Quality improvement of juvenile chondrocyte cell sheets for osteochondral repair: exploring cell culture media volume and basal media supply

Juvenile cartilage-derived chondrocyte (JCC) sheets have been explored as a controlled, scalable method for articular cartilage repair. The current standard preparation method of these sheets uses commercially available temperature-responsive (TR) culture dish inserts in cell culture that allow for easy cell sheet harvest and removal without damaging the cellsheet produced extracellular matrix. A current area-of-focus is augmenting the in vivo efficacy of these sheets in cartilage regeneration; however, no current best-practice exists for sheet fabrication to enable this goal. Two metrics of quality for JCC sheets used in articular repair include harvested sheet thickness and sheet contraction ratio upon detachment from plastic culture surfaces. The purpose of this study is to examine the effect of two different growth variables (basal cell culture media supply and cumulative culture media volume) on these sheet metrics. Cell sheets were prepared in TR plastic inserts with and without basal culture media and in TR dishes, each cultured in conditions with low (0.225 mL/1000 cells seeded) and moderate (0.6 mL/1000 cells) cumulative media (CM) volume. An additional condition was run using basally supplied TR inserts with high (1.31 mL/1000 cells) CM volume. Sheets were stained with Safranin-O to identify matrix and cell sheet thickness was measured. Cell sheet volumes and contraction ratios were calculated. CM volume and basal media supply were found to contribute to both thickness and volume of cell sheets irrespective of cultureware. Increased CM volume conversely produced sheets with less contraction, which is a negative predictor of their chondrogenic capacity. We conclude that use of basal media supply is an encouraging method of improving cell sheet quality while increased CM volume requires additional research to more confidently determine its effect on in vivo efficacy.