Long-term Cultivation of Human Donor Corneas under Serum-free Conditions

Abstract

Abstract: : Purpose: The most frequent applied storage method for corneas in Europe is long-term cultivation for up to 4 weeks in medium MEM supplemented with fetal calf serum (FCS). Culture period is used for serological testing of the donor, proof of sterility and planing of the operation. The quality of the cornea in particular the endothelial cell density can be checked during culture as well as directly before keratoplasty. Previous studies with ten different media indicated that the commercial medium Endothelial-SFM (SFM) seems to be a more suitable medium for cornea cultivation than MEM. Furthermore cultivation of isolated human corneal endothelial cells in SFM was possible even under serum-free conditions. The aim of this study was to compare directly the media SFM and MEM. Furthermore the possibility to culture human donor corneas in SFM without serum supplementation was tested. Methods: Six pairs of corneas exhibiting an endothelial cell density lower than 2000 cells/mm2 after 8 to 14 days of storage in MEM supplemented with 2% FCS were further cultivated in SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In the second series of experiments, the endothelial cell density of 7 pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS versus serum-free SFM. All cultures were maintained at 37ºC. Results: Although endothelial cells densities of the paired corneas were comparable at the beginning of the observation period, culture in MEM led to a significant higher decrease in cell density than culture in SFM. After nine weeks all corneas in MEM had totally lost their endothelium whereas cell density of corneas in SFM had decrease for only about 20% and the corneas could be further cultivated for more than 20 weeks without total loss of endotheium. No difference in endothelial cell density could be observed comparing medium SFM with or without serum supplementation. Conclusion: Medium SFM seems to be suitable for long term cultivation of human donor corneas even under serum-free condition. Dispense of serum reduces possible variation in culture condition and minimises the risk of contamination with infectious agents. Furthermore culture in SFM is superior to conventional culture condition because the decrease in endothelial cell density is reduced and the culture period can be prolonged.

Keywords: 373 cornea: storage • 371 cornea: endothelium • 607 transplantation