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Title:: A new method to isolation and cultivation of human placental ?chorionic-derived mesenchymal stem cells

作者:: 唐书生1; 2; 孙逊沙3; 吴洁莹1; 2; 陆琰1; 2; 陈劲松1; 2; 李发涛1; 2; 唐婕1; 2; 吴韶清1; 2Δ; 1.广州医科大学附属广州市妇女儿童医疗中心，广州 510623；2.中山大学附属广州市妇女儿童医疗中心，广州 510623；3. 中山大学附属第一医院，广州 510080

Author(s):: TANG Shusheng1; 2; SUN Xunsha3; WU Jieying1; 2; LU Yan1; 2; CHEN Jinsong1; 2; LI Fatao1; 2; TANG Jie1; 2; WU Shaoqing1; 2; 1.Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510623, China; 2.Guangzhou Women and Children’s Medical Center, Sun Yat-sen University, Guangzhou 510623; 3.The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080

Keywords:: Mesechymal stem cells; Chorion; Cell isolation; Cell cultivation; Method

摘要:: 建立一种简便、快捷的人胎盘绒毛膜间充质干细胞(human placental chorionic-derived mesenchymal stem cells, hpcMSCs)分离培养方法。用手持电动匀浆器处理人绒毛膜，经酶消化后接种培养，用倒置显微镜观察细胞形态，CCK-8测定细胞生长曲线，流式细胞术检测细胞表面标记，成脂、成骨诱导分化试剂盒鉴定细胞的分化潜能。获得的人胎盘绒毛膜间充质干细胞为成纤维细胞样细胞，呈平行排列或漩涡状生长，高表达CD73、CD105、CD90，而CD34、CD45、CD14、CD19、HLA-DR呈阴性，生长曲线为“S”型，经成骨或成脂诱导后，茜素红染色或油红O染色呈阳性。符合国际细胞治疗学会认可的间充质干细胞标准。建立了人胎盘绒毛膜间充质干细胞的分离培养新方法，结合了组织块法和酶消化法的优点，为绒毛膜间充质干细胞的应用提供基础。

Abstract:: To establish an effective and easy method of isolating and cultivating human placental chorionic-derived mesenchymal stem cells (hpcMSCs). Human placental chorion were isolated by hand held electric homogenizer and enzyme digestion. The morphology of hpcMSCs was observed under the inverted microscope. The cell growth curve was measured by CCK-8 method. The third generation of hpcMSCs were detected with flow cytometer, and incubated in osteogenesis and adipogenesis. Osteogenous potential was assessed by alizarin red staining. Adipogenic potential was evaluated by Oil red O staining. The adherent cells showed fibroblast-cell-like shape. CD73, CD105 and CD90 membrane antigens could be detected in the third generation of hpcMSCs, but CD34, CD45, CD14, CD19 and HLA-DR membrane antigens were not detected. Following induction, alizarin red staining and Oil red O staining produced a strong reaction in cells.The whole isolate and cultivation is an effective and easy method to obtain a largely amount of primary hpcMSCs. It may represent a plentify stem cell source for the regenerative medicine.