Isolation and culture of rat bone marrow mesenchymal stem cells: passage 3 cells exhibit rapid proliferation and high purity

doi:10.3969/j.issn.2095-4344.2014.50.002

Abstract

Abstract:

BACKGROUND: The first step of tissue engineering research is to produce a large number of seed cells with good biological activity, fast proliferation and high purity.
OBJECTIVE: To explore the biological characteristics of rat bone marrow mesenchymal stem cells isolated and cultured by the whole bone marrow adherence method.
METHODS: Rat bone marrow mesenchymal stem cells were isolated and cultured, and then passage 3 cells were enrolled to draw a cell growth curve. Morphological changes of cells at different passages were observed. Cells in passage 2 and passage 3 were characterized by their surface marker expressions of CD29, CD90, CD34 and CD45.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells at passages 1-3 exhibit classic morphology of fibroblast-like cells and presented plastic-adherent property. Phenotypes of P2 generation were detailed as: expressions of CD29, CD90, CD34, CD45 were 91.5%, 96.2%, 1.3%, and 8.0%, respectively. Phenotypes of passage 3 cells were detailed as: expressions of CD29, CD90, CD34, CD45 were 98.2%, 98.1%, 0.6%, 2.0%, respectively. The cell growth curve indicated the cell proliferation reached the peak at an inoculation
concentration of 8×107/L. Overall, bone marrow mesenchymal stem cells at passage 3 with good viability, rapid proliferation and high purity can be used as seed cells for tissue engineering research.

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

全文链接：

Key words: bone marrow, mesenchymal stem cells, cell culture techniques, cells, cultured

CLC Number:

R394.2

Zeng Zhi-mou, Fan Zhong-cheng, Zhang Shou, Liu Yi-heng, Wu Duo-qing, Wang Cong-ren. Isolation and culture of rat bone marrow mesenchymal stem cells: passage 3 cells exhibit rapid proliferation and high purity[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(50): 8042-8047.

Figures/Tables 3

2.1 骨髓间充质干细胞的形态学观察 ①全骨髓培养法刚分离接种的细胞悬液中杂细胞较多，大部分为圆形透亮的红细胞(图1A)，原代培养第2天镜下可见少量梭形细胞开始贴壁生长(图1B)，接种第3天镜下观察可见由几十个细胞组成的多个细胞集落散在分布于培养皿底，单个细胞形态呈长梭形，集落内细胞未出现融合生长，培养液中仍有大量未贴壁的圆形细胞悬浮(图1C)；此后集落内细胞迅速增殖，集落增大并与周边集落逐渐融合生长，培养至第7天左右长梭形细胞均匀铺满培养皿底，融合范围80%-90%，悬浮于培养液的混杂细胞较前减少。②细胞传代后3-5 h可见少量细胞开始贴壁，18-24 h后大部分细胞均匀贴壁生长，未贴壁的杂细胞经过多次换液逐渐被去除，五六天时培养皿内细胞再次融合至80%-90%(图1D)。③按上述方法反复传代至第5代，通过多次换液和传代去除混杂细胞后骨髓间充质干细胞逐渐纯化；第2代和第3代的单个细胞形态呈典型的长梭形或纺锤状，融合后呈放射状或漩涡状排列(图1E，F)；第4代细胞形态开始变得扁平，长梭形细胞减少，形态不均一，排列不如前几代有规律(图1G)；第5代细胞形态改变更加明显，大部分呈扁平宽大状，细胞有多个突起，而且排列无规则，生长速度明显减慢(图1H)。 2.2 第3代骨髓间充质干细胞生长曲线以细胞浓度1×107 L-1，2×107 L-1，1×108 L-1接种培养，3 d内细胞缓慢增长，而3-5 d细胞迅速增殖，表现为生长曲线中的对数期，5 d以后细胞生长速度逐渐减慢至平稳构成曲线的平台期，其最大吸光度值分别为0.39，0.436，0.857；以细胞浓度4×107 L-1，6×107 L-1，8×107 L-1接种培养，3 d内缓慢增殖，构成曲线的潜伏期，3-6 d内迅速增殖，形成曲线的对数期，6 d以后细胞生长速度较前明显减慢，构成曲线的平台期，其最大吸光度值分别为0.682，0.78，0.905；以细胞浓度1×107 L-1，2×107 L-1接种，细胞生长速度慢，最后细胞总数明显偏少；细胞浓度8×107 L-1相比细胞浓度1×108 L-1的对数生长期更长，速度更快，增殖潜力大，最后获得细胞总数多；细胞浓度8×107 L-1相比细胞浓度4×107 L-1，6×107 L-1，细胞增殖能力更强，最后获得的细胞总数更多(图2)。 2.3 骨髓间充质干细胞表面标志物检测结果第2代骨髓间充质干细胞表型检测结果：CD29的阳性率为91.5%，CD90的阳性率为96.2%，CD34的阳性率为1.3%，CD45的阳性率为8.0%，见图3；第3代骨髓间充质干细胞表型检测结果：CD29的阳性率为98.2%，CD90的阳性率为98.1%，CD34的阳性率为0.6%，CD45的阳性率为2.0%，见图4；第3代流式结果与第2代相比，阳性表型的阳性率更高，而阴性表型的阳性率更低。"

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