Standardized Isolation of Human Mesenchymal Stromal Cells with Red Blood Cell Lysis
Standardized Isolation of Human Mesenchymal Stromal Cells with Red Blood Cell Lysis
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DOI：
10.1007/978-1-60761-999-4_3
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Wagner, Wolfgang
中科院分区：
文献类型：
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作者：
Horn, Patrick;Bork, Simone;Wagner, Wolfgang
人间充质基质细胞（MSC）在组织工程和治疗应用方面寄予厚望。到目前为止，还不可能从骨髓中分离出纯的部分，因此MSC细胞制剂众所周知地代表了不同细胞类型的异质混合物。亚群的组成可能已经受到细胞制备初始步骤的影响。通常，MSC的分离涉及密度分级以将单核细胞（MNC）与红细胞和粒细胞分离。然而，这种方法很难标准化，特别是在GMP条件下。在这里，我们描述了一种替代的方法，用于分离人MSC的基础上红细胞（RBC）与氯化铵裂解。这导致成纤维细胞集落形成单位（CFU-F）的数量略高，而CFU-F的形态学分析揭示了MSC培养物的相同异质组成，表明亚群的比例不受RBC溶解的影响。免疫表型（CD 73（+）、CD 90（+）、CD 105（+）、CD 31（-）、CD 34（-）、CD 45（-））、成脂和成骨分化潜能也相似。总之，RBC裂解是从骨髓抽吸物中分离人MSC的有效方法。这种技术更快，更容易标准化，用于MSC的临床应用。
Human mesenchymal stromal cells (MSC) raise high hopes for tissue engineering and therapeutic applications. So far, it is not possible to isolate pure fractions from bone marrow and therefore MSC cell preparations notoriously represent heterogeneous mixtures of different cell types. The composition of subpopulations can already be affected by the initial steps of cell preparation. Usually, isolation of MSC involves density fractionation to separate the mononuclear cells (MNCs) from erythrocytes and granulocytes. However, this method is difficult to standardize especially under GMP conditions. Here, we describe an alternative approach for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. This results in a slightly higher number of fibroblastic colony forming units (CFU-F), whereas morphological analysis of the CFU-F reveals the same heterogeneous composition of MSC cultures indicating that the proportion of subpopulations is not affected by RBC lysis. Immunophenotype (CD73(+), CD90(+), CD105(+), CD31(-), CD34(-), CD45(-)), adipogenic, and osteogenic differentiation potential of MSC were also similar with both methods. In conclusion, RBC lysis comprises an efficient method for the isolation of human MSC from bone marrow aspirate. This technique is faster and can be standardized more easily for clinical application of MSC.