Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis
Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis
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DOI：
10.1080/14653240802398845
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发表时间：
2008-01-01
期刊：
影响因子：
4.5
通讯作者：
Wagner, W.
中科院分区：
文献类型：
--
作者：
Horn, P.;Bork, S.;Wagner, W.
背景人骨髓间充质干细胞（mesenchymal stromal cells，MSC）在组织工程和临床治疗中的应用前景广阔。它们的分离通常涉及单核细胞（MNC）的密度分级，但这很难标准化，特别是在良好生产规范（GMP）条件下。MSC代表细胞类型的异质混合物，并且亚群的组成受到细胞制备的初始步骤的影响。方法采用氯化铵裂解红细胞的方法分离人骨髓间充质干细胞。直接将集落形成与Ficoll密度分级和未处理的全骨髓（BM）抽吸物的培养进行比较。结果7天后，每毫升骨髓穿刺液中的成纤维细胞集落形成单位（CFU-F）数量在红细胞溶解后略高，集落明显大于密度分级，可能是由于血小板的维持。相比之下，在未处理的BM中集落形成低得多。亚群的异质性组成反映了初始集落之间的差异，关于生长模式（紧密或分散）和细胞形态（圆形或细长）。这种异质组合物不受三种不同分离方法的影响。此外，CD 271+细胞的富集导致相同的形态学异质性。所有的细胞制剂使用一组表面标志物证明了相同的免疫表型，并显示出成脂和成骨分化潜力。讨论本研究表明，人MSC可以有效地分离红细胞裂解。这种技术更快，更容易标准化，用于MSC的临床应用。
Background Human mesenchymal stromal cells (MSC) have raised high hopes for tissue engineering and clinical therapy. Their isolation usually involves density fractionation of mononuclear cells (MNC) but this is difficult to standardize, especially under good manufacturing practice (GMP) conditions. MSC represent a heterogeneous mixture of cell types and the composition of subpopulations is affected by the initial steps of cell preparation. Methods This study describes a straightforward method for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. Colony formation was compared directly with Ficoll density fractionation and culture of an untreated whole bone marrow (BM) aspirate. Results After 7 days the number of fibroblastic colony-forming units (CFU-F) per milliliter of BM aspirate was slightly higher upon RBC lysis and the colonies were significantly larger compared with density fractionation, possibly because of maintenance of platelets. In contrast, colony formation was much lower in untreated BM. The heterogeneous composition of subpopulations was reflected by differences between the initial colonies with regard to growth pattern (tight or disperse) and cell morphology (round or elongated). This heterogeneous composition was not affected by the three different isolation methods. Furthermore, enrichment of CD271+ cells resulted in the same morphologic heterogeneity. All cell preparations demonstrated the same immunophenotype using a panel of surface markers and displayed adipogenic and osteogenic differentiation potential. Discussion This study demonstrates that human MSC can be efficiently isolated by RBC lysis. This technique is faster and can be standardized more easily for clinical application of MSC.