Laboratorio Interdipartimentale di Terapia Cellulare Stefano Verri, Azienda Ospedaliera San Gerardo, Monza, Milan, Italy.
J Cell Mol Med. 2010 Jun;14(6B):1619-34. doi: 10.1111/j.1582-4934.2009.00854.x. Epub 2009 Jul 20.
The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.
本研究旨在开发和验证一种符合良好生产规范 (GMP) 的程序，用于制备用于心血管修复的骨髓 (BM) 来源的 CD133(+)细胞。从现有的从人脐带血中纯化 CD133(+)细胞的实验室方案开始，我们将这些程序在 GMP 设施中实施，并应用质量控制条件定义 CD133(+)细胞的纯度、微生物安全性和活力。根据包括释放质量检查（步骤 1）以及其表型完整性和生物学功能的“原理验证”（步骤 2）的两步实验方案，对 CD133(+)细胞分离和释放过程进行验证。该测试程序使用体外培养测定和在免疫抑制的小鼠后肢缺血模型中进行体内测试来完成。这些标准和程序已成功应用于正在进行的缺血性心肌病患者自体干细胞给药的临床试验中，从 BM 生产 GMP 级别的 CD133(+)细胞。我们的结果表明，根据欧洲监管机构的最新质量要求，目前可用于 CD133(+)细胞选择的方案的 GMP 实施是可行且可重复的，并且能够生产具有完整生物学潜力的细胞。
