94 Quality Improvement of Juvenile Chondrocyte Cell Sheets: Exploring Cell Culture Media Volume and Culture Dish Size

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 cell- sheet produced extracellular matrix. A current area-of-focus is augmenting the in vivo efficacy of these sheets while minimizing the cell sheet fabrication cost. Use of TR dishes in place of TR inserts may produce sheets of larger surface area at lower cost, however it has been unclear how different cultureware options and associated culture parameters impact the chondrogenic capacity desired for JCC sheets. This research examines the histology of JCC sheets grown in both TR dish and semipermeable TR insert cultureware to identify the differences in sheet properties.

Sheets were prepared in TR inserts with and without basal media and in TR dishes, each cultured in conditions with low (0.225 mL/1000 cells seeded) and standard (0.6 mL/1000 cells seeded) total media volume. Cell pellets were isolated from each sheet and differentiated using a modified chondrogenic differentiation media. Sheets and pellets were stained with Safranin-O to identify matrix and cell sheet thickness was measured. Surface area and volume were calculated. Media volume was found to contribute to both thickness and volume of cell sheets irrespective of cultureware. Basal media supply was observed to amplify this effect. At the standard media volume, TR inserts with basal exposure to media produced substantially thicker sheets than all other dish conditions. Additionally, all pellets demonstrated glycosaminoglycan (GAG) production. We conclude that use of TR dishes and lower media volumes as a method of cost-reduction in the production of JCC sheets would require sacrifices to the overall sheet thickness or volume.