| Purpose: To optimize and explore culture system of human foetal corneal endothelial cells (hFCECs), including the methods of primary culture, subcultivation and cryopreservation.
Methods: Primary hFCECs were cultured by sticking tissues piece method and passaged by trypsiniz /EDTA and cultured in DMEM/F-12 including 10% FBS and bovine corneal endothelium cells lysates (BCECL),which were determined using BCA protein assay. Cryopreservation of hFCECs was with 90% FBS and 10% DMSO. Cell proliferation was assayed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide(MTT).The immunocytochemistry and semi-quantification reverse transcriptive PCR (RT-PCR) were performed to confirm the origin of hFCECs with anti–NSE, nestin,Ki67,ZO-1,CK-3/12,vimentin and Na+-K+-ATP enzyme. Statistical analysis was finished with SPSS 11.5 software.
Results: The hFCECs were cultured and passaged successfully, cryopreservation and recovery were feasible. Cell proliferation assay manifested that 5% BCECL promoted significantly the proliferation of hFCECs(P<0.05). Immunofluorescence showed hFCECs positive staining of anti-NSE, anti-nestin in the cytoplasm, anti-Ki67 on the nucleus and anti-ZO-1 on the membrane, but with negative staining of anti-CK-3/12 and anti-vimentin. RT-PCR showed that the hFECs expressed high-level of Na+-K+-ATP enzyme and ZO-1, but hFSCs only showed low-level expression.
Conclusions: we confirmed the culture method of hFCECs including the primary, subcultivation and cryopreservation. It was confirmed that the BCECL distintly improve proliferation, maintain morphology and inhibit senescence of hFCECs in vitro.
Key words: foetus; endothelium,corneal; cell proliferation; cryopreservation |
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