Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors

  • Walaa Almutawaa
  • Leili Rohani
  • Derrick E. RancourtEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1502)


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.


Induced pluripotent stem cells Bioreactor Pluripotency Energy metabolism 


  1. 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 131:861–872CrossRefPubMedGoogle Scholar
  2. 2.
    Robinton DA, Daley GQ (2012) The promise of induced pluripotent stem cells in research and therapy. Nature 481:295–305CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bellin M, Marchetto MC, Gage FH, Mummery CL (2012) Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol 13:713–726CrossRefPubMedGoogle Scholar
  4. 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 10:915–929PubMedGoogle Scholar
  5. 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 318:1920–1923CrossRefPubMedGoogle Scholar
  6. 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 363:1397–1409CrossRefPubMedGoogle Scholar
  7. 7.
    Kirouac DC, Zandstra PW (2008) The systematic production of cells for cell therapies. Cell Stem Cell 3:369–381CrossRefPubMedGoogle Scholar
  8. 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 318:1917–1920CrossRefPubMedGoogle Scholar
  9. 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 8:424–429CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 16:573–582CrossRefPubMedGoogle Scholar
  11. 11.
    Kehoe DE, Jing D, Lock LT, Tzanakakis ES (2010) Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng Part A 16:405–421CrossRefPubMedGoogle Scholar
  12. 12.
    dos Santos FF, Andrade PZ, da Silva CL, Cabral JM (2013) Bioreactor design for clinical-grade expansion of stem cells. Biotechnol J 8:644–654CrossRefPubMedGoogle Scholar
  13. 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 1283:23–29CrossRefPubMedGoogle Scholar
  14. 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 20:76–89CrossRefPubMedGoogle Scholar
  15. 15.
    Day B, Rancourt DE (2013) Metabolic status of pluripotent cells and exploitation for growth in stirred suspension bioreactors. Biotechnol Genet Eng Rev 29:24–30CrossRefPubMedGoogle Scholar
  16. 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 9:465–466CrossRefPubMedGoogle Scholar
  17. 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 6:248–259CrossRefPubMedGoogle Scholar
  18. 18.
    Fridley KM, Kinney MA, McDevitt TC (2012) Hydrodynamic modulation of pluripotent stem cells. Stem Cell Res Ther 3:45CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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 8:268–278CrossRefPubMedGoogle Scholar
  20. 20.
    Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646CrossRefPubMedGoogle Scholar
  21. 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 17:413–422CrossRefPubMedGoogle Scholar
  22. 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 19:547–556CrossRefPubMedGoogle Scholar
  23. 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 25:681–686CrossRefPubMedGoogle Scholar
  24. 24.
    Krawetz RJ, Li X, Rancourt DE (2009) Human embryonic stem cells: caught between a ROCK inhibitor and a hard place. Bioessays 31:336–343CrossRefPubMedGoogle Scholar
  25. 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 18:9–24CrossRefPubMedGoogle Scholar
  26. 26.
    O’Connor MD, Kardel MD, Eaves CJ (2011) Functional assays for human embryonic stem cell pluripotency. Methods Mol Biol 690:67–80CrossRefPubMedGoogle Scholar
  27. 27.
    Meisner LF, Johnson JA (2008) Protocols for cytogenetic studies of human embryonic stem cells. Methods 45:133–141CrossRefPubMedGoogle Scholar
  28. 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, e12148CrossRefPubMedPubMedCentralGoogle Scholar
  29. 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 28:721–733CrossRefPubMedGoogle Scholar
  30. 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, e20914CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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 28:661–673CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Walaa Almutawaa
    • 1
  • Leili Rohani
    • 1
  • Derrick E. Rancourt
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of CalgaryCalgaryCanada

Personalised recommendations