. Mooney, G.N. Duda, S. Geissler, Biomaterials that promote cell-cell interactions enhance the paracrine function of MSCs, Biomaterials 140 (2017) 103-114. [4] H.M. Wobma, D. Liu, G. Vunjak-Novakovic, Paracrine Effects of Mesenchymal Stromal Cells Cultured in Three-Dimensional Settings on Tissue Repair, ACS Biomater Sci Eng 4(4) (2018) 1162-1175. [5] M. Nakao, K. Kim, K. Nagase, D.W. Grainger, H. Kanazawa, T. Okano, Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets, Stem Cell Res Ther 10(1) (2019) 353. CHAPTER 6 FUTURE WORK 6.1 Evaluation of MSC Sheet Therapeutic Outcomes for Fibrotic Liver Disease 6.1.1 Introduction The most clinically intriguing characteristic of MSCs is their ability to secrete a wide range of bioactive cytokines and growth factors that can influence nearby cells via paracrine signaling [1, 2]. MSCs have been documented to provide generous source of bioactive factors that orchestrate various biological processes desirable for tissue regeneration, including angiogenesis, immune modulation, and cell recruitment and proliferation [3-7]. An increasing body of clinical evidence suggests that the therapeutic benefits observed following MSC transplantation may be largely attributed to the paracrine function of these cells, rather than their differentiation potential [8, 9]. The current clinical gold standard for administering MSC therapies is by systemic cell injection of single cell suspensions, a valuable method for the clinical ease of use. However, the inability of injection delivered MSCs to localize at the site of tissue injury dilutes potential paracrine benefit, and clinical applications must compensate by administering higher absolute cell doses or repeated cell injections [10-12]. Failed engraftment of inject cells results in poor survival and suboptimal in situ activity [13], contributing to the mismatch in preclinical expectations and realized clinical outcomes. 142 The operational motive guiding the work of this dissertation was to combine clinically approved and paracrine potent hUC-MSCs with clinically validated cell delivery platform, cell sheet technology. Cell sheet technology is documented to support superior cell localization, engraftment, and survival at the target tissue compared to systemically administered MSCs with demonstrated improved therapeutic outcomes across disease targets [14-20]. In addition to fabricating hUC-MSC as sheets, many contemporary research efforts by a number of groups have focused on 3D tissue engineering strategies that can enhance the clinically valuable secretory function of MSCs [21-24], motivating our novel efforts to employ and manipulate physical parameters within cell sheet engineering that could similarly boost MSC cytokine production function. The guiding translational hypotheses of this dissertation, therefore, were that (i) fabricating hUC-MSCs as cell sheets would resolve issues of engraftment, survival, and concentrated paracrine function associated with systemic cell injections, leading to improved therapeutic outcomes in an in vivo disease model, and that (ii) higher MSC sheet secreted concentration of paracrine factors would provide a larger therapeutic paracrine response in an in vivo disease model. Therefore, future work should evaluate single- and multilayer 3D hUC-MSC sheets prepared by centrifugation methods described in the body of this dissertation in an in vivo disease model. Liver fibrosis presents a pathology that stands to benefit from hUC-MSC sheet intervention. Liver fibrosis cascades in hepatocyte damage, recruitment of inflammatory cells to the injured liver, and activation of collagen-producing cells (hepatic stellate cells) [25]. The fibrogenic response is a complex process in which accumulation of ECM proteins, tissue contraction, and alteration in blood flow are prominent [26]. Extensive 143 clinical testing using diverse applications of MSC for liver cirrhosis and liver failure has been conducted under the research premise that MSC operate as promising antifibrotic therapeutics, secreting various immune-modulating, vascularization-mediating, and tissue regenerating trophic factors [27-30]. These clinical attempts have largely depended on systemic or direct injection to deliver MSCs, but long-term outcomes have shown marginal clinical benefits with high inter-patient variability to date [31-33]. hUC-MSC sheets as a 3D cell-delivery platform represent a promising intervention for liver fibrosis, capable of overcoming the therapeutic limitations attributed to poor engraftment due to systemic injection delivery, as well as providing therapeutically relevant pharamacology by paracrine