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Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor

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Bioreactors in Stem Cell Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1502))

Abstract

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface–volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

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References

  1. Daley GQ, Scadden DT (2008) Prospects for stem cell-based therapy. Cell 132:544–548

    Article  PubMed  CAS  Google Scholar 

  2. Koga H, Engebretsen L, Brinchmann JE, Muneta T, Sekiya I (2009) Mesenchymal stem cell-based therapy for cartilage repair: a review. Knee Surg Sports Traumatol Arthrosc 17:1289–1297

    Article  PubMed  Google Scholar 

  3. Meijer GJ, de Bruijn JD, Koole R, van Blitterswijk CA (2007) Cell-based bone tissue engineering. PLoS Med 4:e9

    Article  PubMed  PubMed Central  Google Scholar 

  4. 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

    Article  PubMed  CAS  Google Scholar 

  5. Jung S, Panchalingam KM, Wuerth RD, Rosenberg L, Behie LA (2012) Large-scale production of human mesenchymal stem cells for clinical applications. Biotechnol Appl Biochem 59:106–120

    Article  PubMed  CAS  Google Scholar 

  6. Merten O-W (2015) Advances in cell culture: anchorage dependence. Philos Trans R Soc Lond B Biol Sci 370:20140040

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Malda J, Frondoza CG (2006) Microcarriers in the engineering of cartilage and bone. Trends Biotechnol 24:299–304

    Article  PubMed  CAS  Google Scholar 

  8. Ma T, Tsai A-C, Liu Y (2015) Biomanufacturing of human mesenchymal stem cells in cell therapy: influence of microenvironment on scalable expansion in bioreactors. Biochem Eng J. doi:10.1016/j.bej.2015.07.014

    Google Scholar 

  9. Rowley J, Abraham E, Campbell A, Brandwein H, Oh S (2012) Meeting lot-size challenges of manufacturing adherent cells for therapy. BioProcess Int 10:7

    Google Scholar 

  10. Kehoe D, Schnitzler A, Simler J, DiLeo A, Ball A (2012) Scale-up of human mesenchymal stem cells on microcarriers in suspension in a single-use bioreactor. BioPharm Int 25:28–38

    CAS  Google Scholar 

  11. Jing D, Sunil N, Punreddy S, Aysola M, Kehoe D, Murrel J, Rook M, Niss K (2013) Growth kinetics of human mesenchymal stem cells in a 3-L single-use, stirred-tank bioreactor. BioPharm Int 26:28–38

    CAS  Google Scholar 

  12. Rafiq QA, Brosnan KM, Coopman K, Nienow AW, Hewitt CJ (2013) Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett 35:1233–1245

    Article  PubMed  CAS  Google Scholar 

  13. van Eikenhorst G, Thomassen YE, van der Pol LA, Bakker WA (2014) Assessment of mass transfer and mixing in rigid lab-scale disposable bioreactors at low power input levels. Biotechnol Prog 30:1269–1276

    Article  PubMed  CAS  Google Scholar 

  14. Tsai A-C, Liu Y, Ma T (2015) Expansion of human mesenchymal stem cells in fibrous bed bioreactor. Biochem Eng J. doi:10.1016/j.bej.2015.09.002

    Google Scholar 

  15. Ismadi M-Z, Hourigan K, Fouras A (2014) Experimental characterisation of fluid mechanics in a spinner flask bioreactor. Processes 2:753–772

    Article  Google Scholar 

  16. Liovic P, Šutalo ID, Stewart R, Glattauer V, Meagher L (2012) Fluid flow and stresses on microcarriers in spinner flask bioreactors. In: Proceedings of the 9th international conference on CFD in the minerals and process industries

    Google Scholar 

  17. Scaglione S, Wendt D, Miggino S, Papadimitropoulos A, Fato M, Quarto R, Martin I (2008) Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system. J Biomed Mater Res A 86:411–419

    Article  PubMed  CAS  Google Scholar 

  18. Leung HW, Chen A, Choo AB, Reuveny S, Oh SK (2010) Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures. Tissue Eng Part C Methods 17:165–172

    Article  PubMed  CAS  Google Scholar 

  19. Yi W, Sun Y, Wei X, Gu C, Dong X, Kang X, Guo S, Dou K (2010) Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress. Mol Cell Biochem 341:9–16

    Article  PubMed  CAS  Google Scholar 

  20. Zhao F, Chella R, Ma T (2007) Effects of shear stress on 3-D human mesenchymal stem cell construct development in a perfusion bioreactor system: experiments and hydrodynamic modeling. Biotechnol Bioeng 96:584–595

    Article  PubMed  CAS  Google Scholar 

  21. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  PubMed  CAS  Google Scholar 

  22. 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

    Article  PubMed  CAS  Google Scholar 

  23. Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J (2007) A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog 23:187–193

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Teng Ma Ph.D. .

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© 2016 Springer Science+Business Media New York

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Tsai, AC., Ma, T. (2016). Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor. 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_2016_338

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  • DOI: https://doi.org/10.1007/7651_2016_338

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6476-5

  • Online ISBN: 978-1-4939-6478-9

  • eBook Packages: Springer Protocols

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