A Study On Reconstruction Of Corneal Epithelium By Using Cultured Human Amniotic Epithelial Cells In Vitro

Objectiveâ‘ To establish a method of culturing human amniotic epithelial cells (hAECs) invitro, and to observe its biologic feature.;â‘¡To investigate the effects of EGF, bFGF onhAECs;â‘¢To investigate the possibility of corneal epithelium reconstruction by usinghAECs.Methodsâ‘ A piece of amniotic membrane taken from an uncomplicated elective caesareansection was digested by collagen, trypsin and EDTA respectively. The amniotic epithelial cellswere primary cultured and passaged in DMEM (dulbecco's modified eagle's medium) containing10% fetal bovine serum. The amniotic epithelial cells were seeded in DMEM containing 10%fetal bovine serum to primary culture and passage. The optimal culture condition were studiedand the feature of the amniotic epithelial cells were observed by inverted microscope in vitro. Thecultured cells were investigated morphologically by Hematoxylin-eosin staining, scanningelectron microscope(SEM), transmission electron microscope (TEM) and identified bycytokeratin immunocytochemistry.â‘¡The hAECs were seeded in 96-well microplates, Theoptical density(OD) of hAECs were detected by the methods of MTT after 48 hours on the effectof various concentrations of EGF and bFGF.â‘¢The cultured hAECs of the 2 nd or 3 rdgeneration were continued to culture on fresh corneal stroma. 1 or 2 days later, the hAECsformed a monolayer, then it were carried out air-lifting cultivation in insert culture dish. Thecultured cells on corneal stroma were investigated morphologically by Hematoxylin-eosinstaining, its ultramicrostructures were studied by scanning electron microscope(SEM) andtransmission electron microscopy(TEM), and cytokeratin 12 was detected byimmunohistochemistry.Resultsâ‘ HAECs can be successfully primary cultured and passaged in vitro, and those cellscan live and reproduce for a certain period. It can be successfully cultured and passage in vitro,and can be passaged successively 6-8 times. Most of the cultured cells are polygon and are oftypical slabstone-like appearance, many microvilli can be observed on cell faces by SEM andmany desmosomes were observes by TEM. Cytokeratin monoclonal antibody staining ispositive.â‘¡EGF, bFGF can promote the growth of hAECs in proper concentration.â‘¢HAECs can be successfully reproduced in corneal stroma in vitro, monolayer was formedafter being cultured for 1 or 2 days, stratifications with 4 to 5 layers epithelial cells were developed. The composed tissue was very similar to normal corneal epithelium.Conclusionsâ‘ HAECs can be successfully primary cultured and passaged in vitro, and thosecells can live and reproduce for a certain period;â‘¡EGF, bFGF can stimulate the growth ofhAECs;â‘¢HAECs could be expected to reconstruct the corneal epithelium by tissueengineering technology.