Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review

Panchalingam et al. Stem Cell Research & Therapy (2015) 6:225 DOI 10.1186/s13287-015-0228-5 REVIEW Open Access Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review Krishna M. Panchalingam1, Sunghoon Jung1, Lawrence Rosenberg2,3 and Leo A. Behie1* Abstract Human mesenchymal stem cells (hMSCs), also called mesenchymal stromal cells, have been of great interest in regenerative medicine applications because of not only their differentiation potential but also their ability to secrete bioactive factors that can modulate the immune system and promote tissue repair. This potential has initiated many early-phase clinical studies for the treatment of various diseases, disorders, and injuries by using either hMSCs themselves or their secreted products. Currently, hMSCs for clinical use are generated through conventional static adherent cultures in the presence of fetal bovine serum or human-sourced supplements. However, these methods suffer from variable culture conditions (i.e., ill-defined medium components and heterogeneous culture environment) and thus are not ideal procedures to meet the expected future demand of quality-assured hMSCs for human therapeutic use. Optimizing a bioprocess to generate hMSCs or their secreted products (or both) promises to improve the efficacy as well as safety of this stem cell therapy. In this review, current media and methods for hMSC culture are outlined and bioprocess development strategies discussed. Introduction Human mesenchymal stem cells (hMSCs) were first isolated from bone marrow but have since been found in other tissues in the body, such as adipose tissue, umbilical cord blood, the Wharton jelly of the umbilical cord, synovium, lung, pancreas, and muscle [1–3]. Whereas these other hMSC sources have emerged in the last few years and are being studied, bone marrow-derived hMSCs (BM-hMSCs) have been rigorously studied over many years and are used in the majority of hMSC clinical studies and trials. The clonogenic BM-hMSC fraction ranges from 10 to 100 CFU-F (colony-forming unit—fibroblast) per 106 marrow mononuclear cells (MNCs) and is typically isolated and expanded in classic serum-based media on tissue culture plastic. BM-hMSCs are characterized by (a) their adherence to plastic; (b) multipotency (i.e., adipogenic, osteogenic, and chondrogenic differentiation); (c) positive expression of surface antigens CD73, CD90, and CD105; and (d) lack of CD34, CD45, CD14 or CD11b, CD19 or CD79α, and * Correspondence: 1 Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada Full list of author information is available at the end of the article HLA-DR expression [4]. In addition to their multipotency, hMSCs have been shown to have the ability to secrete bioactive factors which can modulate the immune system (e.g., indoleamine 2,3-dioxygenase and prostaglandin E2) and promote tissue repair (e.g., glial cell linederived neurotrophic factor and vascular endothelial growth factor, or VEGF) [5]. In fact, it is widely accepted that the majority of hMSC-mediated therapeutic benefits are due to their secretion of bioactive molecules as it has been shown that these factors have various therapeutic effects both in vitro and in vivo (i.e., anti-inflammatory, anti-fibrotic, anti-apoptotic, anti-angiogenic, or immunomodulatory) as well as repair/regenerative actions. To generate hMSCs for clinical studies, it is necessary to first expand these cells for several passages in vitro, after which adequate potency testing should be performed before cell infusion. Any bioprocess used to produce therapeutic cells needs to be carefully designed, as this process is distinctly different from the well-known processes used to produce biopharmaceuticals. The first of these differences is that each batch or lot of therapeutic cells generated to treat one patient would be much smaller than © 2015 Panchalingam et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Panchalingam et al. Stem Cell Research & Therapy (2015) 6:225 the cell yields achieved for therapeutic protein production. Although hMSCs can be expanded for more than 40 population doublings (PDs) in culture, it has been suggested that cells of fewer than 20 PDs, particularly BM-hMSCs, be used for clinical applications with regard to safety and efficacy to avoid possible cell transformation [6, 7]. The second difference compared with therapeutic protein production is that hMSCs are the therapeutic product themselves. Thus, it is critical to produce functional hMSCs that retain their therapeutic properties. In this regard, it is important to develop a bioprocess for the expansion of hMSCs in a well-defined environment, where the nutritional, physiochemical, and mechanical requirements are met, controlled, and maintained (i.e., in bioreactors) for the culture period in order to generate consistent quantities of cells with the same desired properties. If variability is present between batches, this could undermine the therapeutic properties of the hMSCs. Hence, it is important to produce hMSCs for therapeutic applications in a well-defined manner (i.e., defined medium formulation) under good process control (i.e., online computer control in bioreactors) which can be operated in a closed system according to Good Manufacturing Practice (GMP). Human mesenchymal stem cell culture Culture media Conventional medium used for isolating and expanding hMSCs is typically a defined basal medium—i.e., Dulbecco’s modified Eagle’s medium (DMEM)—supplemented with fetal bovine serum (FBS): 10–20 % (vol/vol). However, concerns exist with the use of FBS for clinical use: namely (a) the variability of FBS from batch to batch, (b) its ill-defined nature, and (c) the possibility that FBS contains harmful contaminants such as prions, viral, and zoonotic agents. Moreover, when hMSCs are cultured in a medium containing animal proteins, a substantial amount of these proteins is retained in the cytoplasm of hMSCs, which may elicit an immunologic reaction when the cells are transplanted in vivo [8]. It is for this reason that, even though FBS is still widely used in hMSC research, it has been suggested (by our group and others) that the development of a defined serum-free medium is needed for the expansion of quality-assured clinically acceptable hMSCs [9–11]. H (...truncated)