Tozetti Patrícia Aparecida, Caruso Samia Rigotto, Mizukami Amanda, Fernandes Taisa Risque, da Silva Fernanda Borges, Traina Fabiola, Covas Dimas Tadeu, Orellana Maristela Delgado, Swiech Kamilla
Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
Dept. of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
Biotechnol Prog. 2017 Sep;33(5):1358-1367. doi: 10.1002/btpr.2494. Epub 2017 May 30.
Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost-effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno-free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3-D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10 and 1.4 ± 0.3 × 10 cells/cm . UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5-23.0 × 10 cells/cm ) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno-free expansion processes represents an important step toward a GMP compliant large-scale production platform for MSC-based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358-1367, 2017.
选择用于细胞生产的培养系统和培养基是实现用于细胞治疗目的的安全、可扩展且具有成本效益的细胞扩增生物工艺的关键步骤。由于安全和效率方面的原因，使用AB型人血清（AB HS）作为间充质基质细胞（MSC）培养的无动物源替代补充剂越来越受到关注。在此，我们评估了不同的可扩展培养系统，以使用AB HS在扩增和冷冻保存期间补充培养基，从而生产出数量可观的脐带基质来源的MSC（UCM MSC），以实现无动物源生物工艺。UCM MSC在可扩展的平面（紧凑型10层培养瓶和转瓶）和基于3D微载体的培养系统（转瓶培养器和搅拌罐生物反应器）中进行培养。10层培养瓶和转瓶分别能够产生2.6±0.6×10⁶和1.4±0.3×10⁶个细胞/cm²。与平面系统相比，在搅拌条件下基于微载体的UCM MSC扩增能够产生更高的细胞密度（5.5 - 23.0×10⁶个细胞/cm²）。然而，由于收获效率适中（转瓶培养器为80%，生物反应器为46.6%），回收的总细胞数低于预期。在所有评估的培养系统中，细胞扩增后仍保持其功能特性。细胞（使用AB HS）的冷冻保存也成功进行。建立可扩展的无动物源扩增工艺是迈向基于MSC的临床应用的符合GMP的大规模生产平台的重要一步。© 2017美国化学工程师学会生物技术进展，33:1358 - 1367，2017。
