This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers.
Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media.
These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.
方法:这项工作分析了五种广泛使用的培养基配方对菌落形成潜力的影响,增殖动力学,人骨髓来源的MSCs(BM-MSCs)的三系分化潜能和免疫调节潜能。还使用243个标记的高含量流式细胞术筛选组来表征表面标记表达谱。
结果:BM-MSCs的生物学属性存在显著差异,包括克隆性,扩散,响应于培养基的组成,揭示了分化倾向和免疫调节能力。尽管它们的生物学差异,所有细胞制剂均一致且强烈地表达针对BM-MSC提出的标准阳性标志物:CD73、CD90和CD105。免疫表型分析显示,培养基对表面蛋白质组也有显著影响,三分之一的测试标记物表现出可变的表达谱。主成分分析表明,与血小板裂解物和含有胎牛血清的培养基相比,在专有的无异种和无血清培养基中分离和扩增的BM-MSC表现出最一致的细胞表型,供体之间几乎没有变异性。
结论:这些数据表明培养基组成对MSCs的生物学特性具有非常显著的影响,但是标准表面标记测试掩盖了这些差异。结果表明,需要(1)标准化的制造方法,主要关注确定的培养基和(2)MSC表征和产品释放的新的生物学相关测试。