Bone marrow mesenchymal stem cells on Matrigel: growing and changing

doi:10.3969/j.issn.2095-4344.0427

Abstract

Abstract:

BACKGROUND: Matrigel as an extracellular matrix complex can facilitate cell proliferation, differentiation and collagen secretion in a cell culture system.
OBJECTIVE: To establish a three-dimensional culture model of Matrigel combined with bone marrow mesenchymal stem cells (BMSCs) and to observe the morphology, proliferation and survival of BMSCs in the Matrigel three-dimensional culture model.
METHODS: BMSCs were isolated and cultured by the whole bone marrow adherent method, followed by osteogenic and adipogenic induction and identification. The growth curve of passage 3 BMSCs was determined through the CCK-8 experiment. Passage 2 BMSCs were combined with Matrigel, and the morphology and proliferation of BMSCs were observed by hematoxylin-eosin staining under a phase contrast microscope. The viability of the cells was evaluated by the Live/Dead staining.
RESULTS AND CONCLUSION: (1) BMSCs cultured by the whole bone marrow adherent method had the ability to differentiate into osteoblasts and adipocytes. (2) BMSCs exhibited an “S”-shaped growth curve, which was consistent with the growth characteristics of normal cells. (3) Under the phase contrast microscope, BMSCs cultured by the Matrigel model were extended and interconnected in a three-dimensional network growth state with good proliferation. The Matrigel gradually became soft and collapsed over time (7days) and a few cells were still in a network growth after 14 days. Hematoxylin-eosin staining results showed that the cell cytoplasm was larger at 4 days and the cells became thinned and mutually cross-linked at 7 days. (4) The active percentage of BMSCs in the Matrigel model was 92.57%, 95.54% and 97.37% at 1, 4, 7 days of culture, respectively. To conclude, BMSCs cultured on the Matrigel has good proliferation and high viability.

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: Bone Marrow, Mesenchymal Stem Cells, Extracellular Matrix, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Zhang Bin-bin, Gao Quan-wen, Li Bing, Huang Sha, Yao Bin. Bone marrow mesenchymal stem cells on Matrigel: growing and changing[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 1993-1998.

Figures/Tables 1

2.1 骨髓间充质干细胞分离培养及诱导分化鉴定结果原代培养24 h，细胞开始贴壁，48 h后细胞大量贴壁，细胞呈多角形或短小梭形，排列不规则，培养1周左右，细胞快速生长融合成集落，排列呈漩涡状，10-14 d细胞融合成片，排列紧密。传代后，细胞形态均一、胞膜清晰，呈多角形或长梭形(图1A)。成骨诱导培养21 d后茜素红染色阳性，细胞外基质中可见典型红色矿化结节(图1B)。成脂诱导20 d后细胞中可见大小不等的脂滴，油红O染色阳性，细胞核被染成蓝色，脂滴被染成橘红色(图1C)。 2.2 CCK-8检测第3代骨髓间充质干细胞增殖特性第3代骨髓间充质干细胞的生长曲线呈“S”形，骨髓间充质干细胞具有较强的增殖能力。接种第1，2天细胞生长缓慢，生长曲线基本呈平线，为细胞潜伏适应期，接种第3-7天，细胞适应了环境，生长速度加快，生长曲线呈现为线性曲线，表明该段时间为细胞的对数生长期，第8天后培养基营养物质有限，细胞相互间竞争，细胞增殖明显减慢，曲线逐渐变得平缓，进入平台期(图2)。 2.3 骨髓间充质干细胞在Matrigel基质胶中的形态及增殖情况传代后单层培养的骨髓间充质干细胞6-8 h开始贴壁，24 h后达到完全贴壁，细胞呈长梭形或多角形，培养两三天时融合的细胞可达到80%-90%，四五天可达100%融合，培养6 d以后大部分细胞开始老化、脱落。与Matrigel基质胶复合培养的骨髓间充质干细胞均匀分布于基质胶材料中，随后细胞逐渐伸长形成明显的不规则状，第7天时可见细胞相互交联呈三维网状生长，随着时间延长，基质胶开始软化塌陷，14 d时虽仍可见部分网状交联结构，但大部分已软化(图3)。三维培养4 d时苏木精-伊红染色可见细胞胞质较大，开始交联，7 d时细胞胞质伸长更加明显，交联成网状(图4)。提示Matrigel复合骨髓间充质干细胞培养模型的建立使得骨髓间充质干细胞在基质胶微环境中形成了具有三维梯度的多细胞群，细胞增殖能力较强。 2.4 骨髓间充质干细胞在matrigel基质胶中的细胞活力三维培养第1，4，7天进行live/dead染色，第1天时细胞均匀分布于基质胶材料中，随着培养时间的延长细胞开始相互交联呈三维网状生长，多数细胞均呈现绿色荧光(活细胞)(图5)，第1，4，7天细胞活性百分比分别为92.57%，95.54%，97.37%，呈逐渐升高趋势(图6)，提示比格犬骨髓间充质干细胞与Matrigel基质胶复合后，不同梯度的细胞基本能够存活，活性较好，并且具备增殖能力，细胞与材料之间具有良好的生物相容性。"

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