signaling. The goal of this future work is to assert the therapeutic novelty of hUC-MSC sheets over the clinical cell therapy standard of systemic MSC injections for treating fibrotic liver disease, as well as to validate the therapeutic paracrine benefit of hUC-MSC sheets mediated by localized secretion of pro-regenerative cytokines in response to the dynamic disease environment. Fibrosis is the excessive accumulation of extracellular matrix components due to liver injury, with collagens as predominant structural components. Liver fibrosis can progress to cirrhosis, characterized by severe distortion of the delicate hepatic vascular architecture, the shunting of the blood supply away from hepatocytes, and the resultant functional liver failure. Continuous administration of trophic factors provides therapeutics outcomes in liver fibrosis [34]. As detailed in the Chapters 2-4 of this dissertation, cell sheets prepared with hUC-MSCs exhibited significantly higher cytokine (HGF, VEGF, IL-10) production related to tissue regeneration, vascularization, and anti-inflammation, respectively, compared to 144 dissociated hUC-MSCs (Figure 1.4), and 3D hUC-MSC sheets prepared by centrifugation further boosted this secretory function (Figure 3.3). Multilayered hUC-MSC sheets prepared from centrifugation maximize the realized cell dose, and thereby the absolute secreted factor dose, from engraftable cell sheet constructs (Figure 4.4). Therefore, we hypothesize that multilayered hUC-MSC sheets will locally secrete more pro-liver regenerative cytokines to the fibrotic liver and improve therapeutic outcomes in preclinical liver fibrosis compared to single layer hUC-MSC sheets and to injection administered hUC-MSC suspensions. hUC-MSC sheets will be prepared according to centrifugation and centrifugation layering protocols described in this dissertation (Chapters 3 and 4, respectively). To test this hypothesis, fibrotic liver disease will be modeled using the established carbon tetrachloride (CCl4) method in BALB/c-nu/nu mice previously described by our group [15, 35]. This experimentation will require male and female BALB/c-nu/nu mice randomly distributed among experimental groups. The disease model will be qualified with 1) diseased and 2) healthy models at 1, 2, and 4 weeks prior to hUC-MSC sheet transplantation (Figure 6.1). hUC-MSC sheet therapeutic efficacy will be evaluated by comparing 1) dissociated hUC-MSC suspension intraperitoneal (I.P.) injection, 2) single-layer and 3) multilayered hUC-MSC sheet transplantation (Figure 6.2). hUC-MSC sheets (groups 2 and 3) will be transplanted onto the left lobe of the liver surface by direct surgical placement, without any additional suturing, and transplanted hUC-MSC sheets will be covered by the median lobe of the liver. Positive (sham) and negative (disease model without treatment) controls will be compared with the 3 experimental groups to test liver function, excessive ECM accumulation within the liver, paracrine factors secreted from hUC-MSC sheets, and 145 hepatic vascular regeneration at 2, 8, and 14 days following hUC-MSC transplantation. 6.1.2 Animal model justification In vitro testing will narrow down experimental MSC sheet groups and standardize MSC sheet fabrication and preparation methods; however, it is impossible to develop a translational cell therapy without in vivo testing. Liver fibrosis environments are complex, involving a multitude of factors including cells, proteins, and biochemical pathways during liver regeneration [36]. Currently, there is no in vitro platform that fully and reliably reproduces this environment sufficient to predict in vivo responses to cellular transplantation. No known computer model exists that sufficiently predicts interactions between fibrotic liver and transplanted cells. The use of animal models is crucial for determining mechanisms underlying initiation, progression, and resolution of fibrosis and for developing novel therapies. Therefore, without an in vivo animal model, cell sheet fates in vivo cannot be determined, and further translation of this therapy would not be feasible. This study proposes a mouse liver fibrosis model that is well documented to recapitulate fibrogenesis in human liver disease [37]. To test human MSCs in an animal model, immunodeficient models are necessary for a more complete understanding of in situ events during disease progression and treatment. NOD-SCID mice are commonly used for xenograft studies for their impaired T- and B- cell development and deficient NK cell functions that mitigate outright xenogeneic rejection [38-40]. However, B-cells are indispensable for establishing liver fibrosis [41]. Therefore, this study uses BALB/cnu/nu (immunodeficient) for liver fibrosis modeling because they lack thymus 146 responsible for the production of T-cells but are still able to produce B-cells. For liver fibrosis modeling, male BALB/c-nu/nu mice aged 7-9 weeks old will be used [42]. It is well known that sex renders a differential response to chemical toxin treatments and therapeutic interventions [43]. Therefore, male and female mice will be used for efficacy studies with consideration for sex-attributed morphological and functional differences. 