改良法原代培养人脂肪间充质干细胞及其鉴定

Modified primary culture and identification of human adipose-derived mesenchymal stem cells

背景:采用常规酶消化法提取人脂肪间充质干细胞,由于消化的时间过长,导致获得的细胞活性变差,为满足组织工程对种子细胞数量的需求,该实验旨在寻找一种改良的人脂肪间充质干细胞培养方法。目的:胶原酶Ⅰ消化联合组织块培养法获取人脂肪间充质干细胞并进行相关生物学特性的鉴定。方法:将脂肪组织剪碎后分别应用胶原酶Ⅰ消化联合组织块培养法、常规胶原酶消化法进行培养;通过锥虫蓝拒染法计数细胞,比较不同方法获取细胞的产量、活力及增殖能力;利用流式细胞仪检测改良组细胞表面抗原CD105、CD73与CD44的表达;成骨或成脂诱导后,用碱性磷酸酶染色和油红O染色鉴定细胞分化能力。结果与结论:①胶原酶Ⅰ消化联合组织块培养法获取的细胞数量远多于常规酶消化法;②胶原酶Ⅰ消化联合组织块培养法获取的人脂肪间充质干细胞增殖能力强,细胞活力好,比常规酶消化法早1 d进入平台期,传代一次需5 d左右;③第3代改良组人脂肪间充质干细胞CD105、CD73与CD44表达均呈阳性,且纯度均达93%以上;④改良法获取的人脂肪间充质干细胞有良好的成骨与成脂分化能力;⑤结果表明,胶原酶Ⅰ消化法联合组织块培养法较常规酶消化法可获得数量多、活力好、增殖能力强的人脂肪间充质干细胞,且能够稳定传代,具有良好的分化能力,这满足了组织工程学的需求。

BACKGROUND:Human adipose-derived mesenchymal stem cells were extracted by conventional enzymatic digestion.The long-term digestion resulted in poor cell viability.To meet the demand for the number of seed cells in tissue engineering,this experiment aimed to find a modified human adipose-derived mesenchymal stem cell culture method.OBJECTIVE:Human adipose-derived mesenchymal stem cells were obtained by collagenase I digestion combined with tissue block culture method and the relevant biological characteristics were identified.METHODS:The adipose tissue was cut up and cultured with collagenase I digestion combined with tissue block culture and conventional collagenase digestion method.Cells were counted by trypan blue exclusion method.The yield,viability and proliferation ability of cells obtained by different methods were compared.The expression of cell surface antigens CD105,CD73 and CD44 was tested by flow cytometry in the modified group.After osteogenic or adipogenic induction,alkaline phosphatase staining and oil red O staining were used to identify cell differentiation ability.RESULTS AND CONCLUSION:(1)The number of cells obtained by collagenase I digestion combined with tissue block culture method was far more than that obtained by conventional enzyme digestion method.(2)The cells obtained by collagenase I digestion combined with tissue block culture had the strong proliferation ability and good cell viability.They entered the plateau phase 1 day earlier than the conventional collagenase digestion method,and each passage took about 5 days.(3)The expression of CD105,CD73 and CD44 was positive in the modified group of P3 generation,and the purity was over 93%.(4)Human adipose-derived mesenchymal stem cells obtained by the modified method had good osteogenic and adipogenic differentiation capabilities.(5)The results showed that the collagenase I digestion method combined with tissue block culture method could obtain a large number of human adipose-derived mesenchymal stem cells with better viability and proliferation ability than the conventional enzymatic digestion method,and they could be passaged stably with a good differentiation ability,which met the needs of tissue engineering.