人胎盘绒毛膜间充质干细胞分离培养:组织块法的优化  

Isolation and cultivation of human placental chorionic-derived mesenchymal stem cells:optimization of the tissue explants method

作　　者：金宇林[1] 吴洁莹[1] 陆琰[1] 陈劲松[1] 李发涛[1] 唐婕[1] 刘东[1] 梁绮华[1] 李焱[1] 汤雪薇[1] 谢闺娥[1] 吴韶清[1] 

机构地区：[1]广州医科大学附属广州市妇女儿童医疗中心,广东省广州市510623

出　　处：《中国组织工程研究》2017年第21期3388-3393,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81102248);广东省医学科研基金(B2010274);广东省科技计划项目(2014A020 212027);广州市妇女儿童医疗中心儿科研究所基金(YIP-2016-014)~~

摘　　要：背景:关于人胎盘绒毛膜间充质干细胞分离培养方法的研究很多,但是如何简便、快捷获得大量原代培养间充质干细胞的问题尚未解决。目的:探讨优化人胎盘绒毛膜间充质干细胞体外分离培养组织块法的最佳方法。方法:无菌条件下,取正常足月剖宫产胎盘绒毛膜,用匀浆器处理组织块,采用组织块法贴壁分离培养人胎盘绒毛膜间充质干细胞,经初次培养的组织块进行再次接种培养。结果与结论:用电动匀浆器处理人胎盘绒毛膜省时省力,组织分散度和去除红细胞效果好。初次培养和再次培养获得细胞的平均时间分别为(17.73±1.14)d和(10.03±1.30)d。每个Φ100 mm培养皿获得的原代细胞分别为(6.97±0.98)×10~5个和(13.82±1.44)×10~5个。获得的贴壁细胞呈典型的成纤维细胞形态,呈平行或漩涡状生长,高表达CD90、CD73、CD105,而CD45、CD34、CD14、CD19、HLA-DR为阴性。细胞可向成骨、成脂方向诱导分化。结果表明优化的方法能够简便、快捷地处理人胎盘绒毛膜组织,获得大量的原代间充质干细胞。BACKGROUND： There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells（hpc MSCs）, but how to simply and efficiently harvest a large amount of primary MSCs has not been resolved. OBJECTIVE： To optimize the tissue explants method of isolating and culturing hpc MSCs in vitro. METHODS： Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpc MSCs were prepared by tissue explants method. The fluid and tissue of the primary culture flask and douching normal saline of the initial culture were centrifuged and prepared for secondary culture. RESULTS AND CONCLUSION： It saved time and effort to treat human placental chorionic villi with electric homogenizer, with good effects on tissue dispersion and removal of red blood cells. The average time of cell acquisition in initial culture and secondary culture was（17.73±1.14） and（10.03±1.30） days, respectively. The yields of primary cultured cells in initial culture and secondary culture were（6.97±0.98）×10~5 and（13.82±1.44）×10~5 per Φ100 mm culture dish, respectively. The adherent cells showed fibroblast-cell-like shape, which were in parallel or circinate arrangement. Highly expressed CD73, CD105 and CD90 could be detected in the third generation of hpc MSCs, but CD34, CD45, CD14, CD19 and HLA-DR were negative. Following induction, alizarin red staining and oil red O staining produced a strong reaction in cells. In a word, the optimized method is a simple and efficient method for obtaining a large amount of primary hpc MSCs.

关 键 词：胎盘 绒毛膜 间质干细胞 细胞培养技术 组织工程 干细胞 培养 间充质干细胞 细胞培养 组织块法 优化 匀浆器 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]