6.2 Statistical Powering To determine minimum necessary animals, a power analysis was performed according to relevant published literature [44]. An F test ANOVA with a statistical power level of 80%, an α value of 0.05, and a 0.6 effect size resulted in sample sizes of n=8 for each experimental group for each timepoint [44]. This calculation was performed using free sample size calculator software, GPower. Effect size was based on sensitivity calculations and sample sizes required to determine statistically significant differences in histological scoring and quantitative mechanical data [45]. An additional 10% increase (n= ~1 animal/group/timepoint) accounts for unexpected loss of animals during the course of the experiment. No procedures will be done to additional animals unless they must replace an experimental animal due to medical necessity. For qualification of the liver fibrosis model, the study outlined contains the following groups: 1) diseased and 2) healthy models at 1, 2, and 4 weeks prior to MSC sheet transplantation (Figure 6.1). Enzyme activity testing and histological scoring in this study requires 54 nude mice (9 mice (8 experimental + 1 reserve)) × 3 timepoints × 2 groups × 1 analysis). Serum for enzyme activity testing will be collected from the mice used for histological scoring. Evaluation of the therapeutic efficacy of hUC-MSC sheet 147 transplantation requires the following groups: 1) sham (positive control), 2) disease no therapy (negative control), 3) single layer hUC-MSC sheet transplantation, 4) multilayered hUC-MSC sheet transplantation, and 5) dissociated MSC suspension injection at 2-, 8-, and 14-days after cell transplantation (Figure 6.2). The number of nude mice required for serum enzyme activity testing, gene expression level testing, histological scoring, and hepatic vascular regeneration evaluation will be 315 ((gene expression level testing (270): 9 mice (8 experimental + 1 reserve) × 3 timepoints × 5 groups × 2 analysis) + hepatic vascular regeneration evaluation (45): 9 mice (8 experimental + 1 reserve) × 1 timepoints × 5 groups × 1 analysis)). Serum for enzyme activity testing will be collected from the mice for histological scoring. The total number of animals necessary for this study is 369 nude mice. 6.2.1 Experimental plan • Experiment 1. Qualify the murine liver fibrosis model BALB/c-nu/nu mice will be intraperitoneally (IP) administered CCl4 dissolved in corn oil (0.25 µL CCl4/g body weight) twice per week for 4 weeks, producing progressive fibrotic cirrhosis [15, 46, 47]. To design accurate experimental time points, we will generate a database with body weight and serum markers (aspartate aminotransferase/alanine aminotransferase [AST/ALT]) that correlate with diseased histology (Figures 6.1 and 6.2). During the experimental study, disease animals will be evaluated for body weight and serum markers and only successful fibrosis-induced mice will be used for cell transplantation. 148 • Experiment 2. Evaluate liver functions after hUC-MSC sheet transplantation Serum isolated from venous blood will be collected at 2, 4, 8, and 14 days after transplantation. Timepoints were chosen based on our previous study [15, 46]. AST/ALT and bilirubin level, which increase when hepatocytes are damaged, obtained in blood samples will be evaluated [15, 46]. Liver/body weight will be assessed [15, 46]. • Experiment 3. Measure ECM accumulation related to live fibrosis after hUC-MSC sheet transplantation Resected liver tissue samples from three different areas in each lobule (left and median lobes) at 2, 8, and 14 days after transplantation will be H&E stained to determine liver necrosis and hepatocyte injury. Excessive liver collagen accumulation will be measured with hydroxyproline analysis (quantitative test) and Sirius Red (comparative test) staining [15, 46]. Liver myofibroblasts producing ECM in fibrous scarring will be detected by IHC staining α-smooth muscle actin (αSMA) [48]. Additionally, ED1 staining will be performed to detect macrophage infiltration in liver