, Volume 68, Issue 1, pp 45–59 | Cite as

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

  • Priyanka Gupta
  • Mohd-Zulhilmi Ismadi
  • Paul J. Verma
  • Andreas Fouras
  • Sameer Jadhav
  • Jayesh Bellare
  • Kerry Hourigan
Original Research


In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.


Microcarrier Induced pluripotent stem cells Spinner flask 



The authors would like to acknowledge Ms. Joan Clark, Monash Micro Imaging Facility (Monash University), for helping with the SEM imaging, Dr. Trevor Wilson, Medical Genomics Facility, Monash Health and Technology Precinct, for his help in the Array Scan analysis, and Ms. Karla Contreras, Division of Biological Engineering (Monash University) for her assistance. This work was supported by the Australian Research Council Discovery Program (Grant DPDP130100822) and by the Australia India Strategic Research Fund (Grant BF050038).

Conflict of interest

The authors declare that no competing financial interests exist for this work.


  1. Abbasalizadeh S, Larijani MR, Samadian A, Baharvand H (2012) Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor. Tissue Eng Part C Methods 18:831–851. doi: 10.1089/ten.TEC.2012.0161 CrossRefGoogle Scholar
  2. Abranches E, Bekman E, Henrique D, Cabral JM (2007) Expansion of mouse embryonic stem cells on microcarriers. Biotechnol Bioeng 96:1211–1221. doi: 10.1002/bit.21191 CrossRefGoogle Scholar
  3. Alfred R, Radford J, Fan J, Boon K, Krawetz R, Rancourt D, Kallos MS (2011) Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium. Biotechnol Prog 27:811–823. doi: 10.1002/btpr.591 CrossRefGoogle Scholar
  4. Bardy J, Chen AK, Lim YM, Wu S, Wei S, Weiping H, Chan K, Reuveny S, Oh SK (2013) Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells. Tissue Eng Part C Methods 19:166–180. doi: 10.1089/ten.TEC.2012.0146 CrossRefGoogle Scholar
  5. Chen AK, Chen X, Choo AB, Reuveny S, Oh SK (2011) Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Res 7:97–111. doi: 10.1016/j.scr.2011.04.007 CrossRefGoogle Scholar
  6. Cormier JT, Nieden NIZ, Rancourt DE, Kallos MS (2006) Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors. Tissue Eng 12:3233–3245. doi: 10.1089/ten.2006.12.3233 CrossRefGoogle Scholar
  7. Dusting J, Sheridan J, Hourigan K (2006) A fluid dynamics approach to bioreactor design for cell and tissue culture. Biotechnol Bioeng 94:1196–1208. doi: 10.1002/bit.20960 CrossRefGoogle Scholar
  8. Fernandes AM, Fernandes TG, Diogo MM, da Silva CL, Henrique D, Cabral JM (2007) Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system. J Biotechnol 132:227–236. doi: 10.1016/j.jbiotec.2007.05.031 CrossRefGoogle Scholar
  9. Fernandes AM, Marinho PA, Sartore RC, Paulsen BS, Mariante RM, Castilho LR, Rehen SK (2009) Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system. Braz J Med Biol Res 42(6):515–522CrossRefGoogle Scholar
  10. Fluri DA, Tonge PD, Song H, Baptista RP, Shakiba N, Shukla S, Clarke G, Nagy A, Zandstra PW (2012) Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures. Nat Methods 9:509–516. doi: 10.1038/nmeth.1939 CrossRefGoogle Scholar
  11. Fok EY, Zandstra PW (2005) Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 23:1333–1342. doi: 10.1634/stemcells.2005-0112 CrossRefGoogle Scholar
  12. Ismadi MZ, Higgins S, Samarage CR, Paganin D, Hourigan K, Fouras A (2013) Optimisation of a stirred bioreactor through the use of a novel holographic correlation velocimetry flow measurement technique. PLoS ONE 8:e65714. doi: 10.1371/journal.pone.0065714 CrossRefGoogle Scholar
  13. Ismadi ZM, Gupta P, Fouras A, Verma P, Jadhav S, Bellare J, Hourigan K (2014) Flow characterization of spinner flask for induced pluripotent stem cell culture application. Plos One (Submitted)Google Scholar
  14. 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. doi: 10.1089/ten.TEA.2009.0454 CrossRefGoogle Scholar
  15. 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. doi: 10.1089/ten.TEC.2009.0228 CrossRefGoogle Scholar
  16. Leung HW, Chen A, Choo AB, Reuveny S, Oh SK (2011) Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures. Tissue Eng Part C Methods 17:165–172. doi: 10.1089/ten.TEC.2010.0320 CrossRefGoogle Scholar
  17. Lock LT, Tzanakakis ES (2009) Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng Part A 15:2051–2063. doi: 10.1089/ten.tea.2008.0455 CrossRefGoogle Scholar
