Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay

Background: To evaluate the ability of different commercially available cel l Culture media to induce proliferation and morphological changes in primar y cultures of human corneal endothelial cells (HCEC). This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas. Methods: A total of I I different culture media enric hed with 0%, 2%, 5%, and 10% fetal calf serum (FCS) were compared. The test media were divided into three groups: Group 1: Media based on minimal esse ntial medium (MEM), currently used for long-term corneal organ culture in E uropean eye banks; Group 2: F99-based media, enriched for growth of corneal endothelial cells at serum-reduced conditions; and Group 3: Media designed for growth of special cell types or for short-term corneal organ culture. The growth-promoting capacity of each test medium was quantified using an H CEC proliferation assay, whereas changes in cell morphology were evaluated by phase-contrast microscopy. Results: The morphological characteristics of HCEC were best maintained in the group of F99-based media, which also indu ced the highest level of cell proliferation under serum-reduced conditions. Specifically, the medium F99-Sr (F99 enriched with ascorbic acid, insulin, bFGF, transferrin. selenium, and lipids) induced a two- to three-fold high er HCEC density at both 0% and 2% FCS when compared to all other test media , and it also maintained the most endothelial cell-like morphology. Also, a t higher serum concentrations (5% and 10% FCS), the cell growth was most pr ominent in F99-Sr, as well as in the medium SFM that originally was designe d for serum-free growth of vascular endothelial cells. Conclusion: This stu dy suggests that the media F99-Sr and SFM should be further tested and refi ned as potential new storage solutions for long-term corneal organ culture at physiological temperatures.