Xin Yi, Li Na, Huang Yimin, Cui Wei, Liu Sa, Xu Xiufang, Zhang Zhaoguang
Department of Molecular Biology, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Oct;29(10):1087-93.
To establish a reliable method of isolation, culture and characterization of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and study its multiple differentiation potency.
HUCMSCs were isolated and cultured using Trypsin-type II collagen and hyaluronidase digestion method and tissue explant culture method, respectively. The cell growth of hUCMSCs was observed under an inverted microscope. Cell viability rate of the different passages was evaluated by trypan blue staining. The proliferation profile of hUCMSCs was analyzed by growth curve and MTT assay. Flow cytometry was used to study the cell cycle and immunophenotypage change. The differentiation potency of hUCMSCs towards the osteoblasts, adipocytes was assayed using the differentiation kits. The differentiation towards the cardiomyocytes and endothelial cells was tested by immunofluoresence staining with the specific markers.
After 1-day culture of the enzyme digested cells, under the inverted microscope, the adherent cells were round, and 4 days later, they grew quickly and presented fusiform. Seven days later, the cells proliferated from the center to the peripheral and fused by 80% on day 10. With the tissue explant culture method, the cells started to proliferate gradually from the periphery of the tissue and grew quickly and arrayed closely in monolayer after 10 days. The cell viability in both isolation methods were more than 96% as tested by trypan blue staining. The growth curve of the third passage presented an "S" shape. MTT assay showed that the optimal cell proliferation occured on day 3 to 5. The ratios of G0/G1 phase and S+G2/M phase was 88.78% and 10.21% respectively by enzyme digestion, and 84.82% and 13.87% respectively by explant culture method. There was no significant difference in cell cycle. The positive rates of CD90, CD105, CD73 were more than 99% and the expressions of CD45, CD34, CD14, CD11b, CD79a, CD19, HLA-DR were lower than 1%. The hUCMSCs isolated by the two methods could efficiently differentiate towards the osteoblasts, lipocytes, cardiac myocytes and endothelial cells, and the positive rates were all above 90%.
The hUCMSCs can be effectively isolated by both enzyme digestion and explant culture methods. The enzyme isolation method presents a better method regarding the cell number obtained. This study showed the enzyme isolation method may be an optimal method to isolate the hUCMSCs for the cellular therapy and stem cell bioengineering.
建立可靠的人脐带间充质干细胞（hUCMSCs）分离、培养及鉴定方法，并研究其多向分化潜能。
分别采用Ⅱ型胶原酶和透明质酸酶消化法及组织块培养法分离培养hUCMSCs。在倒置显微镜下观察hUCMSCs的细胞生长情况。通过台盼蓝染色评估不同传代细胞的活力。采用生长曲线和MTT法分析hUCMSCs的增殖情况。运用流式细胞术研究细胞周期和免疫表型变化。使用分化试剂盒检测hUCMSCs向成骨细胞、脂肪细胞的分化能力。通过特异性标志物免疫荧光染色检测向心肌细胞和内皮细胞的分化情况。
酶消化法培养1天后，倒置显微镜下可见贴壁细胞呈圆形，4天后细胞快速生长并呈梭形。7天后细胞从中心向周边增殖，10天时融合率达80%。采用组织块培养法，细胞从组织周边开始逐渐增殖，10天后快速生长并紧密排列成单层。台盼蓝染色检测两种分离方法的细胞活力均超过96%。第三代细胞生长曲线呈“S”形。MTT法显示细胞在第3至5天增殖最佳。酶消化法G0/G1期和S+G2/M期比例分别为88.78%和10.21%，组织块培养法分别为84.82%和13.87%。细胞周期无显著差异。CD90、CD105、CD73阳性率均超过99%，CD45、CD34、CD14、CD11b、CD79a、CD19、HLA-DR表达低于1%。两种方法分离的hUCMSCs均可高效分化为成骨细胞、脂肪细胞、心肌细胞和内皮细胞，阳性率均高于90%。
酶消化法和组织块培养法均可有效分离hUCMSCs。酶分离法在获得细胞数量方面表现更佳。本研究表明酶分离法可能是用于细胞治疗和干细胞生物工程的hUCMSCs分离的最佳方法。
