In vitro culture of human fetal corneal endothelial cells

Abstract

To explore and optimize a proper culture system for human fetal corneal endothelial cells (hFCECs), including the methods of primary culture, passage and cryopreservation. Fresh fetal corneas were explanted to propagate primary corneal endothelial cells. The cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) in the absence or presence of the extracts from bovine corneal endothelium cells (bCECs), and the cells were identified with immunocytochemical staining. The passage and cryopreservation of hFCECs were optimized according to previous reports on adult corneal endothelial cells. Using the explant culture method, hFCECs migrated successfully within 3 days and assumed polygonal-shaped corneal endothelial morphology. The optimizing methods were 0.125% trypsin + 0.02% EDTA for passage and 10% DMSO + 90% FBS for cryopreservation. Recovered hFCECs from cryopreservation remained typical morphology and immunological markers of corneal endothelial cells, including positive staining of NSE, Nestin, Ki67 and ZO-1, and negative staining of CK3/12, which demonstrated that they retained the characterizations of corneal endothelial cells and proliferative capacity. Moreover, the extracts from bCECs can promote the proliferative capacity of hFCECs significantly, while maintaining their typical endothelial morphology. The culture conditions of human fetal corneal endothelial cells were firstly optimized, including the primary culture, passage and cryopreservation. Meanwhile, we confirmed that the extracts from bovine corneal endothelium promoted the proliferative capacity while maintaining the morphology of hFCECs in vitro.