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Standardized Isolation of Human Mesenchymal Stromal Cells with Red Blood Cell Lysis

  • Patrick Horn
  • Simone Bork
  • Wolfgang Wagner
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 698)

Abstract

Human mesenchymal stromal cells (MSC) raise high hopes for tissue engineering and therapeutic ­applications. So far, it is not possible to isolate pure fractions from bone marrow and therefore MSC cell preparations notoriously represent heterogeneous mixtures of different cell types. The composition of ­subpopulations can already be affected by the initial steps of cell preparation. Usually, isolation of MSC involves density fractionation to separate the mononuclear cells (MNCs) from erythrocytes and ­granulocytes. However, this method is difficult to standardize especially under GMP conditions. Here, we describe an alternative approach for isolation of human MSC based on red blood cell (RBC) lysis with ammonium chloride. This results in a slightly higher number of fibroblastic colony forming units (CFU-F), whereas morphological analysis of the CFU-F reveals the same heterogeneous composition of MSC cultures indicating that the proportion of subpopulations is not affected by RBC lysis. Immunophenotype (CD73+, CD90+, CD105+, CD31, CD34, CD45), adipogenic, and osteogenic differentiation potential of MSC were also similar with both methods. In conclusion, RBC lysis comprises an efficient method for the isolation of human MSC from bone marrow aspirate. This technique is faster and can be standardized more easily for clinical application of MSC.

Key words

Mesenchymal stromal cells Mesenchymal stem cells Isolation Red blood cell lysis Density gradient centrifugation Ammonium chloride CFU-F 

Notes

Acknowledgments

We like to thank Anke Diehlmann for assistance in MSC culture. This work was supported by the German Ministry of Education and Research (BMBF) within the supporting program “cell based regenerative medicine” (START-MSC and CB-HERMES), the German Research Foundation DFG (HO 914/7-1), the Joachim Siebeneicher-Stiftung, Germany, and the Academy of Sciences and Humanities, Heidelberg (WIN-Kolleg) and the Stem Cell Network North Rhine Westphalia.

References

  1. 1.
    Reyes M., Lund T., Lenvik T., Aguiar D., Koodie L., Verfaillie C. M. (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98, 2615–25.PubMedCrossRefGoogle Scholar
  2. 2.
    Jiang Y., Jahagirdar B. N., Reinhardt R. L. et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Petersen B. E., Bowen W. C., Patrene K. D. et al. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284, 1168–70.PubMedCrossRefGoogle Scholar
  4. 4.
    Schwartz R. E., Reyes M., Koodie L. et al. (2002) Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109, 1291–302.PubMedGoogle Scholar
  5. 5.
    Dominici M., Le Blanc K., Mueller I. et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–7.Google Scholar
  6. 6.
    Wagner W., Wein F., Seckinger A. et al. (2005) Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 33, 1402–16.PubMedCrossRefGoogle Scholar
  7. 7.
    Wagner W., Horn P., Castoldi M. et al. (2008) Replicative senescence of mesenchymal stem cells – a continuous and organized process. PLoS ONE 5, e2213.CrossRefGoogle Scholar
  8. 8.
    Le Blanc K., Frassoni F., Ball L. et al. (2008) Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371, 1579–86.PubMedCrossRefGoogle Scholar
  9. 9.
    Nagaya N., Kangawa K., Itoh T. et al. (2005) Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 112, 1128–35.PubMedCrossRefGoogle Scholar
  10. 10.
    Gastens M. H., Goltry K., Prohaska W. et al. (2007) Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy. Cell Transplant 16, 685–96.PubMedGoogle Scholar
  11. 11.
    Bartmann C., Rohde E., Schallmoser K. et al. (2007) Two steps to functional mesenchymal stromal cells for clinical application. Transfusion 47, 1426–35.PubMedCrossRefGoogle Scholar
  12. 12.
    Parekkadan B., Sethu P., van P. D., Yarmush M. L., Toner M. (2007) Osmotic selection of human mesenchymal stem/progenitor cells from umbilical cord blood. Tissue Eng 13, 2465–73.PubMedCrossRefGoogle Scholar
  13. 13.
    Horn P., Bork S., Diehlmann A. et al. (2008) Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis. Cytotherapy 10, 676–85.PubMedCrossRefGoogle Scholar
  14. 14.
    Schallmoser K., Bartmann C., Rohde E. et al. (2007) Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47, 1436–46.PubMedCrossRefGoogle Scholar
  15. 15.
    Kocaoemer A., Kern S., Kluter H., Bieback K. (2007) Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 25, 1270–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Pittenger M. F., Mackay A. M., Beck S. C. et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–7.PubMedCrossRefGoogle Scholar
  17. 17.
    Colter D. C., Class R., DiGirolamo C. M., Prockop D. J. (2000) Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA 97, 3213–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Wagner W., Ho A. D. (2007) Mesenchymal stem cell preparations-comparing apples and oranges. Stem Cell Rev 3, 239–48.PubMedCrossRefGoogle Scholar
  19. 19.
    Ho A. D., Wagner W., Franke W. (2008) Heterogeneity of mesenchymal stromal cell preparations. Cytotherapy 10, 320–30.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Helmholtz Institute for Biomedical Engineering, Department of Cell BiologyRWTH Aachen University Medical SchoolAachenGermany

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