| Objective:Corneal endothelial cells were isolated and cultured in vitro.Primar y and subcultured cells were cultured,identified,as.well as preserved,and this study intended to seek out the most available method for the isolation and cu lture of corneal endothelial cells in vitro.To analyze and compare the differenc es and characteristics of corneal endothelial cells of rhesus monkes and tree sh rews,and animal model with high similarity to human corneal endothelial cells were explored,which could be optimized the engineering endothelial transplan tation.Methods:Eight rhesus and eight tree shrews were sacrificed and 16 eyes were randomLy divided into 2 groups in each group:experimental group(8 eyes)a nd control group(8 eyes).In experiment group:the cells were isolated and cul tivated by a modified "peel-and-digest" procedure.In the rhesus monkeys contr ol group:the CECs ware isolated by tissue-piece inoculation.In the tree shrew s control group:it was cultivated by the stripping film method.The morpholog y and function of the cells were observed under the inverted aberration micros cope,and the feasibility of separating the corneal endothelial seed cells by diff erent methods were analyzed.The primary and passaged cells were cultivated,in order to observe and record the cell growth status,passage time,proliferatio n and cell viability.The cultured cells were identified by neuron-specific enolas e(NSE)immunostaining.In addition,the traditional cryopreservation and res uscitation method for long time preservation of corneal endothelial cells was st udied.At the same time,the study could be compare the differences of the gr owth characteristics and proliferative abilities of corneal endothelial cells from t wo different species.Results:In the rhesus monkey experimental group,primary corneal endothelial cells obtained from modified "peel-and-digest" procedure were grown slowly.T he cells were connected closely,and the morphology of cells was polygonal,w hich could form an intact monolayer and consisted of paving-stone shape witho ut stroma cells in 21 days.The CECs of the tree shrews experimental group gr ew faster than the rhesus monkeys experimental group.The shapes of cells we re polygonal or short spindle,and it reached the intact monodermic state on d ay 11.The tissues of the rhesus monkeys control group which isolated by tiss ue-piece inoculation were disintegrated at the 5th day,and no cells were obser ved.The tissues of the rhesus monkeys control group which isolated by tissue-piece inoculation were disintegrated at the 5th day,and no cells were observed.The films of the tree shrews control group which detached by the stripping fi lm method were disintegrated 5 days later.There were no cells removed.The seed cells were harvested,and then Subculture MCECs proliferated fast,which could be passage in 6～7 days averagely in the rhesus monkey group.The morphology of cells was maintained well.The tree shrews group could be su bculture in 3 to 4 days.With the increase of the passages,the cells’ volume a nd the pleomorphism were increased obviously.The CECs stopped proliferate a t the 5th passage.Positive NSE staining showed that two different cells express ed NSE.However,the consequent of recovery from cryopreservation wasn’t sat isfactory.The cells in the rhesus monkeys group showed cell were dissolved within 4 days after recovery.The tree shrews group cells could proliferate well,but morphological diversity and cell volume increased.Conclusion:The modified "peel-and-digest" method could harvest numbers of available CECs efficiently in vitro.The CECs of the rhesus monkey and tree s hrew could be divide,proliferate and passage in vitro,which lay a basis on fu rther study of the construction of tissue-engineered corneal endothelium.The rh esus monkey corneal endothelial cells are closer to human corneal endothelial c ells,including morphology maintenance,cell proliferation and function of expre ssion,when compared with tree shrews corneal endothelial cells. |
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