Abstract
Mesenchymal stem cells (MSCs) have emerged as an important tool for tissue engineering, thanks to their differentiation potential and their broad trophic activities. However, for clinical purposes or for relevant in vitro applications, large quantities of MSCs are required, which could hardly be reached using conventional cultivation in plastic dishes. Microcarriers have high surface to volume ratio, which enables the easy scale-up of the expansion and differentiation of MSCs. In addition, the agitation in stirred tank bioreactors limits the diffusion gradient of nutrients or morphogens, thus providing a physiologically relevant environment to favor MSC production at large scale. This work describes a simple method for the mass expansion and differentiation of MSCs, including the procedures to monitor the proliferation, metabolic status and phenotype of MSCs during suspension culture. Moreover, this work proposes suitable materials for cGMP compliant culture conditions enabling the clinical grade production of MSCs.
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References
Linero I, Chaparro O (2014) Paracrine effect of mesenchymal stem cells derived from human adipose tissue in bone regeneration. PLoS One 9:e107001
Gao X et al (2014) Bone marrow mesenchymal stem cells promote the repair of islets from diabetic mice through paracrine actions. Mol Cell Endocrinol 388:41–50
Wu L et al (2011) Trophic effects of mesenchymal stem cells increase chondrocyte proliferation and matrix formation. Tissue Eng Part A 17:1425–1436
Chan JKY, Lam PYP (2013) Human mesenchymal stem cells and their paracrine factors for the treatment of brain tumors. Cancer Gene Ther 20:539–543
Crisan M et al (2008) A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3:301–313
Sacchetti B et al (2007) Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131:324–336
Van Dijk CGM et al (2015) The complex mural cell: pericyte function in health and disease. Int J Cardiol 190:75–89
Lozito TP et al (2013) Three-dimensional osteochondral microtissue to model pathogenesis of osteoarthritis. Stem Cell Res Ther 4:S6
Alexander PG, Gottardi R, Lin H, Lozito TP, Tuan RS (2014) Three-dimensional osteogenic and chondrogenic systems to model osteochondral physiology and degenerative joint diseases. Exp Biol Med (Maywood) 239:1080–1095
Chen AK-L, Reuveny S, Oh SKW (2013) Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction. Biotechnol Adv 31:1032–1046
Sart S, Schneider Y-J, Li Y, Agathos SN (2014) Stem cell bioprocess engineering towards cGMP production and clinical applications. Cytotechnology 66:709–722
Van Wezel AL (1967) Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature 216:64–65
Sart S, Agathos SN, Li Y (2013) Engineering stem cell fate with biochemical and biomechanical properties of microcarriers. Biotechnol Prog 29:1354–1366
Martin Y, Eldardiri M, Lawrence-Watt DJ, Sharpe JR (2011) Microcarriers and their potential in tissue regeneration. Tissue Eng Part B Rev 17:71–80
Bertolo A et al (2015) Injectable microcarriers as human mesenchymal stem cell support and their application for cartilage and degenerated intervertebral disc repair. Eur Cell Mater 29:70–80, discussion 80–81
Sart S, Ma T, Li Y (2013) Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation. Biotechnol Prog 29:143–153
Baxter MA et al (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells Dayt (Ohio) 22:675–682
Sepúlveda JC et al (2014) Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal endotoxemia model. Stem Cells Dayt (Ohio) 32:1865–1877
Dominici M et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317
Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP (1997) Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295–312
Sart S, Schneider Y-J, Agathos SN (2009) Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J Biotechnol 139:291–299
Sart S, Errachid A, Schneider Y-J, Agathos SN (2013) Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors. J Tissue Eng Regen Med 7:537–551
Sart S, Ma T, Li Y (2014) Extracellular matrices decellularized from embryonic stem cells maintained their structure and signaling specificity. Tissue Eng Part A 20:54–66
Sart S, Schneider Y-J, Agathos SN (2010) Influence of culture parameters on ear mesenchymal stem cells expanded on microcarriers. J Biotechnol 150:149–160
Ragni E, Viganò M, Rebulla P, Giordano R, Lazzari L (2013) What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes. J Cell Mol Med 17:168–180
Sart S, Tsai A-C, Li Y, Ma T (2014) Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev 20:365–380
Frith JE, Thomson B, Genever PG (2010) Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods 16:735–749
Sucosky P, Osorio DF, Brown JB, Neitzel GP (2004) Fluid mechanics of a spinner-flask bioreactor. Biotechnol Bioeng 85:34–46
Liovic P, Sutalo ID, Stewart R, Glattauer V, Meagher L (2012) Fluid flow and stresses on microcarriers in spinner flask bioreactors. In: Ninth International Conference on CFD in the Minerals and Process Industries CSIRO, Melbourne, Australia 10–12 December 2012
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Sart, S., Agathos, S.N. (2015). Large-Scale Expansion and Differentiation of Mesenchymal Stem Cells in Microcarrier-Based Stirred Bioreactors. In: Turksen, K. (eds) Bioreactors in Stem Cell Biology. Methods in Molecular Biology, vol 1502. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_314
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DOI: https://doi.org/10.1007/7651_2015_314
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