Mesenchymal Stem Cell Assays and Applications pp 175-188

Part of the Methods in Molecular Biology book series (MIMB, volume 698) | Cite as

Dynamic Expansion Culture for Mesenchymal Stem Cells

  • Hicham Majd
  • Thomas M. Quinn
  • Pierre-Jean Wipff
  • Boris Hinz
Protocol

Abstract

To be applied in sufficient numbers for regenerative medicine, primary mesenchymal stem cells (MSCs) need to be amplified in culture. Standard cell culture involves regular passing because MSC proliferation in size-limited culture vessels stagnates due to contact inhibition of growth. The use of harmful enzymes for passaging and the mechanical properties of standard culture vessels change the MSC phenotype. Initially, fast growing multipotent and regenerative MSCs will turn into slowly growing cells with reduced multipotency and fibrotic character. We here describe an innovative culture system that maintains overall constant cell densities which are near-optimal for proliferation, while preventing contact-inhibition of cell growth. This is achieved by dynamically enlarging a novel highly elastic culture dish using a motorized mechanical device and adapting the culture surface to the increasing cell numbers. Dynamic MSC culture expansion reduces the number of enzymatic passages by a factor of 3 and delivers higher MSC yields than conventional culture. On the expanded culture surface, MSCs maintain stem cell characteristics and high growth rates over months and are still inducible to follow different lineages thereafter.

Key words

Highly elastic silicone rubber MSC culture Passaging Surface functionalization Fibrosis 

