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

Abstract

Aim: To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" 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 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial 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) changes 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 and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05).

Conclusion: Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.

Figures

Figure 1

Autoradiograph of human donor endothelial cells following 48 hours of 3H-uridine incorporation during corneal organ culture. (A) Dense accumulations of silver grains cover three radiolabelled nuclei undergoing RNA synthesis (haematoxylin and eosin counterstain). Profiles of the same nuclei (B) and the area of the autoradiographic silver grains covering these profiles (C) as assessed by digital image analysis.