Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-β1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-β1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.