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Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells

机译:不同细胞分离方法对滑膜间充质干细胞活力和细胞表面抗原表达的影响

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摘要

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.
机译:细胞表面抗原的流式细胞仪分析是用于分离和鉴定成人组织中干细胞的有力工具。与造血干细胞的收集相反,酶消化过程通常是制备间充质干细胞(MSCs)悬浮液所必需的,这可能会影响细胞表面标志物的表达。在这项研究中,我们检查了各种细胞分离试剂和消化时间对干细胞相关表面抗原和MSC功能表达的影响。使用四种不同的试剂:胰蛋白酶,TrypLE,胶原酶和非酶细胞解离试剂(C5789; Sigma-Aldrich)将人MSC从培养皿中分离。解离试剂孵育5至120分钟后,通过流式细胞仪分析细胞表面标志物。胰蛋白酶和TrypLE在5分钟内迅速使细胞解离,而胶原酶和C5789需要60分钟才能获得最大的细胞产量。 C5789在120分钟时显着降低了细胞活力。胰蛋白酶治疗可显着降低CD44 + ,CD55 + ,CD73 + ,CD105 + ,CD140a + < / sup>,CD140b + 和CD201 + 在30分钟内的细胞数。胶原酶处理使CD140a表达减少了30分钟。相比之下,TrypLE处理并未影响到30分钟测试的任何细胞表面抗原的表达。尽管用胰蛋白酶处理60分钟后表面抗原表达明显丧失,但酶消化对MSCs多能性的不利影响还是有限的。总体而言,我们的数据表明,TrypLE优于经测试可快速制备可行MSC悬浮液的其他细胞解离试剂。

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