The Research Of Cornea Endothelium's Culture In Vitro

The morphologic and functional integrality is the primary index of the clinic utility and preservation quality evaluation of the cornea endothelium. The observation of the medication's effect on the cornea endothelium's vitality relies on the in cell culture in vitro, which is also the vital premise of successful experiment in vivo. Cornea endothelium dysfunction is most common in clinic. So, cornea endothelium's successful culture in vitro is very significant to cornea's pathology, pharmacology and transplant. Besides, it is also the central point which constitute histological engineering man-made cornea.This study is concentrated on the screening and optimization of culture method and conditions to confirm successful cultivation of cornea.Membrane-uncovering method: cut the cornea endothelium cell layer along the edge of the cornea and attach the layer to the bottom of the 48-well plate. Hexagon single layer cells are achieved. This method is Trypsin free and prevents the endothelium from damage.Enzyme method: When the cornea tissues are abundant, enzyme method is adopted. The content and time of the enzyme are important. The cells inoculated are easy to attach to the plate using this method.Tissue culturing method: When the cornea tissues are not plenty, tissue culturing method is adopted. This method is convenient and thecells grow quickly. It avoids the use of Trypsin and the cell growths normally.The research shows that the optimal medium is DMEM/F12(1:1)Subculture: Taken into account of the essential effect of the enzyme such as content, amount and time, we do some research on the appropriate method of subculture.In order to overcome the lagged growth of the cells and the difficulty of subculture, marine bioactive extracts are firstly used on the endothelium. The results indicate that chito-oligosaccharide and NAG could expedite the growth of the cells significantly(p<0.0005).They have the similar effect but low cost when compared with EGF. So they have a promising future in the cornea endothelium's culture in vitro.Cornea endothelium are firmly attached on the 631s polysaccharide biomembrane and growth well. According to the result, we conclude that cornea endothelium have a good bio-compatibility with the 631s polysaccharide. The co-application of the chito-oligosaccharide, NAG and the EGF during the cultivation can expedite the growth of the cornea endothelium. It has significant shortened the formation time of the confluence of man-made bioactive cornea.The cryopreservation experiment indicate that 15% glycerol is the optimal cryoprotective liquid. The living rate is comparatively high and the morphology of the cell is normal. In the preservation, it is better whenusing the cold equilibration time of 15cm/hr and the living rate is comparatively high, the morphology of the cell is normal. The resuscitation experiment indicates that: 60and 40 water bath can-both get high survive rate except the damage on the cell when using the former condition.