tissue [49]. Tissue samples from healthy and diseased liver models will be collected each week for fibrotic indicators as positive and negative controls; histology will be evaluated and scored for fibrotic stage by a pathologist according to the Ishak system [50]. • Experiment 4. Evaluated the paracrine effect from hUC-MSC sheets Liver tissues adjacent to the transplant area from each experimental group will be collected at 2, 8, and 14 days after hUC-MSC transplantation. mRNA will be extracted from the collected liver tissue samples and cDNA will be synthesized from the mRNA. Then, human-specific gene expression related to hepatocyte regeneration (HGF, PGE2, 149 IL-6, and EGF) and to vascularization (VEGF and angiopoietin) will be assessed with GAPDH or b-actin as a house keeping gene by qPCR analysis [15, 51]. Disease model without treatment samples will be used as a negative control. Transplanted hUC-MSC sheet groups will be used as a positive control. • Experiment 5. Measure hepatic vascular regeneration mediated by hUCMSC sheets Resected liver tissue samples from three different areas in each lobule (left and median lobes) at 2, 8, and 14 days after transplantation will be used for histology. At necropsy, paraffin-embedded liver tissue specimens will be stained with CD31 antibody [52]. For high-resolution µCT analysis, a catheter will be inserted into the portal vein and Microfil (silicon rubber) will be perfused at a rate of 3-4 ml/min per 100 g body weight [53]. After fixation, liver will be scanned using high-resolution µCT to visualize vascular regeneration [53]. 6.3 Statistical Analysis All data sets will be tested for normality using a Shapiro-Wilk test. For quantitative analysis sets, one-way ANOVA will be used to determine statistical significance among multiple groups over multiple time points. Post-hoc analysis will include either Tukey’s testing (for normal data) or Bonferroni testing (for non-normal data). Histological scoring of liver pathologies in Experiment 1 and 3 will be analyzed using the Ishak system [50]. Significance will be calculated using GraphPad Prism 9 software and is generally defined as p ≤ 0.05. 150 6.3.1 Expected outcomes Given previous experience by our group to induce staged liver fibrosis using CCl4 in immunodeficient mice, we expect to observe liver fibrosis within 4 weeks of CCl4 treatment [15, 46]. If our hypothesis is valid, hUC-MSCs in multilayered sheets should demonstrate higher cytokine gene expression levels related to liver regeneration (hepatocyte and hepatic vascular regeneration) than in the single-layer sheet and dissociated hUC-MSC suspension injection groups. Increased cytokine secretion will induce hepatic vascularization in sheet groups. Improved blood supply through regenerated hepatic vascular architectures will reduce excessive accumulation of ECM proteins (reduced collagen deposition, αSMA+, and ED1+) and enhance liver functions (reduced AST, ALT, and bilirubin concentration in the blood). We anticipate multilayered hUC-MSC sheets to show higher therapeutic efficacy compared to singlelayered hUC-MSC sheets and dissociated hUC-MSC suspension injections, given localized and retained high doses of highly secretory hUC-MSCs as layered sheets [15, 46, 51]. With these data, a relationship can be determined between hUC-MSC cytokine production function in multilayered hUC-MSC sheets in vitro with relative improved therapeutic efficacy in vivo, providing a criterion for functional hUC-MSC sheet preparation before transplantation. 6.3.2 Potential pitfalls and alternative strategies Based on our previous in vitro findings that MSC sheet multilayering increases the absolute secreted cytokine dose (see Chapter 4), we anticipate multilayered hUCMSC sheets will contribute the largest therapeutic efficacy in treating liver fibrosis 151 relative to our proposed comparison groups. However, an inappropriate disease model can obfuscate the therapeutic effects of hUC-MSC sheet transplantation. If the mouse mortality rate is too great to analyze any effect of treatment, CCl4 concentration will be reduced. If transplanted hUC-MSCs and cytokines secreted from hUC-MSC sheets cannot penetrate the fibrotic liver capsule, we expect cytokines secreted from hUC-MSC sheets to activate the mesothelium to promote proliferation of parenchymal cells related to liver regeneration [54]. Additionally, micro holes of only mesothelium depth can be made on the liver surface (i.e., via micro needle punch) to improve hUC-MSC secreted cytokine penetration into the fibrous capsule. 