Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15 Street, 44-102, Gliwice, Poland.
Kardio-Med Silesia, Maria Skłodowska-Curie 10C Street, 41-800, Zabrze, Poland.
Stem Cell Res Ther. 2019 Aug 5;10(1):235. doi: 10.1186/s13287-019-1331-9.
Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare.
ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated.
All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest.
Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.
脂肪组织来源的间充质基质细胞（ASCs）已被证明在再生医学中具有一些有前途的特性，可作为先进治疗药物产品（ATMP）使用。然而，其来源的不同、分离方法和扩增程序导致实验室和临床结果难以比较。
从脂肪抽吸物中分离出 ASCs，并在三种不同的培养基配方中进行培养：αMEM 和 DMEM 作为基础培养基，添加 10%的人血小板裂解物（hPL）；DMEM 补充 20%胎牛血清（FBS）和 bFGF 作为金标准培养基。随后，评估培养基对 ASCs 生长动力学、形态和免疫表型、分化能力、克隆形成潜力和分泌谱的影响。
所有培养的 ASC 系均表现出相似的形态和相似的克隆形成潜力，并具有分化为三种谱系的能力：脂肪细胞、成骨细胞和软骨细胞。所有培养的 ASC 的免疫表型均符合国际细胞治疗协会（ISCT）的指南，可将细胞定义为间充质基质细胞（MSC）（≥95%CD105、CD73、CD90，≤2%CD45、CD34、CD14、CD19、HLA-DR）。免疫表型在第二代后稳定，且在不同条件下培养的 ASC 之间没有差异。例外是在 FBS 和 bFGF 存在下培养的 ASC，其表达 CD146 抗原。在不同培养基中培养的 ASC 的分泌谱相似。主要分泌的细胞因子是 IL-6，其水平具有供体特异性。然而，我们观察到培养基配方对 ASC 生长动力学有很强的影响。补充 hPL 的培养基中 ASC 的增殖率最高。
不含动物源性抗原（无动物源）的培养基可用于培养被定义为 MSC 的细胞。无动物源培养基是生产作为先进治疗药物产品的临床级 MSC 的安全替代品。此外，在这种培养条件下，MSC 可以根据良好生产规范（GMP）要求轻松扩增到临床应用所需的细胞数量。
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