Evaluation of potential organ culture media for eye banking using human donor corneas

Aim-To evaluate the ability of different commercially available cell cultur e solutions to preserve human donor corneas during 3 weeks of "closed syste m" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum -free organ culture medium for eye banking. Methods-72 normal human donor corneas were organ cultured for 21 days at 31 degreesC in eight different test media (nine corneas in each group). The b asic culture solutions included: minimal essential medium (MEM), MEM with s tabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid , insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All med ia were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the end othelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) c hanges in storage medium pH, glucose, and lactate content. Results-SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS . Corneas stored in SFM also showed the least leakage of keratan sulphate a nd the highest glucose consumption and lactate production. In five media (M EM with 2% FCS, MEM with stabilised L-glutanuine, M199, F99, and F99-Sr), c omparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium w as lost after storage in DIF1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highes t RNA synthetic activity combined with a cell loss of only 11% (4%), compar ed with 19% (6%) at 2% FCS (p <0.05). Conclusion-Among the present test solutions, SFM appears to be the most pro minent candidate for a new corneal organ culture medium and should be furth er tested and possibly refined to effectively substitute serum addition.