6.4 Quantitative Estimate of MSC Sheet Bioactive Factor Diffusion into Liver Tissue The proposed liver fibrosis model assumes cell sheet therapeutic value via the diffusion of MSC-derived bioactive factors into the liver tissue to stimulate a proregenerative response from host cells. Cytokines VEGF, HGF, and IL-10 are highlighted in this dissertation for their therapeutic value in liver fibrosis, both as stem cell therapy secreted factors as well as biologic drug candidates, functioning to stimulate liver tissue regeneration and suppress chronic immune damage [10, 30]. In the MSC sheet case, we anticipate that MSCs will respond to signals from the disease environment that stimulates MSC sheets to release VEGF, HGF, and IL-10 into the tissue. Chapter 4 and 5 demonstrated that MSC sheets diffuse these molecules into their aqueous media environment in static culture, in the absence of disease stimulation. The efficacy of bioactive factor transport from MSC sheets to the liver tissue can 152 be estimated by diffusion profile modeling. Of course, MSCs secrete myriad bioactive factors and molecules that contribute to tissue regeneration and the release profile is regulated by dynamic environmental stimuli. However, for simplicity of modeling and given the prominence of HGF-mediated liver tissue regeneration described in clinical efforts and in research literature [55], HGF alone was selected for diffusion profiling to estimate the efficacy of release from MSC sheets into the liver tissue and the therapeutic relevance of delivered HGF concentrations over time. Furthermore, accounting only for secreted factor diffusion of course discounts therapeutic mechanisms related to direct MSC-to-host cell-cell contact [32]. The in vitro focus of this dissertation, though, has been on MSC sheet-secreted soluble factors to gauge therapeutic potency. Estimating the bio-transport efficacy of HGF based on empirical sheet-secreted static concentrations provides baseline justification that the secreted factor concentrations reported in this dissertation are on the order of pre-clinical relevance. In Chapters 3 and 4, MSC sheets were shown to secrete HGF daily in static culture over the course of 4 days. The proposed MSC sheet would operate as a live-cell bioactive delivery depot, whereby live cells would continually secrete necessary proregenerative agents in response to real-time environmental stimuli. For this reason, MSC sheet diffusion was modeled as an infinite source, whereby the concentration of secreted HGF is constant and steady state once it is placed onto liver tissue. Following MSC sheet transplantation onto the liver surface, HGF diffusion away from the cell sheet is only considered in the direction toward the liver tissue and assumes that 50% of HGF will diffuse away from each side of the cell sheet. This model considers the therapeutic timepoints established in the proposed in vivo liver fibrosis model described in Chapter 6, 153 calculating HGF concentration within the liver tissue as a function of distance from the cell sheet at 24 hours, 1 week, and 2 weeks following transplantation. HGF is assumed to not be filtered or removed from the liver tissue by cells or ECM components in the tissue or by blood circulation. Given these parameters, the following constant source diffusion function was implemented: 𝐶(𝑥, 𝑡) = 𝐶! × 𝑒𝑟𝑓𝑐 . 𝑥 2√𝐷𝑡 2 Whereby, 𝐶(𝑥, 𝑡) is the concentration of HGF as a function of diffusion distance and time, 𝑥 is protein diffusion distance (µm) into the liver tissue, 𝑡 is time (hours), 𝐶! is a constant source surface concentration of HGF produced by the MSC sheet, determined empirically as mass of HGF secreted by a single cell sheet in static culture over 24 hours (Figure 3.3) per cell sheet area (Figure 2.2), 𝐶! = 3.4x10-5 pg/µm2 𝑒𝑟𝑓𝑐 indicates an error function, And 𝐷 is the diffusion coefficient for HGF through tissue [56, 57]. 𝐷 = 2.736x105 µm2/hour This function was plotted to achieve the HGF diffusion profile, reported as HGF concentration per unit area at 𝑥 distance in the liver at a predetermined time, from a surface transplanted MSC sheet into liver tissue (Figure 6.3). We assume at distance 𝑥 in the liver tissue, MSC sheet secreted HGF will permeate the full tissue cross-section along the dorsoventral diameter with a constant 154 concentration. Taking the dorsoventral diameter of the rat liver to be 5.2 cm [58], we can calculate an assumed circular surface area at any distance 𝑥 in the tissue to be 2.124x109 µm2. Therefore, at 1.0 cm within the liver tissue, we can expect 0.20, 10, and 16 ng of MSC sheet secreted HGF to have diffused across that tissue cross-sectional area by 1, 7, and 14 days, respectively. This assumption allows us to