Skip to main content
SpringerLink
Log in

Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors

  • Protocol
  • First Online:
Bioreactors in Stem Cell Biology

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

Abstract

Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872

    Article  CAS  PubMed  Google Scholar 

  2. Robinton DA, Daley GQ (2012) The promise of induced pluripotent stem cells in research and therapy. Nature 481:295–305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bellin M, Marchetto MC, Gage FH, Mummery CL (2012) Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol 13:713–726

    Article  PubMed  Google Scholar 

  4. Grskovic M, Javaherian A, Strulovici B, Daley GQ (2011) Induced pluripotent stem cells—opportunities for disease modelling and drug discovery. Nat Rev Drug Discov 10:915–929

    CAS  PubMed  Google Scholar 

  5. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM, Jaenisch R (2007) Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318:1920–1923

    Article  CAS  PubMed  Google Scholar 

  6. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Goedel A, Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A, Laugwitz KL (2010) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363:1397–1409

    Article  CAS  PubMed  Google Scholar 

  7. Kirouac DC, Zandstra PW (2008) The systematic production of cells for cell therapies. Cell Stem Cell 3:369–381

    Article  CAS  PubMed  Google Scholar 

  8. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920

    Article  CAS  PubMed  Google Scholar 

  9. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Krawetz R, Taiani JT, Liu S, Meng G, Li X, Kallos MS, Rancourt DE (2010) Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng Part C Methods 16:573–582

    Article  CAS  PubMed  Google Scholar 

  11. Kehoe DE, Jing D, Lock LT, Tzanakakis ES (2010) Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng Part A 16:405–421

    Article  CAS  PubMed  Google Scholar 

  12. dos Santos FF, Andrade PZ, da Silva CL, Cabral JM (2013) Bioreactor design for clinical-grade expansion of stem cells. Biotechnol J 8:644–654

    Article  PubMed  Google Scholar 

  13. Badenes SM, Fernandes TG, Rodrigues CA, Diogo MM, Cabral JM (2015) Scalable expansion of human-induced pluripotent stem cells in xeno-free microcarriers. Methods Mol Biol 1283:23–29

    Article  CAS  PubMed  Google Scholar 

  14. Hunt MM, Meng G, Rancourt DE, Gates ID, Kallos MS (2014) Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters. Tissue Eng Part C Methods 20:76–89

    Article  PubMed  Google Scholar 

  15. Day B, Rancourt DE (2013) Metabolic status of pluripotent cells and exploitation for growth in stirred suspension bioreactors. Biotechnol Genet Eng Rev 29:24–30

    Article  CAS  PubMed  Google Scholar 

  16. Shafa M, Day B, Yamashita A, Meng G, Liu S, Krawetz R, Rancourt DE (2012) Derivation of iPSCs in stirred suspension bioreactors. Nat Methods 9:465–466

    Article  CAS  PubMed  Google Scholar 

  17. Amit M, Chebath J, Margulets V, Laevsky I, Miropolsky Y, Shariki K, Peri M, Blais I, Slutsky G, Revel M, Itskovitz-Eldor J (2010) Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells. Stem Cell Rev 6:248–259

    Article  PubMed  Google Scholar 

  18. Fridley KM, Kinney MA, McDevitt TC (2012) Hydrodynamic modulation of pluripotent stem cells. Stem Cell Res Ther 3:45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gareau T, Lara GG, Shepherd RD, Krawetz R, Rancourt DE, Rinker KD, Kallos MS (2014) Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors. J Tissue Eng Regen Med 8:268–278

    Article  CAS  PubMed  Google Scholar 

  20. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646

    Article  CAS  PubMed  Google Scholar 

  21. Meng G, Liu S, Krawetz R, Chan M, Chernos J, Rancourt DE (2008) A novel method for generating xeno-free human feeder cells for human ES cell culture. Stem Cells Dev 17:413–422

    Article  CAS  PubMed  Google Scholar 

  22. Meng G, Liu S, Li X, Krawetz R, Rancourt DE (2010) Extra-cellular matrix isolated from foreskin fibroblasts supports long term xeno-free human embryonic stem cell culture. Stem Cells Dev 19:547–556

    Article  CAS  PubMed  Google Scholar 

  23. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Muguruma K, Sasai Y (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686

    Article  CAS  PubMed  Google Scholar 

  24. Krawetz RJ, Li X, Rancourt DE (2009) Human embryonic stem cells: caught between a ROCK inhibitor and a hard place. Bioessays 31:336–343

    Article  CAS  PubMed  Google Scholar 

  25. Heng BC, Heinimann K, Miny P, Iezzi G, Glatz K, Scherberich A, Zulewski H, Fussenegger M (2013) mRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient. Metab Eng 18:9–24

    Article  CAS  PubMed  Google Scholar 

  26. O’Connor MD, Kardel MD, Eaves CJ (2011) Functional assays for human embryonic stem cell pluripotency. Methods Mol Biol 690:67–80

    Article  PubMed  Google Scholar 

  27. Meisner LF, Johnson JA (2008) Protocols for cytogenetic studies of human embryonic stem cells. Methods 45:133–141

    Article  CAS  PubMed  Google Scholar 

  28. Emre N, Vidal JG, Elia J, O’Connor ED, Paramban RI, Hefferan MP, Navarro R, Goldberg DS, Varki NM, Marsala M, Carson CT (2010) The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLoS One 5, e12148

    Article  PubMed  PubMed Central  Google Scholar 

  29. Prigione A, Fauler B, Lurz R, Lehrach H, Adjaye J (2010) The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells 28:721–733

    Article  CAS  PubMed  Google Scholar 

  30. Varum S, Rodrigues AS, Moura MB, Momcilovic O, Easley CA, Ramalho-Santos J, Van Houten B, Schatten G (2011) Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS One 6, e20914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Armstrong L, Tilgner K, Saretzki G, Atkinson SP, Stojkovic M, Moreno R, Przyborski S, Lako M (2010) Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells 28:661–673

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada, AB T2N 1N4

    Walaa Almutawaa, Leili Rohani & Derrick E. Rancourt

Authors
  1. Walaa Almutawaa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leili Rohani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Derrick E. Rancourt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Derrick E. Rancourt .

Editor information

Editors and Affiliations

  1. Ottawa Hospital Research Institute , Ottawa, Ontario, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Almutawaa, W., Rohani, L., Rancourt, D.E. (2015). Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension 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_311

Download citation

  • DOI: https://doi.org/10.1007/7651_2015_311

  • Published:

  • 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

Publish with us

Policies and ethics

Search

Navigation