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

doi:10.3969/j.issn.2095-4344.2017.21.018

Abstract

Abstract:

BACKGROUND: There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells (hpcMSCs), 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 hpcMSCs in vitro.

METHODS: Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpcMSCs 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)×10⁵ and (13.82±1.44)×10⁵ 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 hpcMSCs, 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 hpcMSCs.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Placenta, Chorion, Mesenchymal Stem Cells, Cell Culture Techniques, Tissue Engineering

CLC Number:

R394.2

Jin Yu-lin, Wu Jie-ying, Lu Yan, Chen Jin-song, Li Fa-tao, Tang Jie, Liu Dong, Liang Qi-hua, Li Yan, Tang Xue-wei, Xie Gui-e, Wu Shao-qing. Isolation and cultivation of human placental chorionic-derived mesenchymal stem cells: optimization of the tissue explants method[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3388-3393.

Figures/Tables 1

2.1 原代人胎盘绒毛膜间充质干细胞细胞形态、获得时间和细胞得率镜下观察人胎盘绒毛膜间充质干细胞贴壁生长。初次培养的细胞，1周后，就能够看到极少数成纤维样细胞从组织块上爬出来，在第10天能够看到细胞呈旋涡状生长(图1A)，形态均一，获得细胞的平均时间为(17.73± 1.14) d，每个Φ100 mm培养皿获得原代细胞为(6.97± 0.98)×105个。再次接种培养的细胞，在第10天能够看到组织块上爬出的细胞较多(图1B)，获得细胞的平均时间为(10.03±1.30) d，每个培养皿获得原代细胞为(13.82± 1.44)×105个。结果表明，2种接种情况下，接种培养的细胞形态无明显差异，获得时间上，再次接种培养的细胞收获时间快，差异有显著性意义(P=0.000 < 0.05)；获得细胞数量上，再次接种培养获得细胞约初次培养的2倍，差异有显著性意义(P=0.000 < 0.05)。达到90%融合度的细胞形态为长梭形成纤维细胞状，呈平行或漩涡状生长(图1C)。 2.2 人胎盘绒毛膜间充质干细胞免疫表型分析流式细胞术检测第3代人胎盘绒毛膜间充质干细胞，表面标记抗体CD90、CD105、CD73的表达率分别(98.54±0.21)%，(99.62±0.12)%，100%，均高于95%，而CD34、CD45、CD14、CD19、HLA-DR检测为阴性，表达率分别为(0.82± 0.25)%，(1.22±0.27)%，(1.15±0.14)%，(0.11±0.09)%，(1.21±0.31)%，均低于2%，见图2。 2.3 人胎盘绒毛膜间充质干细胞增殖能力分析 CCK-8法绘制第3代人胎盘绒毛膜间充质干细胞的生长曲线，1-2 d间充质干细胞增殖不明显；3-5 d细胞进入对数生长期，细胞增殖加速；6-7 d细胞进入平台期，生长缓慢，见图3。 2.4 人胎盘绒毛膜间充质干细胞诱导分化潜能分析第3代人胎盘绒毛膜间充质干细胞经成骨、成脂分化培养基诱导4周后，油红O染色可见胞浆内脂滴染成红色(图4A)，而茜素红染色，可见红色矿化沉着物(图4B)。说明人胎盘绒毛膜间充质干细胞在体外特定环境下具有向成骨细胞或脂肪细胞分化的潜能。"

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