calculate the mass of diffused HGF as a function of time and diffusion distance to compare against doses used in preclinical studies efficacy studies for the treatment of liver fibrosis with HGF. In a preclinical study for the treatment of CCl4-induced liver cirrhosis by human recombinant HGF intraportal injections, Matsuda et al (1997) found a soluble HGF dose on the order of 30 µg/kg injected daily intraperitoneally for 3 weeks over 12 weeks of biweekly treatment with CCl4 resulted in significantly accelerated recovery from liver fibrosis/cirrhosis [59]. With an average rat weight of 100 g, Matsuda et al (1997) administered approximately 3 µg of HGF daily to achieve therapeutic efficacy [59]. To gauge the relevance of our predicted HGF secreted doses by MSC sheet diffusion, an ideal comparison would be a dose-response curve for a steady-state HGF-releasing hydrogel transplanted to the liver surface with known initial starting concentrations. However, in the Matsuda et al (1997) case and throughout the literature, soluble HGF is primarily administered intravenously or intraperitoneally [59]. The starting concentrations for venous or peritoneal injections must be high enough to account for an HGF half-life in circulation of 2.4 minutes [60]. In a pharmacokinetic study of IV administered soluble HGF, a 0.1 mg/kg initial concentration only rendered 90 ng/mL serum HGF within 5 minutes, declining overtime according to the 2.4-minute half-life [60]. If we assume a 10-fold dilution from administered concentration to serum-level 155 HGF concentration, 300 ng of soluble HGF may have been responsible for the measured therapeutic outcomes against liver fibrosis in Matsuda et al (1997) [59]. In the predicted case of MSC sheet HGF diffusion, protein masses on the order of 10 ng at 1.0 cm within the tissue by 7 and 14 days are achieved (Figure 6.1). Assuming at least an additional 10fold dilution from serum-level to tissue-level HGF concentrations, an order of 10 ng of soluble HGF diffused within the tissue could be extrapolated to be a feasible dose for liver regeneration following CCl4-induced fibrosis. Per the HGF diffusion profile from MSC sheets, HGF doses on the order of 10 ng could be achieved over 2.0 cm into the liver tissue at least by day 7, and over 14 days following MSC sheet transplantation. Extrapolating from previous studies, this represents a feasible pre-clinical dose for observing therapeutic outcomes in liver fibrosis. Of course, the diffusion profile modeled is based on the HGF production of a single cell sheet under static conditions. As shown in Chapter 4, MSC sheet bilayers secreted twice the soluble HGF concentration under static culture over 4 days relative to single MSC sheets (Figure 4.4). Cell sheet multilayering should at least double the hypothetical secreted HGF concentrations with the same profile as a function of distance diffused and time (Figure 6.3). Chapter 6 details an experimental outline to empirically determine the therapeutic impact of single and multi-layered MSC sheet transplantation on pre-clinical liver fibrosis outcomes. 156 6.5 MSC Sheet Exosomal Consideration as a Mediator of Therapeutic Paracrine Effect The clinical understanding that MSC paracrine function, rather than direct tissue replacement through differentiation, is primarily responsible for their regenerative potential triggered a new wave of research investigation into the MSC secretome as a practical therapeutic [61, 62]. Extensive applications of conditioned medium from MSCs reveal that the conditioned medium alone shows therapeutic potential, in some cases demonstrating at least equal effectiveness as transplanted MSCs [63-67]. The MSC secretome has been well documented to contain bioactive factors such as VEGF, fibroblast growth factor (FGF), HGF, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), interleukins (ILs), and matrix metalloproteinases (MMPs), and it was commonly conceptualized that these secreted growth factors and cytokines stimulate host tissues to regenerate (i.e., the paracrine effect) [5, 6, 68, 69]. However, a new mechanism for MSC therapeutic potential has been put forth, and that is that the transfer of MSC-secreted exosomes/macrovesicles packaging various bioactive factors is stimulating host-tissue regeneration, mediating the observed paracrine effect [70-76]. Extracellular vesicles (EVs) are small vesicular nanoparticles secreted by cells, first observed in normal plasma as a platelet-derived particle and were referred to as “platelet dust” in 1967 [77]. Within the EV population, exosomes are classified by their diameter (40 – 150 µm) and their