Cell Sheet Technology for Corneal Surface Reconstruction

Corneal blindness affects more than 6 million people worldwide, of which the most common cause is limbal stem cell deficiency. Autologous regeneration using sources that can be easily harvested with minimal morbility to the patient would be ideal. The in vitro reconstruction of corneal epithelium by cultivation of primary oral mucosal epithelial cells (OMECS) is a potential approach for regenerative therapies of ocular surface diseases. However, cell culture conditions, culture medium, and tissue culture surface for growing these primary cells to a multilayered cell sheet are challenging. The purpose of the study is to optimize cell culture protocols and conditions to grow human OMECS. We investigated the influence of different medium supplements on the formation of the cell sheet. Human buccal tissue biopsy was used to isolate and culture the primary OMECS without a feeder and with clinical grade reagents. As fetal bovine serum was not used in the cell culture medium, KO serum replacement, and human serum albumin (HSA) were used as a protein and amino acid supplement source. EGF was also investigated in combination with Rock inhibitor supplementation to improve cell growth. Live cell imaging showed that in the first week of cell culture, colony forming units were visible throughout the entire surface of the 6W-plate, indicating a great pool of progenitor stem cells in the isolated OMECS. However, cell attachment was not efficient suggesting non-functional, incompatible, or deficit of extra cellular matrix (ECM) components. Different type of extracellular matrix substrates (CELLstart and Biolaminin521) along with VitC were used to improve cell attachment. The results showed that combination of VitC and ECM substrates promoted cell attachment to cell culture surface. Combination of KOSR with human serum albumin at a low concentration promoted cell growth. Cells were scraped off and analyzed in Western blot, and the results showed that cells expressed Fibronectin, PCNA as well as corneal progenitor marker K15, indicating potential cell attachment and growth. The study shows that cell culture of OMECS with a low amount of protein and with ECM substrates combined with VitC represents a promising approach for an autologous, feeder free clinical grade cell sheet for corneal surface diseases.