| Description |
The blood-brain barrier (BBB) limits entry of most molecules into the brain and complicates the development of brain-targeting compounds, necessitating novel BBB models. This dissertation describes the first microfluidic BBB model allowing the study of BBB properties in relation to various chemical compounds by enabling tunable wall shear stress (WSS) via dynamic fluid flow, cell-cell interaction through a thin co-culture membrane, time-dependent delivery of test compounds, and integration of sensors into the system, resulting in significant reduction of reagents and cells required and shorter cell seeding time. Use of parallel channels first enabled simultaneous monitoring of multiple cell populations under a wide range (~x15) of WSS. The microfluidic model formed the BBB by incorporating brain endothelial (b.End3) and glial (C6/C8D1A) cells at the intersection of two crossing microchannels, respectively representing luminal and abluminal sides, fabricated in a transparent polydimethylsiloxane (PDMS) substrate utilizing high-precision soft lithography techniques. The utilized cells were adopted from immortalized cells for high consistency over repeated passages and pure and proliferative culture. The developed microfluidic BBB model was validated by (1) expression of tight junction protein ZO-1 and glial protein GFAP by fluorescence imaging, and P-gp activity by Calcein AM, confirming key BBB proteins; (2) high trans-endothelial electrical resistance (TEER) of co-cultures exceeding 250Ωcm2 confirming sufficiently contiguous cell layer formation; (3) chemically-induced barrier modulation, with transient TEER loss by 150μM histamine (~50% for 8-15min), and increase in permeability at elevated pH (10.0); (4) size-dependent (668-70,000Da) compound permeability mimicking in vivo trends; and (5) highly linear correlation (R2>0.85) of clearance rates of seven selected neural drugs with in vivo brain/plasma ratios. We demonstrated the effects of WSS (0-86dyn/cm2) on bEnd.3 properties under increasing WSS, including increase in (6) TEER, (7) cell re-alignment toward flow direction, and (8) protein expression of ZO-1/P-gp, and (9) decrease in tracer permeability. The developed in vitro microfluidic BBB model provides distinct advantages for monitoring and modulating barrier functions and prediction of compound permeability. Thus, it would provide an innovative platform to study mechanisms and pathology of barrier function as well as to assess novel pharmaceuticals early in development for their BBB clearance capabilities. |
