The Experimental Study On The Tissue Engineering Corneal Epithelium Reconstruction By Using Human Amniotic Epithelial Cells

Objectiveâ‘ To establish a method of culturing and labelling human amniotic epithelial cells (HAECs) in vitro.â‘¡To explore the feasibility of HAECs differentiated into corneal epithelium cells as "seed cells".â‘¢To investigate the transdifferentiation of HAECs into corneal epithelium cells using atomic force microcopy (AFM) on morphology.â‘£To evaluate the effect of the HAECs-rabbit corneal stroma tissue engineering cornea on ocular reconstruction in animal models.Methodsâ‘ HAECs were isolated from serologically screened donor human placenta using enzyme digestion and cultivated in culture medium for primary culture and passage. The cultured HAECs were observed on morphology, identified by immunocytochemical methods and investigated the effect of DAPI fluorescence labelling for 2 weeks.â‘¡HAECs were co-cultured with rabbit corneal stroma cells for 2 weeks, identified by immunofluorescence staining for CK3+12. HAECs were seeds onto rabbit or human corneal stroma, and incubated in culture medium for 2 weeks using air-liquid interface culture methods, identified by immunohistostaining for CK3+12.â‘¢The characteristic of morphology and ultra-microstucture of HAECs before and after co-culture were investigated using AFM, compared to human corneal epithelial cells (HCECs).â‘£HAECs-rabbit corneal stroma tissue engineering cornea transplantation were operated for ocular surface restoration in different animal models with limbal stem cell deficiency. The effect was assesses by ocular surface observation, histopathology and cell biology.Resultsâ‘ HAECs immediately harvested from the fresh placenta could be cultured in culture media. HAECs became a cobble-stone-like shape with a low N/C ratio and formed a sheet with obviously discernible intercellular junctions. The cultured polygon HAECs displayed positive immunoreactivity to keratin and negative to CK3+12. DAPI fluorescence labelling ratio of the cultured cell was 100%, the ratio had no change but fluorescence intensity decreased after 2 weeks.â‘¡The co-cultured HAECs displayed positive immunoreactivity to CK3+12. HAECs on the corneal stroma have 4-5 layers, same as normal corneal epithelium, displayed positive immunoreactivity to CK3+12.â‘¢The ATM images revealed that the nucleus center shape of the co-cultured HAEC changed from valley appearance to HCEC-like mountain peak appearance.â‘£HAECs-rabbit corneal stroma tissue engineering cornea grafts were transparency during observation period, with corneal epithelium-like stratified epithelium and CK3+12 positive expression.Conclusionsâ‘ HAECs can be successfully primary cultured and passaged in vitro. DAPI is an effective fluorescence marker for HAECs.â‘¡HAECs could be transformed into HCECs induced by rabbit corneal stroma cells or rabbit/human corneal stroma.â‘¢The visualized data obtained from AFM for HAECs can prove further the co-cultured HAECs with corneal stroma cells transformed into HCECs successfully on morphology.â‘£HAECs-rabbit corneal stroma tissue engineering cornea grafts can reconstruct corneal epithelium.