effect of amniotic fluid on the invitro culture of human corneal endothelial cells

purpose: to evaluate the effect of human amniotic fluid (haf) on the growth of human corneal endothelial cells (hcecs) and to establish an in vitro method for the expansion of hcecs. methods: hcecs were cultured in dmem-f12 supplemented with 20% fetal bovine serum (fbs). confluent monolayer cultures were trypsinized and passaged using 20% fbs- or 10%, 20%, or 30% haf-containing media. cell proliferation and cell death elisa assays were performed to assess the effect of haf on cell growth and viability. the identity of cells cultured with 20% haf were studied using immunocytochemistry (icc) and real-time rt-pcr techniques for markers presenting specific characteristics of hcecs including ki-67, vimentin, na/k-atpase and zo-1. results: primary cultures of hcecs were successfully established under haf-containing media which led to rapid cell proliferation according to cell proliferation and death elisa assays. real-time pcr and icc revealed that expression of na/k-atpase and zo-1 in 20% haf cell cultures was more compared to controls (20% fbs). conclusion: haf containing medium exhibited an invaluable stimulatory effect on hcec growth. it can be regarded as an enriched supplement in hcecs regeneration studies.