组织工程学血管构建中脐带内皮细胞分离及冻存技术的初步研究

目的为制备组织工程学血管随时提供种子细胞,对人脐静脉内皮细胞分离、体外培养、传代及鉴定方法进行研究,并研究其冻存技术,探讨建立脐带血管内皮细胞库的可能性.方法新鲜脐带49务,脐静脉内灌注消化酶消化,获得内皮细胞,进行体外培养,相差显微镜下观察酶消化结果及所获内皮细胞贴壁生长规律;采用VⅢ因子免疫荧光染色,鉴定所获内皮细胞及其纯度;加入冻存液,置于液氮进行保存,复苏后,分别进行台酚蓝拒染试验及MTT还原试验,绘制细胞生长曲线.结果血管内灌注消化液法可获得取高纯度的内皮细胞,胰酶和胶原酶混合灌注法消化结果优于单纯胰酶及胰酶和EDTA组,各组消化时间均以10～15min为佳;混合酶消化组细胞获得率及贴壁结果均较其他二组为优.与未冻存细胞相比,复苏的细胞活力保持在95%以上.复苏后的内皮细胞的生长曲线与未冻存细胞的生长曲线无差异.结论脐静脉灌注酶消化法可获取足够数量及纯度的内皮细胞,混合酶消化法消化效果最佳,经体外培养扩增后,可提供足够数量及纯度的内皮细胞.冻存的内皮细胞复苏后仍保持较高的活力及体外增殖能力,能及时提供制备组织工程学血管的种子细胞来源.

HUMAN UMBILICAL ENDOTHELIAL CELL CRYOPRESERVING TECHNIQUE AND CELL CULTURE DURING CONSTRUCTION OF TISSUE ENGINEERED VASCULAR GRAFT

Objective To provide suitable source cells in time to construct tissue engineered vascular grafts, and to investigate the isolation, identification, culture and cryopreservation techniques of human umbilical vein endothelial cells in vitro. Digestive effects of three different digestive solutions were also investigated in this study. Methods 49 human newborn umbilical cords were used in this study. Endothelial cells were isolated by means of filling digestive enzyme solution into the lumen of umbilical veins. Three kinds of digestive solution(trypsin only; trypsin and collagenase, trypsin and ethylenedi-amine tetra acetic acid)were applied in this study. The digestion results and endothelial cell's growth were monitered by phase contrast microscope. The identity and purity of endothelial cells were proved by von Willebrand factor immunofluorescence staining. Endothelial cells were suspended and cryopre-served in liquid nitrogen. One part of postthawed endothelial cells and non - frozen cells were used to MTT(3 - (4, 5 - dimethylthiazol - 2 - yl) - 2, 5 - diphenylt tetrazolium bromide) test, Relative spreading was studied by daily observation and photography by phase contrast microscope. Postthawed endothelial cells vitability were determined by tyophenol blue test. Results Extremely highly purified endothelial cells could be isolated by infusing digestive solutions to the lumen of human umbilical veins. Among the three digestive solutions, digestive effect of the mixture of trypsin and collagenase was proved to be better than that of the mixture of trypsin and ethylenediamine tetra acetic acid(EDTA). It was proved that the best digestive time is 10 to 15 minutes in all the three groups. Human umbilical endothelial cells acquiring rate and attaching efficiency of in the the trypsin and collagenase group was higher than that of the other two groups. Compared with non - frozen endothelial cells, postthawed endothelial cells showed more than 95% of vitality. Growth curves were found not significantly different between postthawed endothelial cells and non- frozen ones. Conclusion Enough highly pure endothelial cells could be isolated by infusion digestive solutions to the lumen of human umbilical veins. The best digestive results were the trypsin and collagenase group. The endothelial cells were cultured and amplified. Postthawed endothelial cells were proved to have high vitality and growth potential in vitro. Amplified postthawed endothelial cells could be qualified for the construction of tissue engineered vessels and provided an alterative source cell choice.