biogenesis from multivesicular body fusion with the cell membrane [78]. It has been demonstrated that EVs play a key role in intercellular communication by transfer of their cargoes, such as proteins, nucleic acids, and lipids [78]. A large body of preclinical and clinical studies have investigated whether the 157 therapeutic potential of MSCs may be largely mediated by paracrine factors contained in EVs [75, 76]. The first preclinical study of MSC-EVs was reported using a mouse model of myocardial ischemia-reperfusion injury in 2010 [79]. Subsequently, therapeutic effects of MSC-EVs have been reported in various organs including lung, kidney, liver, CNS, cartilage, bone, and heart [80]. Direct comparison of the efficacy of MSC therapy with MSC-EV therapy is difficult, due to the potential of MSCs to provide a long-term source of EVs at the injured site. However, many studies showed that MSC-EVs appear to be as effective as their parental MSCs [75]. EVs boast several translational advantages over MSC therapies, including less risk of side effects related to embolism, immune rejection, and tumorigenesis, encouraging clinical investigation of MSC-EV therapies [81]. The first case of MSC-EV therapy in human was the administration of allogenic bone marrow MSC-EVs for a patient with therapy-refractory acute GvHD in 2014 [82]. GvHD symptoms recovered within a week after administration of MSC-EV and no significant side effects were observed [82]. To date, several clinical studies of MSC-EVs have been conducted or are ongoing [83]. Phase II/III clinical trial of human cord blood derived EVs for patients with chronic kidney disease showed safety, anti-inflammatory effects, and amelioration of kidney dysfunction [84]. In addition, clinical trial of MSC-EVs for macular holes (NCT03437759), bronchopulmonary dysplasia (NCT03857841), stroke (NCT03384433) and diabetes mellitus type I (NCT02138331) are ongoing. More recently, three clinical trial for MSC-EVs for acute respiratory distress syndrome due to COVID-19 is ongoing in China (NCT04276987, ChiCTR2000030261, ChiCTR2000030484). In summary, the potential of MSC-EVs is widely explored for 158 various diseases. 6.5.1 Evaluation of MSC-sheet EVs This dissertation measures the value of an engineered tissue effect by the stimulation of MSC pro-regenerative cytokine production in vitro, operating under the mechanistic understanding that these secreted factors contribute well documented therapeutic paracrine effects in vivo. With growing research, clinical, and industry understanding that MSC-EVs, containing these pro-regenerative proteins in addition to bioactive lipids, RNAs, and small molecules, are responsible for MSC therapeutic potential, it is important that future measurements of the value of the in vitro tissue effect, as well as of mechanisms supporting therapeutic efficacy in proposed in vivo future studies, reflect this new knowledge. This section will outline future studies that consider EV production, characterization, and therapeutic function within the scope of the work described in this dissertation. It should be noted, though, that cell sheet delivery is a live cell therapy, where live cells can situationally respond in real time to the disease environment and continuously modulate necessary bioactive factor release over time; exosome therapies harness only the molecular cargo present at the time of exosome harvest and are unable to adapt or respond to regenerative cues after implantation. This live cell response is one key advantage of MSC sheet therapy over MSC sheet-EV potential. 159 • In vitro isolation and characterization of MSC sheet-EVs Ultracentrifugation (UC) for EV isolation is widely used for its utility in a smallscale, research lab setting [75]. Very high forces in excess of 100,000 g for prolonged periods are needed to separate small particles less than 200 nm in size [75]. Generally, starting conditioned media volumes range from 0.5 – 1 liters and go through a series of filtrations to achieve a final volume under 500 µL [85]. From this volume, size exclusion chromatography isolates exosome from non-exosome fractions [85]. The MSC sheets described in this dissertation were fabricated using 3.5 cm dishes containing 2 mL of volume. Under these circumstances, large quantities of MSC sheets would need to be fabricated to collect 0.5 L of conditioned medium. Alternatively, larger diameter TRCD could be employed using the same initial seeding densities to achieve larger conditioned medium volumes from less individual cell sheets. Exosomes are representative of the origin cell proteomics, and therefore provide a novel mode