Skip to main content

Isolation, Expansion, and Immortalization of Human Adipose-Derived Mesenchymal Stromal Cells from Biopsies and Liposuction Specimens

  • Protocol
  • First Online:
Mesenchymal Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1416))

Abstract

Human adipose tissue has proven to be an abundant, accessible, and rich source of adult mesenchymal stromal cells, suitable for tissue engineering and regenerative medicine. However, a major complication in fully investigating these cells may derive from their limited life span.

Although methods to isolate, expand, and immortalize these cells have been widely reported in the literature, exhaustive explanations on the problems that can be encountered during these processes and how these can be solved have never been described. It is of fundamental importance to follow a common protocol to achieve reliable and reproducible results. Here, we describe a protocol to isolate and expand human adipose stromal cells from specimens obtained from tissue biopsies and liposuction surgical interventions. Finally, we broadly describe the cell immortalization technique, and particular attention is paid to some of the apparently “secondary” aspects.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
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. Zuk PA, Zhu M, Ashjian P et al (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279–4295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Friedenstein AJ, Petrakova KV, Kurolesova AI et al (1968) Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissue. Transplantation 6:230–247

    Article  CAS  PubMed  Google Scholar 

  3. Alessandri G, Pagano S, Bez A et al (2004) Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet 364:1872–1883

    Article  CAS  PubMed  Google Scholar 

  4. Toma JG, McKenzie IA, Bagli D et al (2005) Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells 23:727–737

    Article  CAS  PubMed  Google Scholar 

  5. Seo BM, Miura M, Gronthos S et al (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–155

    Article  CAS  PubMed  Google Scholar 

  6. Gimble J, Guilak F (2003) Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5:362–369

    Article  PubMed  Google Scholar 

  7. Bourin P, Bunnell BA, Casteilla L et al (2013) Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy. Cytotherapy 15:641–648

    Article  PubMed  PubMed Central  Google Scholar 

  8. Mizuno H, Tobita M, Uysal AC (2012) Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells 30:804–810

    Article  CAS  PubMed  Google Scholar 

  9. Zuk PA, Zhu M, Muzuno H et al (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228

    Article  CAS  PubMed  Google Scholar 

  10. Astori G, Vignati F, Bardelli S et al (2007) “In vitro” and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. J Transl Med 5:55

    Article  PubMed  PubMed Central  Google Scholar 

  11. Li H, Zimmerlin L, Marra KG et al (2011) Adipogenic potential of adipose stem cell subpopulations. Plastic Recon Surg 128:663–672

    Article  CAS  Google Scholar 

  12. Erickson GR, Gimble JM, Franklin DM et al (2002) Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 290:763–769

    Article  CAS  PubMed  Google Scholar 

  13. Serrano M, Blasco MA (2001) Putting the stress on senescence. Curr Opin Cell Biol 13:748–753

    Article  CAS  PubMed  Google Scholar 

  14. Campisi J (2001) Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 11:S27–S31

    Article  CAS  PubMed  Google Scholar 

  15. Balducci L, Blasi A, Saldarelli M et al (2014) Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors. Stem Cell Res Ther 5:63

    Article  PubMed  PubMed Central  Google Scholar 

  16. Clynes M (2014) Cell culture models for study of differentiated adipose cells. Commentary Stem Cell Res Ther 5:137

    Article  PubMed  Google Scholar 

  17. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M et al (2006) Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 8:166–177

    Article  CAS  PubMed  Google Scholar 

  18. Yoshimura K, Shigeura T, Matsumoto D et al (2006) Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 208:64–76

    Article  CAS  PubMed  Google Scholar 

  19. Bunnell BA, Flaat M, Gagliardi C et al (2008) Adipose-derived stem cells: isolation, expansion and differentiation. Methods 45:115–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Spees JL, Gregory CA, Singh H et al (2004) Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 9:747–756

    Article  CAS  PubMed  Google Scholar 

  21. Shay JW, Wright WE, Werbin H (1991) Defining the molecular mechanisms of human cell immortalization. Biochim Biophys Acta 1072:1–7

    CAS  PubMed  Google Scholar 

  22. Halvorsen TL, Leibowitz G, Levine F (1999) Telomerase activity is sufficient to allow transformed cells to escape from crisis. Mol Cell Biol 19:1864–1870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zwerschke W, Jansen-Durr P (2000) Cell transformation by the E7 oncoprotein of human papillomavirus type 16: interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 78:1–29

    Article  CAS  PubMed  Google Scholar 

  24. Kiyono T, Foster SA, Koop JI et al (1998) Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396:84–88

    Article  CAS  PubMed  Google Scholar 

  25. O’Hare MJ, Bond J, Clarke C et al (2001) Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells. Proc Natl Acad Sci U S A 98:646–651

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luigi Balducci .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Balducci, L., Alessandri, G. (2016). Isolation, Expansion, and Immortalization of Human Adipose-Derived Mesenchymal Stromal Cells from Biopsies and Liposuction Specimens. In: Gnecchi, M. (eds) Mesenchymal Stem Cells. Methods in Molecular Biology, vol 1416. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3584-0_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3584-0_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3582-6

  • Online ISBN: 978-1-4939-3584-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics