符合 GMP 标准的骨髓间充质干细胞的分离和大规模扩增。

BACKGROUND: Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. METHODS AND FINDINGS: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. CONCLUSIONS: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

背景：近年来，间充质基质细胞（MSC）在组织修复、组织工程和免疫抑制治疗方面的重要性日益增加。由于骨髓中 MSC 的可用性有限，因此在临床应用前需要进行体外扩增，以获得治疗适用的细胞剂量。将临床前研究转化为临床级别的 MSC 大规模扩增，需要精确定义和标准化所有程序参数，包括细胞接种密度、培养基和培养设备。虽然胎牛血清等异种添加物仍广泛用于细胞培养，但在临床环境中使用存在许多风险，如朊病毒和病毒传播或对异种成分的免疫反应。

方法和发现：我们使用血小板裂解液作为培养基补充物建立了无动物扩增方案，从而可以确认其在大规模 MSC 分离和扩增中的安全性和可行性。设计了五个不同的符合 GMP 标准的标准化方案，用于安全、可靠、高效和经济地分离和扩增 MSC，并通过 qPCR 和组织化学分析来分析 MSC 的分化能力。通过流式细胞术分析了国际细胞治疗学会定义的标准 MSC 标志物的表达，以及额外的 MSC 标志物和各种趋化因子和细胞因子受体的表达。使用 LUMINEX 平台分析了培养基中代谢标志物和细胞因子的变化。

结论：本研究中描述的五种不同的 MSC 分离和扩增系统都适合生产至少 1 亿个 MSC，这通常被认为是单个临床剂量。最终产品根据 ISCT 定义的 MSC 的最低标准是相等的。我们表明，分析的趋化因子和整合素受体具有相同的表达模式，这表明来自任何一种系统的 MSC 都具有相同的归巢和黏附特性。