References

  1. 1.
    Segers, V.F. and Lee, R.T. (2008) Stem-cell therapy for cardiac disease. Nature 451, 937–942.PubMedCrossRefGoogle Scholar
  2. 2.
    Caplan, A.I. (2005) Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng 11, 1198–1211.PubMedCrossRefGoogle Scholar
  3. 3.
    Braccini, A., Wendt, D., Jaquiery, C., Jakob, M., Heberer, M., Kenins, L., Wodnar-Filipowicz, A., Quarto, R., and Martin, I. (2005) Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts. Stem Cells 23, 1066–1072.PubMedCrossRefGoogle Scholar
  4. 4.
    Caplan, A.I. (2007) Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 213, 341–347.PubMedCrossRefGoogle Scholar
  5. 5.
    Giordano, A., Galderisi, U., and Marino, I.R. (2007) From the laboratory bench to the patient’s bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol 211, 27–35.PubMedCrossRefGoogle Scholar
  6. 6.
    Javazon, E.H., Beggs, K.J., and Flake, A.W. (2004) Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 32, 414–425.PubMedCrossRefGoogle Scholar
  7. 7.
    Bruder, S.P., Jaiswal, N., and Haynesworth, S.E. (1997) Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 64, 278–294.PubMedCrossRefGoogle Scholar
  8. 8.
    Vacanti, V., Kong, E., Suzuki, G., Sato, K., Canty, J.M., and Lee, T. (2005) Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture. J Cell Physiol 205, 194–201.PubMedCrossRefGoogle Scholar
  9. 9.
    Kinner, B., Zaleskas, J.M., and Spector, M. (2002) Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 278, 72–83.PubMedCrossRefGoogle Scholar
  10. 10.
    Hinz, B. (2010), The myofibroblast – friend or foe in regenerative medicine ? In Regenerative medicine and biomaterials for the repair of connective tissues, C.A.J. Ralphs, Editor. Woodhead: Cambridge.Google Scholar
  11. 11.
    Galmiche, M.C., Koteliansky, V.E., Briere, J., Herve, P., and Charbord, P. (1993) Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 82, 66–76.PubMedGoogle Scholar
  12. 12.
    Breitbach, M., Bostani, T., Roell, W., Xia, Y., Dewald, O., Nygren, J.M., Fries, J.W., Tiemann, K., Bohlen, H., Hescheler, J., Welz, A., Bloch, W., Jacobsen, S.E., and Fleischmann, B.K. (2007) Potential risks of bone marrow cell transplantation into infarcted hearts. Blood 110, 1362–1369.PubMedCrossRefGoogle Scholar
  13. 13.
    Ortiz, L.A., Gambelli, F., McBride, C., Gaupp, D., Baddoo, M., Kaminski, N., and Phinney, D.G. (2003) Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A 100, 8407–8411.PubMedCrossRefGoogle Scholar
  14. 14.
    Yan, X., Liu, Y., Han, Q., Jia, M., Liao, L., Qi, M., and Zhao, R.C. (2007) Injured microen­vironment directly guides the differentiation of engrafted Flk-1(+) mesenchymal stem cell in lung. Exp Hematol 35, 1466–1475.PubMedCrossRefGoogle Scholar
  15. 15.
    Ninichuk, V., Gross, O., Segerer, S., Hoffmann, R., Radomska, E., Buchstaller, A., Huss, R., Akis, N., Schlondorff, D., and Anders, H.J. (2006) Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice. Kidney Int 70, 121–129.PubMedCrossRefGoogle Scholar
  16. 16.
    di Bonzo, L.V., Ferrero, I., Cravanzola, C., Mareschi, K., Rustichell, D., Novo, E., Sanavio, F., Cannito, S., Zamara, E., Bertero, M., Davit, A., Francica, S., Novelli, F., Colombatto, S., Fagioli, F., and Parola, M. (2008) Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut 57, 223–231.PubMedCrossRefGoogle Scholar
  17. 17.
    Eisenstein, M. (2006) Thinking outside the dish. Nat Methods 3, 1035–1043.CrossRefGoogle Scholar
  18. 18.
    Martin, I., Wendt, D., and Heberer, M. (2004) The role of bioreactors in tissue engineering. Trends Biotechnol 22, 80–86.PubMedCrossRefGoogle Scholar
  19. 19.
    Majd, H., Wipff, P.J., Buscemi, L., Bueno, M., Vonwil, D., Quinn, T.M., and Hinz, B. (2009) A novel method of dynamic culture surface expansion improves mesenchymal stem cell proliferation and phenotype. Stem Cells 27, 200–209.PubMedCrossRefGoogle Scholar
  20. 20.
    Tan, W. and Desai, T.A. (2003) Microfluidic patterning of cells in extracellular matrix biopolymers: effects of channel size, cell type, and matrix composition on pattern integrity. Tissue Eng 9, 255–267.PubMedCrossRefGoogle Scholar
  21. 21.
    Wipff, P.J., Majd, H., Acharya, C., Buscemi, L., Meister, J.J., and Hinz, B. (2009) The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications. Biomaterials 30, 1781–1789.PubMedCrossRefGoogle Scholar
  22. 22.
    Simmons, C.A., Matlis, S., Thornton, A.J., Chen, S., Wang, C.Y., and Mooney, D.J. (2003) Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech 36, 1087–1096.PubMedCrossRefGoogle Scholar
  23. 23.
    Friedl, G., Schmidt, H., Rehak, I., Kostner, G., Schauenstein, K., and Windhager, R. (2007) Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro. Osteoarthritis Cartilage 15, 1293–1300.PubMedCrossRefGoogle Scholar
  24. 24.
    Kurpinski, K., Park, J., Thakar, R.G., and Li, S. (2006) Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain. Mol Cell Biomech 3, 21–34.PubMedGoogle Scholar
  25. 25.
    Frank, O., Heim, M., Jakob, M., Barbero, A., Schafer, D., Bendik, I., Dick, W., Heberer, M., and Martin, I. (2002) Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro. J Cell Biochem 85, 737–746.PubMedCrossRefGoogle Scholar
  26. 26.
    Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.PubMedCrossRefGoogle Scholar
  27. 27.
    Smith-Clerc, J. and Hinz, B. (2010) Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells. Methods Mol Biol 611, 43–57.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hicham Majd
  • Thomas M. Quinn
  • Pierre-Jean Wipff
  • Boris Hinz
    • 1
  1. 1.Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of DentistryUniversity of TorontoTorontoCanada

Personalised recommendations