Bioprocess and Biosystems Engineering

, Volume 27, Issue 6, pp 381–387 | Cite as

Perfusion cultures of human embryonic stem cells

  • Wey Jia Fong
  • Heng Liang Tan
  • Andre Choo
  • Steve Kah Weng OhEmail author
Original papers


Human embryonic stem cells (hESC) are self-renewing pluripotent cells capable of differentiating into cells representative of all three embryonic germ layers. Hence, they hold great potential for regenerative medicine. However, significant cell numbers are required to fulfill their potential therapeutic applications. In this study, perfusion with supplemented conditioned media (SCM), produced by mouse embryonic fibroblasts (MEF), was adopted to improve cell densities of hESC cultures. Perfusion enhanced hESC numbers by 70% compared to static conditions, on both organ culture dish (OCD) and petridish cultures. All cultures maintained healthy expression of the pluripotent marker, Oct-4 transcription factor. In vivo, perfused hESC formed teratomas in severe combined immunodeficiency (SCID) mice models that represent the three embryonic germ layers. When SCM was produced with lower concentrations of MEF, hESC densities and Oct-4 levels were reduced. Hence, perfusion with SCM is a potential feeding method for scale-up production of hESC.


Perfusion Human embryonic stem cell Conditioned media Pluripotency Oct-4 



Human embryonic stem cell


Severe combined immunodeficiency


Conditioned media


Supplemented conditioned media


Mouse embryonic fibroblast


Polyethylene terepthalate


Monoclonal antibody


Mouse embryonic stem cell


Fluorescence activated cell sorter



This work was generously supported by the Agency for Science Technology and Research (A*STAR), Singapore. We thank Angela Chin and Jayanthi Padmanabhan very much for their help with teratoma sectioning and FACS. We thank Dr. Victor Wong for critical review of this manuscript.


  1. 1.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147Google Scholar
  2. 2.
    Odorico JS, Kaufman DS, Thomson JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19(3):193–204Google Scholar
  3. 3.
    Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J (2004) Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22(3):265–274Google Scholar
  4. 4.
    Gerecht-Nir S, Fishman B, Itskovitz-Eldor J (2004) Cardiovascular potential of embryonic stem cells. Anat Rec Part A 276A(1):58–65Google Scholar
  5. 5.
    Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS (2001) Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172(2):383–397Google Scholar
  6. 6.
    Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68(6):2150–2156Google Scholar
  7. 7.
    Xu C, Inokuma SM, Denham J, Golds K, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19(10):971–979Google Scholar
  8. 8.
    Amit M, Shariki C, Margulets V, Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70(3):837–845Google Scholar
  9. 9.
    Dang SM, Gerecht-Nir S, Chen J, Itskovitz-Eldor J, Zandstra PW (2004) Controlled, scalable embryonic stem cells differentiation culture. Stem Cells 22(3):275–282Google Scholar
  10. 10.
    Gerecht-Nir S, Cohen S, Itskovitz-Eldor J (2004) Bioreactor cultivation enhances the efficiency of human embryoid body formation and differentiation. Biotechnol Bioeng 86(5):493–502Google Scholar
  11. 11.
    Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12(2):180–187Google Scholar
  12. 12.
    Choo AB, Padmanabhan J, Chin ACP, Oh SKW (2004) Expansion of pluripotent human embryonic stem cells on human feeders. Biotechnol Bioeng 88:321–332Google Scholar
  13. 13.
    Robertson EJ (1987) Teratocarcinomas and embryonic stem cells. IRL Press, OxfordGoogle Scholar
  14. 14.
    Ryan EA, Lakey JR, Rajotte RV, Korbutt GS (2001) Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50:710–719Google Scholar
  15. 15.
    Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379–391Google Scholar
  16. 16.
    Martin MJ, Muotri A, Gage F, Varki A (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 11:228–232Google Scholar
  17. 17.
    Yang JD, Angelillo Yale, Mina Chaudhry, Cindy Goldenberg, DM Goldenberg (2000) Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. Biotechnol Bioeng 69(1):74–82Google Scholar
  18. 18.
    Li Y, Lasky LC, Yang ST, Kniss DA (2003) Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix. Cytotechnology 41:23–35Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Wey Jia Fong
    • 1
  • Heng Liang Tan
    • 1
  • Andre Choo
    • 1
  • Steve Kah Weng Oh
    • 1
    Email author
  1. 1.Bioprocessing Technology InstituteCentrosSingapore

Personalised recommendations