Purpose: To review and update the experience of our laboratory in culturing human corneal endothelial cells (HCEC) from young and older donors.
Methods: Corneas were obtained from National Disease Research Interchange, Philadelphia, PA. Data from the past 3 years were reviewed to develop criteria for selecting donor corneas to be used for endothelial cell culture. Immunocytochemical localization using mAb 9.3.E identified endothelial cells, and Ki67 staining demonstrated actively cycling cells. Cell counts demonstrated the effect of growth-promoting agents on proliferation of cells from young (<30 years old) and older (>50 years old) donors. Phase-contrast microscopy documented morphologic characteristics of cells in primary culture and the effect of growth factors on cell morphology.
Results: Exclusion criteria were developed to increase the chance of successful culture of HCEC. Isolation methods to remove Descemet membrane with attached endothelial cells avoided contamination with other corneal cell types. EDTA treatment combined with mechanical disruption facilitated isolation of cells. Culture medium containing FBS, EGF, NGF, and bovine pituitary extract stimulated maximal growth and facilitated normal monolayer formation. Age-related differences were detected in the density of confluent cells in primary culture and in the proliferative response to growth-promoting agents.
Conclusions: Untransformed HCEC can be successfully cultured from the corneas of both young and older donors by using care in the selection of donor material. Care must also be taken in the early phases of endothelial cell isolation to obtain maximal numbers of healthy cells for culture. There appear to be true age-related differences in overall proliferative capacity; however, the relative response to specific growth factors was similar in cells from young and older donors. Results of these studies provide guidelines for successful growth of untransformed HCEC for use in regenerative medicine.