  18. Marinho PA, Fernandes AM, Cruz JC, Rehen SK, Castilho LR (2010) Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures. Biotechnol Prog 26:548–555. doi: 10.1002/btpr.328 Google Scholar
  19. Marinho PA, Vareschini DT, Gomes IC, Paulsen Bda S, Furtado DR, Castilho Ldos R, Rehen SK (2013) Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novel animal/human-component-free medium. Tissue Eng Part C Methods 19:146–155. doi: 10.1089/ten.TEC.2012.0141 CrossRefGoogle Scholar
  20. Meunier P, Hourigan K (2013) Mixing in a vortex breakdown flow. J Fluid Mech 731:195–222. doi: 10.1017/jfm.2013.226 CrossRefGoogle Scholar
  21. Nie Y, Bergendahl V, Hei DJ, Jones JM, Palecek SP (2009) Scalable culture and cryopreservation of human embryonic stem cells on microcarriers. Biotechnol Prog 25:20–31. doi: 10.1002/btpr.110 CrossRefGoogle Scholar
  22. Oh SK, Chen AK, Mok Y, Chen X, Lim UM, Chin A, Choo AB, Reuveny S (2009) Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res 2:219–230. doi: 10.1016/j.scr.2009.02.005 CrossRefGoogle Scholar
  23. Olmer R, Haase A, Merkert S, Cui W, Palecek J, Ran C, Kirschning A, Scheper T, Glage S, Miller K, Curnow EC, Hayes ES, Martin U (2010) Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem Cell Res 5:51–64. doi: 10.1016/j.scr.2010.03.005 CrossRefGoogle Scholar
  24. Olmer R, Lange A, Selzer S, Kasper C, Haverich A, Martin U, Zweigerdt R (2012) Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors. Tissue Eng Part C Methods 18:772–784. doi: 10.1089/ten.TEC.2011.0717 CrossRefGoogle Scholar
  25. Phillips BW, Horne R, Lay TS, Rust WL, Teck TT, Crook JM (2008) Attachment and growth of human embryonic stem cells on microcarriers. J Biotechnol 138:24–32. doi: 10.1016/j.jbiotec.2008.07.1997 CrossRefGoogle Scholar
  26. Serra M, Brito C, Sousa MF, Jensen J, Tostoes R, Clemente J, Strehl R, Hyllner J, Carrondo MJ, Alves PM (2010) Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J Biotechnol 148:208–215. doi: 10.1016/j.jbiotec.2010.06.015 CrossRefGoogle Scholar
  27. Shafa M, Sjonnesen K, Yamashita A, Liu S, Michalak M, Kallos MS, Rancourt DE (2012) Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors. J Tissue Eng Regen Med 6:462–472. doi: 10.1002/term.450 CrossRefGoogle Scholar
  28. Steiner D, Khaner H, Cohen M, Even-Ram S, Gil Y, Itsykson P, Turetsky T, Idelson M, Aizenman E, Ram R, Berman-Zaken Y, Reubinoff B (2010) Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28:361–364. doi: 10.1038/nbt.1616 CrossRefGoogle Scholar
  29. Storm MP, Orchard CB, Bone HK, Chaudhuri JB, Welham MJ (2010) Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells. Biotechnol Bioeng 107:683–695. doi: 10.1002/bit.22850 CrossRefGoogle Scholar
  30. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. doi: 10.1016/j.cell.2006.07.024 CrossRefGoogle Scholar
  31. Tat PA, Sumer H, Jones KL, Upton K, Verma PJ (2010) The efficient generation of induced pluripotent stem (iPS) cells from adult mouse adipose tissue-derived and neural stem cells. Cell Transplant 19:525–536. doi: 10.3727/096368910X491374 CrossRefGoogle Scholar
  32. Thouas GA, Sheridan J, Hourigan K (2007) A bioreactor model of mouse tumor progression. J Biomed Biotechnol 2007:32754. doi: 10.1155/2007/32754 CrossRefGoogle Scholar
  33. Tielens S, Declercq H, Gorski T, Lippens E, Schacht E, Cornelissen M (2007) Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in vitro study. Biomacromolecules 8:825–832. doi: 10.1021/bm060870u CrossRefGoogle Scholar
  34. Want AJ, Nienow AW, Hewitt CJ, Coopman K (2012) Large-scale expansion and exploitation of pluripotent stem cells for regenerative medicine purposes: beyond the T flask. Regen Med 7:71–84. doi: 10.2217/rme.11.101 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Priyanka Gupta
    • 1
    • 2
    • 3
    • 4
  • Mohd-Zulhilmi Ismadi
    • 4
    • 5
  • Paul J. Verma
    • 4
    • 6
  • Andreas Fouras
    • 5
  • Sameer Jadhav
    • 2
  • Jayesh Bellare
    • 2
  • Kerry Hourigan
    • 4
    • 5
  1. 1.IITB Monash Research AcademyMumbaiIndia
  2. 2.Department of Chemical EngineeringIIT BombayMumbaiIndia
  3. 3.Department of Chemical EngineeringMonash UniversityMelbourneAustralia
  4. 4.Division of Biological EngineeringMonash UniversityMelbourneAustralia
  5. 5.Department of Mechanical and Aerospace EngineeringMonash UniversityMelbourneAustralia
  6. 6.South Australian Research and Development InstituteRosedaleAustralia

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