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Well-characterized adipose stem cells and chemically defined culture media are important factors that control the production of the cell sheet, used in translational medicine. In this study, we have developed and engineered multilayer adipose stem cell cell sheets (ASCCSs) using chemically defined/serum-free culture media: undifferentiated or differentiated into osteoblasts and chondrocytes. In addition, using the cell sheet transmittance, we estimated the number of cells per cell sheet. Undifferentiated ASCCSs were engineered in 10 days, using serum-free/xeno-free culture media. They were CD29+, CD73+, CD90+, CD105+, HLA-A+, and HLA-DR-. ASCCSs differentiated into chondrocytes and osteoblasts were also engineered using chemically defined and animal-free culture media, in only 14 days. The addition of an ROCK inhibitor improved the chondrocyte cell sheet engineering. The decrease in the cell sheet transmittance rate was higher for the osteoblast cell sheets due to the intracellular Ca2+ accumulation. The estimation of cell number per cell sheet was carried out with the transmittance, which will provide important information for cell sheet posology. In conclusion, three types of ASCCSs were engineered using serum-free, xeno-free culture media, expressing their specific markers. Their transmittance measurement allowed estimating the number of cells per cell sheet, with a non-invasive methodology.

The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface.