Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells
Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells
复制标题
DOI：
10.1016/j.jcyt.2016.05.012
复制
发表时间：
2016-09-01
期刊：
影响因子：
4.5
通讯作者：
Garcia-Lopez, Joan
中科院分区：
文献类型：
--
作者：
Codinach, Margarita;Blanco, Margarita;Garcia-Lopez, Joan
背景资料。多能间充质基质细胞(MSC)在再生医学领域取得了显著的成就，尽管在生产过程中缺乏共同的标准和与GMP相兼容的适当的质量控制。在此，我们描述了一种分离和扩增骨髓(BM)来源的MSC的生物工艺的设计，它的验证和连续48批临床使用的生产。方法：研究方法。从患者的髂骨采集骨髓标本进行自体治疗。生产程序包括：(I)通过自动密度梯度离心法和平板法分离有核细胞(NC)；(Ii)胰酶消化和继代培养；(Iii)收获和配制含有40+/-10×10(6)活细胞的悬浮液。质量控制定义为：(I)细胞计数和活性评估；(Ii)免疫表型；(Iii)无菌试验、支原体检测、内毒素试验和革兰氏染色。结果。在生产48批临床使用的BM-MSC之前，首先设计了一个为期3周的制造生物工艺，然后在3个连续的模拟生产中进行验证。验证包括对MSC特性和遗传稳定性的评估。在生产方面，以含有2.53+/-0.92x10(9)活NC的139.0+/-17.8mLBM为原料，最终产品中产生38.8+/-5.3x10(6)活细胞。表面抗原表达与MSC的预期表型一致，表现为CD73、CD90和CD105高表达，CD31和CD45缺乏表达，低水平的HLA-DR。所有病例的无菌、支原体、革兰氏染色和内毒素检测均为阴性。讨论。在此，我们证明了建立一种可行的、一致的和可重复性的生物工艺来生产临床使用的安全的骨髓来源的MSC。
Background. Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM) derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. Methods. BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 +/- 10 x 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. Results. A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 +/- 17.8 mL of BM containing 2.53 +/- 0.92 x 10(9) viable NC were used as starting material, yielding 38.8 +/- 5.3 x 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. Discussion. Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use.