GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC
GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC
复制标题
DOI：
10.1371/journal.pone.0043255
复制
发表时间：
2012-08-14
期刊：
影响因子：
3.7
通讯作者：
Schrezenmeier, Hubert
中科院分区：
文献类型：
--
作者：
Fekete, Natalie;Rojewski, Markus T.;Schrezenmeier, Hubert
背景资料：间充质基质细胞（MSC）在组织修复，组织工程和免疫抑制治疗中的重要性，在过去的几年中。由于骨髓中MSC的可用性有限，因此在临床应用之前的离体扩增对于获得治疗适用的细胞剂量是必要的。将临床前MSC扩增转化为临床级大规模MSC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、培养基和培养装置。虽然异种添加剂，如胎牛血清仍被广泛用于细胞培养，其在临床上的使用是与许多风险，如朊病毒和病毒传播或不良免疫反应对异种components.Methods和调查结果：我们建立了无动物扩增协议，使用血小板裂解液作为培养基补充，从而可以确认其安全性和可行性，大规模MSC分离和扩增。进行了五种不同的符合GMP的标准化方案，其设计用于安全、可靠、有效和经济地分离和扩增MSC，并通过qPCR和组织化学分析获得的MSC的分化能力。通过流式细胞术分析由国际细胞治疗学会定义的标准MSC标志物的表达以及另外的MSC标志物和各种趋化因子和细胞因子受体的表达。使用LUMINEX platform.Conclusions：本研究中描述的用于分离和扩增MSC的五种不同系统都适合于产生至少1亿个MSC，这通常被视为单次临床剂量。根据ISCT定义的MSC最低标准，最终产品是相等的。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，这表明来自两种系统的MSC显示出相同的归巢和粘附特征。
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.