Gastens Martin H, Goltry Kristin, Prohaska Wolfgang, Tschöpe Diethelm, Stratmann Bernd, Lammers Dirk, Kirana Stanley, Götting Christian, Kleesiek Knut
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, 32545 Bad Oeynhausen, Germany.
Cell Transplant. 2007;16(7):685-96. doi: 10.3727/000000007783465172.
Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.
体外扩增正被用于增加用于自体细胞治疗的干细胞和祖细胞数量。由于确保安全和高质量细胞生产面临挑战，启动测试这些细胞用于组织修复功效的关键临床试验受到了阻碍。本文描述了一种策略，用于在完全封闭的过程中，从细胞采集到培养后处理，使用无菌可连接装置，在混合细胞群体中对骨髓（BM）来源的干细胞进行临床规模的扩增。分离人BM单个核细胞（BMMNC），培养12天，并在收获后使用层流罩中的标准开放程序或自动封闭系统进行洗涤。这些研究的条件类似于长期BM培养，其中造血和基质成分一起培养。然后评估骨髓来源的干细胞和祖细胞的扩增情况。比较了单程灌注生物反应器和标准培养瓶培养的细胞产量、集落形成单位（CFU）数量、表型、稳定性和多谱系分化能力。与手动处理分别为100%和70%相比，使用封闭的Ficoll梯度法纯化BMMNC导致98%的红细胞和87%的粒细胞减少。在封闭系统培养后，以CD105+CD166+CD14-CD45-和成纤维细胞CFU衡量的间充质祖细胞分别扩增了317倍和364倍，而与起始BMMNC相比，CD34+造血祖细胞减少了10倍。培养的细胞通过在体外表现出成脂、成骨和内皮特征而呈现多谱系分化。手动培养和生物反应器培养之间未观察到显著差异。细胞产品的自动培养和洗涤产生了181×10⁶个活细胞，这些细胞含有成纤维细胞CFU，至少可储存24小时。封闭、自动化技术的组合能够生产符合药品生产质量管理规范（GMP）的细胞治疗产品，可在临床环境中随时使用，微生物污染风险最小。