of MSC characterization than the methods used to characterize MSCs in this dissertation (see Section 1.4). Multiplex RNA-sequencing previously described can be used to compare relative quantities of exosome RNA material; protein content within exosomes can also be measured. Comparisons of key pro-regenerative proteins of interest, such as VEGF, HGF, and IL-10, can be evaluated within the MSC-secreted exosome rather than from the MSC or MSC conditioned medium. In this sense, EV fractions can be compared to evaluate the tissue effect in 2D monolayer versus 3D cell sheet groups, or between centrifuged and non-centrifuged cell sheets, as a stand in for the PCR- and ELISA-based analysis described in this dissertation. 160 • Functional evaluation of MSC sheet-EVs Having previously defined the liver as a target organ of interest for applications of MSC sheet therapy, in vitro functional analysis of MSC sheet-EVs on cultures of hepatocytes can inform MSC-EV therapeutic potential. Hepatocytes cultured with and without MSC sheet-EVs can be compared for proliferation, albumin production, and HGF secretion in vitro. Further, to compare the therapeutic impacts of MSC sheets and MSC-sheet EVs, hepatocytes in indirect co-culture with MSC sheets can also be evaluated. However, the cell-based co-culture model is more complex in optimization, requiring adequate mixed media conditions and the ability to distinguish proteins secreted from the hepatocytes versus those from the MSCs. The fibrotic liver model detailed in Section 6.1 can be used exactly as described with the addition of MSC-sheet EVs as an experimental group to determine the therapeutic significance of EVs relative to the origin cell sheet direct transplantation as a therapy. MSC sheet-EVs would be injection administered, therefore method of administration (i.e., IP or directly into tissue), as well as dose and frequency of administration, must be considered for fair comparison to transplanted cells. Quantified outcomes of diseased liver function and paracrine-mediated tissue regeneration following treatment with MSC sheets or MSC sheet-EVs could be compared; if therapeutic outcomes are nearly equal or are improved by EV administration rather than by sheet engraftment, we can assert that the therapeutic mechanism of MSC sheets is largely mediated by a secreted EV paracrine effect. 161 Figure 6.1 Study design for generating a CCl4-induced disease model. Progressive fibrotic disease will be induced by CCl4 injection over 4 weeks prior to cell sheet transplantation. The image was created using BioRender. Figure 6.2 Study design for therapeutic evaluation of hUC-MSC transplantation. Time points for analysis are 2, 8, and 14 days after cell sheet transplantation. 162 Figure 6.3 The diffusion profile from MSC sheet secreted HGF into liver tissue. 163 6.6 References [1] P. Bianco, X. Cao, P.S. Frenette, J.J. Mao, P.G. Robey, P.J. Simmons, C.Y. 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Adel, Umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases, Biomater Res 20 (2016) 21. [85] S. Sjoqvist, T. Ishikawa, D. Shimura, Y. Kasai, A. Imafuku, S. Bou-Ghannam, T. Iwata, N. Kanai, Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing, J Extracell Vesicles 8(1) (2019) 1565264. APPENDIX A collation of my author contributions to published journal articles, critical reviews, and book chapters. 1. S. Bou-Ghannam, K. Kim, D.W. Grainger, T. Okano, 3D cell sheet structure augments mesenchymal stem cell cytokine production, Sci Rep 11(1) (2021) 8170. 2. K. Kim, S. Bou-Ghannam, S. Kameishi, M. Oka, D.W. Grainger, T. Okano, Allogeneic cell sheet therapy: a new frontier in drug delivery systems, J Control Release 330 (2021) 696-704. 3. K. Kim, S. Bou-Ghannam, T. Okano, Cell sheet tissue engineering for scaffoldfree three-dimensional (3D) tissue reconstruction, Methods Cell Biol 157 (2020) 143-167. 4. K. Kim, H. Thorp, S. Bou-Ghannam, D.W. Grainger, T. Okano, Stable cell adhesion affects mesenchymal stem cell sheet fabrication: Effects of fetal bovine serum and human platelet lysate, J Tissue Eng Regen Med 14(5) (2020) 741-753. 5. M. Yamato, S. Bou-Ghannam, T. Okano, Cell Sheet Therapy Applications in Human Clinical Settings, Encyclopedia Tissue Eng Regen Med Vol. 1 (2019) 7175. 6. Sjoqvist, T. Ishikawa, D. Shimura, Y. Kasai, A. Imafuku, S. Bou-Ghannam, T. Iwata, N. Kanai, Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing, J Extracell Vesicles 8(1) (2019) 1565264. 7. K. Kim, S. Bou-Ghannam, H. Thorp, D.W. Grainger, T. Okano, Human mesenchymal stem cell sheets in xeno-free media for possible allogenic applications, Sci Rep 9(1) (2019) 14415.