人脐静脉内皮细胞分离及冷冻保存的技术

目的 研究分离、培养、扩增人脐静脉内皮细胞及进行冷冻保存技术，探讨建立脐带血管内皮细胞库的可能性。方法 新鲜脐带42条，脐静脉内灌注消化酶消化，获得内皮细胞：以含20％的胎牛血清M199液进行培养。采用Ⅷ因子免疫荧光染色及扫描电镜检查，鉴定所获内皮细胞及其纯度：内皮细胞加入含10％DMSO冷冻保存液，置于液氮进行保存，复苏后进行锥虫蓝试验，四氮唑盐(MTT)试验以及细胞凋亡的流式细胞仪检测。结果 血管内灌注消化液法可获取高纯度的内皮细胞，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。冻存内皮细胞的凋亡率较未冻存细胞高，但无统计学意义。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。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，可作为制备组织工程学血管的种子细胞来源。

Practice of Human Umbilical Endothelial Cell Isolation and Cryopreserving Technique

Purpose In order to provide suitable source cells in time to construct tissue engineered vascular grafts,the present study was designed to investigate the isolating and cryopreserving techniques of human umbilical vein endothelial cells in vitro. Methods Forty two human umbilical cords were used in this study.They were isolated by means of filling digestive enzyme solution into the lumen of umbilical veins.They were then cultured in M199 with 20% fetal bovine serum followed by immunofluorescence staining and scanning electron microscope examination.Then endothelial cells were suspended in cryopreserviong solution which contains 10% DMSO and 10% fetal bovine serum in M199 and were cryopreserved in liquid nitrogen.The proliferation of post-thawed cells and non-frozen cells were evaluated by measuring the metabloic activity of tetrazolium compound.The endothelial cell growth characteristics were determined by daily observation using phase contrast microscope.Post-thawed endothelial cells viability was determined by trypan blue staining test.Flow cytometry was used applied in this study to determin the apoptosis rate of post-thawed cells. Results Extremely high purified endothelial cells could be isolated by infusing digestive solutions to the lumen of human umbilical veins.Compared with non-frozen endothelial cells,postthawed endothelial cells showed 95% of vitality.No difference was found in growth curves of postthawed endothelial cells and non-frozen ones.The apoptosis rate of post-thawed endothelial cells was higher than that of non-frozen ones;however,there was no statistical difference. Conclusions Highly pure endothelial cells can be isolated by infusing digestive solutions to the lumen of human umbilical veins.Post-thawed endothelial cells are proved to have high vitality and growth potential in vitro.They may be an alterative choice of source cells for the construction of tissue engineered vascular graft.