Preliminary Study On Construction Of Corneal Posterior Lamellar With Small Intestinal Submucosa Compound Of Rabbit Corneal Endothelial Cells In Vitro

Objective:(1) To research the characteristics of rabbit corneal endothelial cells(CECs) which were cultured in vitro, and to lay a foundation for tissue engineering of corneal posterior lamellar. (2)To investigate the effects of EDTA on the proliferation of cultu-red rabbit cornea endothelial cell in vitro. (3) To investigate the biocompatibility of small intestinal submucosa (SIS) with rabbit CECs and to explore the possibility to construct tissue engineering corneal posterior lamellar with SIS as the scaffold and rabbit CECs as the seed cells.Methods:(1) Descemet's membrane was stripped from rabbit cornea under the dissection microscope, Descemet's membrane with endothelium was incubated in trypsin and EDTA solution at 37℃and then purified for rabbit CECs subculture in vitro. (2) The second passages of the rabbit CECs were treated with different concentrations of EDTA(0.5,2.5,5mmol·L-1) for 1h. The cell biologic activities in different time period and different doses of EDTA were detected with CCK-8. The flow cytometer was used to evaluate the cell cycle of negative control group and EDTA-treated groups. (3) After physical and chemical preparation, the SIS compounded with rabbit CECs for culture in vitro. Histological observation was done under phase contrast microscope and scanning electron microscope respectively.Result:(1) Descemet's membrane with endothelium through enzymic digestion was effective in the isolation of purified rabbit CECs. The culture cells quickly attached grew,proliferated and became confluence in 4-5 days. (2) EDTA can promote the proliferation of rabbit cornea endothelial cells from 24 through 96 hour at the conce-ntration of 0.5 mmol·L-1. (3) The physically and chemically treated SIS was pure with more hollows in it and undamaged collagen fibers. The rabbit CECs can grow,adhere to on the surface of SIS and into the hollows.Conclusion:(1) This research explores the characteristics of rabbit CECs which are cultured in vitro, and establishes a simple and efficient method for culture of rabbit CECs in vitro. (2) EDTA can promote the proliferation of rabbit cornea endothelial cells from 24 through 96 hour at the concentration of 0.5mmol·L"1. (3) SIS can be used as scaffold to construct tissue engineering corneal posterior lamellar as it has good biocompatibility with rabbit CECs.