1 Institute for Transfusion Medicine, University of Ulm , Ulm, Germany .
Tissue Eng Part C Methods. 2014 Feb;20(2):129-39. doi: 10.1089/ten.TEC.2013.0061. Epub 2013 Jul 5.
Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.
间充质基质细胞 (MSCs) 是再生医学临床应用中非常有前途的候选细胞。由于其在人体组织中的含量较低，因此需要进行广泛的体外扩增，以获得足够数量的细胞，作为细胞治疗的临床剂量。目前用于分离和扩增 MSCs 的细胞培养培养基配方包括胎牛血清 (FCS)、人 AB 血清 (ABS) 或人血小板裂解物 (PL) 作为补充物。然而，这些现有的补充物是固有定义不明确的配方，其中含有各种生物活性分子，其批次组成各不相同，并且存在传播常规筛选程序无法检测到的病原体的风险。在这项研究中，我们比较了常用基础培养基（如最低必需培养基 alpha (αMEM)、杜尔贝科改良伊格尔培养基 (DMEM)、伊斯科夫改良杜尔贝科培养基 (IMDM) 和 RPMI 1640）以及人和动物来源的补充物（PL、ABS 和 FCS）刺激细胞增殖的能力。在补充 PL 的 αMEM 中观察到 MSC 增殖最佳。然后，我们使用组合方法评估了可溶性因子文库，包括有丝分裂原（TGF-β1、激活素 A、bFGF、EGF、IGF-I、PDGF-BB 和 VEGF）、趋化因子（CCL21、CCL25、CXCL12 和 RANTES）、蛋白质（人血清白蛋白）、脂质（如油酸、亚油酸和花生四烯酸）和激素（地塞米松、胰岛素和 TSH），以创建一种定义明确的培养基，并对细胞培养表面进行涂层，以促进 MSC 在体外的大量增殖。重组人因子的组合部分满足骨髓来源 MSCs 的营养需求，如果补充辅助 0.6%-1.2% PL，能够促进细胞增殖，与大约 5% PL 相当。通过将 5% PL 与重组因子鸡尾酒组合使用，可实现 MSC 的最大增殖，并且不依赖于细胞培养表面的涂层。
