Primary culture of corneal endothelial cells in vitro and biological identification / 中华实验眼科杂志

ABSTRACT

Background Corneal blindness is one of the major blinding eye diseases in China.With the development and progress of tissue engineering technology,tissue-engineered corneas offers a new approach to the treatment of corneal diseases.To select and cultivate ideal seed cells is a foundation of construction of tissueengineered corneas.Objective This study was to evaluate the efficiency of stripe off the Descemet membrane with endothelium plus enzymic digestion in the isolation of corneal endothelial cells and analyze the bionomics of rabbit corneal endothelial cells (CECs) in vitro.Methods Descemet membrane was stripped from fresh cornea of New Zealand rabbit under the dissection microscope.Descemet membrane with endothelium was incubated in trypsin and EDTA solution at 37 ℃ and then purified for CECs subculture in vitro.The morphology of the cultured cells was observed under the inverted microscope and marked by CM-Dil dye solution.Then the shape of the cells was observed by hematoxylin and eosin staining and the cells were identified for the expression of neuron specific enolase (NSE) using immunochemistry.The viability of the cells were evaluated by trypan blue staining.The surface structure of the cells were examined under the scanning electron microscope.Intercellular zonula occludens-1 (ZO-1) was identified by immunofluorecsence staining.Results A large number of purified CECs were obtained from Descemet membrane with endothelium through enzymic digestion.Cultured cells grew well and formed monolayer 5-7 days later with the cobblestone stone-like arrangement.The survival rate of the cells was 95％.CECs presented with the red annular fluorescence for CM-Dil with the labeling rate ＞90％.NSE was positively expressed in the cytoplasm.Polygon CECs were seen by hematoxylin and eosin staining and showed the brown staining.Abundant microvilli on the cellular surface and interconnected foot process were seen under the scanning electron microscope.ZO-1 showed the green fluorescence.Conclusions The method of striping off the corneal Descemet membrane with endothelium plus enzymic digestion can obtain abundant CECs.Cultured cells have good biological properties.This study may offer a feasible application in the engineering of corneal transplant membrane.