Human adipose-derived stem cells: in vitro isolation, culture, identification and immunological properties

doi:10.3969/j.issn.2095-4344.2014.14.006

Abstract

Abstract:

BACKGROUND: Adipose-derived stem cells have more broad application prospects than bone marrow mesenchymal stem cells in tissue engineering because of the characteristics such as easy to obtain, and thus become one of the research hotspots today.
OBJECTIVE: To observe the growth characteristics of human adipose derived stem cells and to test their stem cell features and immunology characters.
METHODS: The subcutaneous fat tissue were extracted from three healthy volunteers through liposuction, and then digested with collagenase type I followed by in vitro primary culture and passage culture. Then, the biological characteristics of passaged cells were observed. Surface antigens and human leukocyte antigens were detected by flow cytometry. And then, peripheral blood lymphocytes were cultured with allogeneic adipose-derived stem cells to observe whether adipose-derived stem cells can accelerate the proliferation of lymphocytes.
RESULTS AND CONCLUSION: The harvested adipose-derived stem cells had a strong expansion in vitro with “S” shape growth curve. The third generation cells were positive for CD29, CD44, CD90 and human leukocyte antigen I, and negative for CD34, CD45, CD31 and human leukocyte antigen II. Besides, the lymphocytes had no response induced by allogeneic adipose-derived stem cells. The differentiation potential of adipose-derived stem
cells used in experiments was confirmed, and they had a weak rejection to the host, which indicates that the adipose-derived stem cells may be a candidate for cell treatment and tissue engineering.

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: stem cells, lipectomy, subcutaneous fat, antigens, surface, histocompatibility antigens

CLC Number:

R394.2

Eliyar Elham, Wang Yun-hai, Wang Li, Gao Hua, Xu Xin-cai, Zhang Wen-bin. Human adipose-derived stem cells: in vitro isolation, culture, identification and immunological properties[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(14): 2167-2172.

Figures/Tables 1

2.1 人脂肪干细胞的形态学观察将原代培养的脂肪干细胞在倒置显微镜下观察，接种后24 h可见50%细胞基本贴壁，形状为圆形或菱形，原代培养48 h，细胞开始克隆样增生，呈多角形或梭形，此时在培养皿中可见大量红细胞飘浮，用培养基每日清洗换液，到9 d可见细胞形态均一，细胞增殖能力活跃，细胞呈成簇分布，方向性好(图1A)。当细胞融合到85%-90%时，行细胞传代，传代后的脂肪干细胞生长良好，所培养的第3代人脂肪干细胞生长在含10%胎牛血清，含青、链霉素的DMEM-L培养基中，置于含体积分数5% CO2的37 ℃培养箱中培养，细胞生长良好(图1B)。 2.2 人脂肪干细胞的生长曲线生长曲线呈典型的“S”形，前2 d的A值略有降低，可能与部分细胞死亡有关。从第3天开始，进入对数生长期，第7天达高峰；随后逐渐降低(图1C)。 2.3 人脂肪干细胞分化群表面抗原的表达应用流式细胞术检测脂肪干细胞分化群表面抗原表达的结果显示，本实验所用的第3代脂肪干细胞呈呈CD29、CD44、CD90阳性表达；同时呈CD31、CD45、CD34阴性表达，间接排除了造血系统和内皮细胞的污染(表1，图2A-F)。 2.4 人脂肪干细胞的组织相容性抗原表达人脂肪干细胞表面主要组织相容性抗原Ⅰ图2G，H：本实验所分离的脂肪干细胞表面主要组织相容性抗原Ⅰ分子表达呈阳性，而主要组织相容性抗原Ⅱ分子表达呈阴性。和主要组织相容性抗原Ⅱ分子进行检测的结果见 2.5 异体脂肪干细胞与淋巴细胞共培养培养6 d时，实验组和对照组的CPM值差异无显著性意义(表2)。说明异体脂肪干细胞与淋巴细胞共培养时，不刺激淋巴细胞增殖，再次证实了脂肪干细胞较弱的异体移植排斥反应。 "

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