Skip to main content

Expansion of Pluripotent Stem Cells in Defined, Xeno-Free Culture System

  • Protocol
  • First Online:
Book cover Human Embryonic and Induced Pluripotent Stem Cells

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 1551 Accesses

Abstract

The hallmark of pluripotent stem cells is their nearly unlimited self-renewal capacity, and their potential to differentiate into a diverse range of specialized cell types. These unique properties make stem cells important research tools, in vitro models for pharmaceutical testing, and an attractive source of various cell types for regenerative therapies. For stem cell technology to be fully exploited, however, culture systems must be improved to enable large-scale production, and safety ensured. Most stem cell culture systems developed to date utilize undefined, xenogeneic products that pose a risk of a severe immune response and the transmission of infections. In this chapter, we describe a robust method for the expansion of pluripotent stem cells in defined and xeno-free culture conditions. Both mechanical and single-cell enzymatic passaging can be applied with this method. This procedure can be adopted for both basic research purposes and clinical applications.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
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

References

  1. Peerani, R., Rao, B.M., Bauwens, C., Yin, T., Wood, G.A., Nagy, A., Kumacheva, E., Zandstra, P.W. (2007) Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J 26, 4744–4755.

    Article  PubMed  CAS  Google Scholar 

  2. Thomson, J., Itskovitz-Eldor, J., Shapiro, S., Waknitz, M., Swiergiel, J., Marshall, V., Jones, J. (1998) Embryonic stem cell lines derived from human blastocysts Science 282, 1145–7.

    Article  PubMed  CAS  Google Scholar 

  3. Skottman, H., Stromberg, A.M., Matilainen, E., Inzunza, J., Hovatta, O., Lahesmaa, R. (2006) Unique gene expression signature by human embryonic stem cells cultured under serum-free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage. Stem Cells 24, 151–67.

    Article  PubMed  CAS  Google Scholar 

  4. Bradley, J.A., Bolton, E.M., Pedersen, R.A. (2002) Stem cell medicine encounters the immune system. Nat Rev Immunol 2, 859–71.

    Article  PubMed  CAS  Google Scholar 

  5. Selvaggi, T.A., Walker, R.E., Fleisher, T.A. (1997) Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood 89, 776–9.

    PubMed  CAS  Google Scholar 

  6. Martin, M., Muotri, A., Gage, F., Varki, A. (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat med 11, 228–232.

    Article  PubMed  CAS  Google Scholar 

  7. Heiskanen, A., Satomaa, T., Tiitinen, S., Laitinen, A., Mannelin, S., Impola, U., Mikkola, M., Olsson, C., Miller-Podraza, H., Blomqvist, M., Olonen, A., Salo, H., Lehenkari, P., Tuuri, T., Otonkoski, T., Natunen, J., Saarinen, J., Laine, J. (2007) N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells 25, 197–202.

    Article  PubMed  CAS  Google Scholar 

  8. Sakamoto, N., Tsuji, K., Muul, L.M., Lawler, A.M., Petricoin, E.F., Candotti, F., Metcalf, J.A., Tavel, J.A., Lane, H.C., Urba, W.J., Fox, B.A., Varki, A., Lunney, J.K., Rosenberg, A.S. (2007) Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans. Blood 110, 501–508.

    Article  PubMed  CAS  Google Scholar 

  9. Hisamatsu-Sakamoto, M., Sakamoto, N., Rosenberg, A.S. (2008) Embryonic stem cells cultured in serum-free medium acquire bovine apolipoprotein B-100 from feeder cell layers and serum replacement medium. Stem Cells 26, 72–8.

    Article  PubMed  CAS  Google Scholar 

  10. Cobo F, Stacey G, Hunt C, Cabrera C, Nieto A, Montes R, Cortes J, Catalina P, Barnie A, Concha A (2005): Microbiological control in stem cell banks: approaches to standardisation. Appl Microbiol Biotechnol 68, 456–66.

    Article  PubMed  CAS  Google Scholar 

  11. Rajala, K., Lindroos, B., Hussein, S.M., Lappalainen, R.S., Pekkanen-Mattila, M., Inzunza, J., Rozell, B., Miettinen, S., Narkilahti, S., Kerkelä, E., Aalto-Setälä, K., Otonkoski, T., Suuronen, R., Hovatta, O., Skottman, H. (2010) A Defined and Xeno-free Culture Method Enabling the Establishment of Clinical-grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells. Plos One 5, e10246.

    Article  PubMed  Google Scholar 

  12. Ellerström, C., Strehl, R., Noaksson, K., Hyllner, J., Semb, H. (2007) Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation. Stem Cells 25, 1690–6.

    Article  PubMed  Google Scholar 

  13. Hovatta, O., Mikkola, M., Gertow, M., Stromberg, A., Inzunza, J., Hreinsson, J., Rozell, B., Blennow, E., Andang, M., Ahrlund-Richter, L.(2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 18, 14041409.

    Article  PubMed  Google Scholar 

  14. 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, 21502156.

    Article  PubMed  CAS  Google Scholar 

  15. Prasad, S., Czepiel, M., Cetinkaya, C., Smigielska, K., Weli, S., Lysdahl, H., Gabrielsen, A., Petersen, K., Ehlers, N., Fink, T., Minger, S., Zachar, V. (2009) Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif 1, 63–74.

    Article  Google Scholar 

  16. Westfall, S., Sachdev, S., Das, P., Hearne, L., Hannink, M., Roberts, R., Ezashi, T. (2008) Identification of oxygen-sensitive transcriptional programs in human embryonic stem cells. Stem cells dev 5, 869–881.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kristiina Rajala .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Humana Press

About this protocol

Cite this protocol

Rajala, K. (2011). Expansion of Pluripotent Stem Cells in Defined, Xeno-Free Culture System. In: Ye, K., Jin, S. (eds) Human Embryonic and Induced Pluripotent Stem Cells. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1007/978-1-61779-267-0_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-267-0_6

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-266-3

  • Online ISBN: 978-1-61779-267-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics