人脐带非酶解法培养MSCs的方法建立

Culture of mesenchymal stem cells derived from human umbilical cord without enzymatic treatment

目的建立从人脐带Wharton's jelly组织中直接培养间充质干细胞(MSCs)的方法。方法将人脐带切成5 cm左右的片段,纵向剖开,剔除血管,把富含Wharton's jelly组织的脐带面置于10 cm2塑料培养皿的培养面上,加入含20%FBS的LG-DMEM培养基连续培养10 d;移去脐带组织后,培养皿中的细胞继续培养至80%-90%融合。传代和扩增3代(次)后,以倒置光学显微镜观察细胞形态,流式细胞仪检测细胞免疫表型及细胞周期,用含有成骨细胞诱导剂、脂肪细胞诱导剂对传代培养至第3代的细胞分别定向诱导分化为成骨细胞、脂肪细胞,并对诱导后细胞用碱性磷酸酶检测试剂、Von-Kossa染色检测试剂和茜苏红染色试剂作细胞生物学检测。结果脐带组织在贴壁培养5 d,组织周围可见有少量细胞贴壁生长,主要呈"成纤维样",形状不规则,移去脐带组织后继续培养至14-15 d时,细胞达到80%-90%汇合;细胞表面表达CD73、CD105、CD90、CD44、CD29、CD71及CD13,不表达CD34、CD14、CD133、CD45、HLA-DR;培养至第4代时约72.724%的细胞处在G1期,S期细胞占18.069%,第6代时,G1期细胞约为83.875%、S期细胞仅为9.606%左右。细胞经成骨细胞诱导分化后,碱性磷酸酶染色显示呈强阳性,茜苏红染色和Von-Kossa染色显示细胞有钙盐沉积并形成钙结节;细胞经成脂细胞诱导分化后,油红O染色呈红色,显示胞浆内有大量甘油三酯的聚集。结论采用非酶解法从人脐带Wharton's jelly中分离及培养扩增的细胞具备MSCs的基本特性,具有分化为成骨细胞和脂肪细胞的能力。

Objective To establish a method to culture mesenchymal stem cells （MSCs） derived from Wharton＇s jelly in human umbilical cord without enzymatic treatment and characterize the related biological properties. Methods Human umbilical cord sample segments （5cm each） were cut open longitudinally to expose the Wharton＇s jelly while blood vessels were removed, The Wharton＇s jelly surface of the umbilical cord were placed against a plastic dish surface for 10d in LG- DMEM medium with 20% FBS. After removing the cord segment, the culture was carried on using in petri dishes until the cells reached 80-90% subconfluence. These cells were passaged and proliferated to the third generation for cell morphology observation with an inverted microscope. Cell phenotype and cycle were examined by flow cytometry. The third generation cells were then induced to differentiate into osteoblasts or adipocytes with conditioned culture medium and evaluated the in- duced cells＇ bin-characteristics with ALP detection reagent, Von-Kossa Staining detection reagent and Xisu red staining reagent. Results 5d into the adherent culture of the umbilical cord tissue, representative samples of the typical ＂ fibroblast- like＂ cells can be observed with irregularly shaped cell surface. After removing the cord segment, the culture was carried on to d14-15 till the cells reached 80%-90% subconfluence. Cells surface expressions of CD73, CD105, CD90, CD44, CD29 and CD13 were detected with no expressions of CD34,CD14, CD133, CD45, HLA-DR; The cell cycle test showed that the cells of P4 were about 72. 724% in the G1 phase, 18. 069% in the S phase; the cells of P6 were about 83. 875% in the G1 phase and 9. 606% ceils were in the S phase. Alkaline phosphatase staining presented strong positive results, the Xisu red stained and Von-Kossa stained cells showed calcium deposits and formation of calcium nodules. The Oil red O staining was red, which showed large triglyceride accumulation within the cytoplasm.Conclusion The cells isolation from Wharton＇s jelly in human umbilical cord without enzymatic treatment present the basic characteristics of mesenchymal stem cells and the ability to differentiate into osteoblasts and adipocytes.