Expansion of Mesenchymal Stem Cells Derived from Umbilical Cord in Media Containing Human Serum (Method)

  • Sonja Kress
  • Anne Neumann
  • Tim Hatlapatka
  • Antonina Lavrentieva
  • Cornelia Kasper
Part of the Stem Cells and Cancer Stem Cells book series (STEM, volume 9)


The regeneration of damaged or diseased tissues or organs is one of the most ­ambitious and challenging fields in modern medicine. In this context, stem cells and especially mesenchymal stem or stromal cells (MSC) have proven to offer great promise as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Over the last decade the human umbilical cord and other birth- associated tissues have been found to be a rich and valuable source of MSC. The production of therapeutically significant cell numbers still remains to be one of the major challenges in clinical applications. Therefore biotechnological protocols need to be established to guarantee a reproducible and safe isolation and expansion of the cells. In this chapter an overview of techniques for the isolation of MSC from the tissue of the human umbilical cord is given and different expansion ­strategies are presented. In this context the composition of the culture media with regard to xeno-free culture conditions and the usage of growth factors as a proliferation trigger are discussed. Furthermore, different strategies for MSC expansion are briefly described, whereby the expansion in bioreactor systems using microcarriers is highlighted.


Vascular Endothelial Growth Factor Fibroblast Growth Factor Family Bone Morphogenetic Protein Umbilical Cord Telomere Length 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Bianchi G, Banfi A, Mastrogiacomo M, Notaro R, Luzzatto L, Cancedda R, Quarto R (2003) Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 287:98–105PubMedCrossRefGoogle Scholar
  2. Bonewald LF, Dallas SL (1994) Role of active and latent transforming growth factor beta in bone formation. J Cell Biochem 55:350–357. doi: 10.1002/jcb.240550312 PubMedCrossRefGoogle Scholar
  3. Dai ZQ, Wang R, Ling SK, Wan YM, Li YH (2007) Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Prolif 40:671–684. doi: 10.1111/j.1365-2184.2007.00461.x PubMedCrossRefGoogle Scholar
  4. Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J (2007) A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog 23:187–193. doi: 10.1021/bp060155w PubMedCrossRefGoogle Scholar
  5. Gao H, Ayyaswamy PS, Ducheyne P (1997) Dynamics of a microcarrier particle in the simulated microgravity environment of a rotating-wall vessel. Microgravity Sci Technol 10:154–165PubMedGoogle Scholar
  6. Hatlapatka T, Moretti P, Lavrentieva A, Hass R, Marquardt N, Jacobs R, Kasper C (2011) Optimization of culture conditions for the expansion of umbilical cord-derived mesenchymal stem or stromal cell-like cells using xeno-free culture conditions. Tissue Eng Part C Methods 17:485–493. doi: 10.1089/ten.TEC.2010.0406 PubMedCrossRefGoogle Scholar
  7. Javazon EH, Beggs KJ, Flake AW (2004) Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 32:414–425PubMedCrossRefGoogle Scholar
  8. Klein-Nulend J, Bacabac RG, Veldhuijzen JP, Van Loon JJWA (2003) Microgravity and bone cell mechanosensitivity. Adv Space Res 32:1551–1559PubMedCrossRefGoogle Scholar
  9. Koliakos I, Tsagias N, Karagiannis V (2011) Mesenchymal cells isolation from Wharton’s jelly, in perspective to clinical applications. J Biol Res 16:194–201Google Scholar
  10. Lavrentieva A, Majore I, Kasper C, Hass R (2010) Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells. Cell Commun Signal 8:18. doi: 1478-811X-8-18 [pii]  10.1186/1478-811X-8-18 PubMedCrossRefGoogle Scholar
  11. Lepperdinger G, Brunauer R, Jamnig A, Laschober G, Kassem M (2008) Controversial issue: is it safe to employ mesenchymal stem cells in cell-based therapies? Exp Gerontol 43:1018–1023. doi: 10.1016/j.exger.2008.07.004 PubMedCrossRefGoogle Scholar
  12. Levine DW, Wong JS, Wang DIC, Thilly WG (1977) Microcarrier cell culture: new methods for research-scale application. Somatic Cell Genet 3:149–155. doi: 10.1007/bf01551811 PubMedCrossRefGoogle Scholar
  13. Longobardi L, O’Rear L, Aakula S, Johnstone B, Shimer K, Chytil A, Horton WA, Moses HL, Spagnoli A (2006) Effect of igf-i in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling. J Bone Miner Res 21:626–636. doi: 10.1359/jbmr.051213 PubMedCrossRefGoogle Scholar
  14. Luu HH, Song WX, Luo X, Manning D, Luo J, Deng ZL, Sharff KA, Montag AG, Haydon RC, He TC (2007) Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res 25:665–677. doi: 10.1002/jor.20359 PubMedCrossRefGoogle Scholar
  15. Majore I, Moretti P, Stahl F, Hass R, Kasper C (2011) Growth and differentiation properties of mesenchymal stromal cell populations derived from whole human umbilical cord. Stem Cell Rev Rep 7:17–31. doi: 10.1007/s12015-010-9165-y CrossRefGoogle Scholar
  16. Moretti P, Hatlapatka T, Marten D, Lavrentieva A, Majore I, Hass R, Kasper C (2010) Mesenchymal stromal cells derived from human umbilical cord tissues: primitive cells with potential for clinical and tissue engineering applications. Adv Biochem Eng Biotechnol 123:29–54. doi: 10.1007/10_2009_15 PubMedGoogle Scholar
  17. Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, Choong C, Yang Z, Vemuri MC, Rao MS, Tanavde V (2008) PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCS into adipogenic, chondrogenic, and osteogenic lineages. Blood 112:295–307. doi: blood-2007-07-103697 [pii]  10.1182/blood-2007-07-103697 PubMedCrossRefGoogle Scholar
  18. Pons J, Huang Y, Arakawa-Hoyt J, Washko D, Takagawa J, Ye J, Grossman W, Su H (2008) VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochem Biophys Res Commun 376:419–422. doi: S0006-291X(08)01741-5 [pii]  10.1016/j.bbrc.2008.09.003 PubMedCrossRefGoogle Scholar
  19. Reddig PJ, Juliano RL (2005) Clinging to life: cell to matrix adhesion and cell survival. Cancer Metastasis Rev 24:425–439PubMedCrossRefGoogle Scholar
  20. Rodrigues M, Griffith LG, Wells A (2010) Growth factor regulation of proliferation and survival of multipotential stromal cells. Stem Cell Res 1:32. doi: scrt32 [pii]  10.1186/scrt32 CrossRefGoogle Scholar
  21. Rodrigues ME, Costa AR, Henriques M, Azeredo J, Oliveira R (2011) Wave characterization for mammalian cell culture: residence time distribution. New Biotechnol 29:402–408CrossRefGoogle Scholar
  22. Rosland GV, Svendsen A, Torsvik A, Sobala E, McCormack E, Immervoll H, Mysliwietz J, Tonn JC, Goldbrunner R, Lonning PE, Bjerkvig R, Schichor C (2009) Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation (this article contains errors due to a cross contamination of the cell lines we used. To correct this issue we published a letter in cancer res. 2010 Aug 1, 70(15):6393–6396). Cancer Res 69:5331–5339. doi:10.1158/0008-5472.Can-08-4630Google Scholar
  23. Rubin JP, Bennett JM, Doctor JS, Tebbets BM, Marra KG (2007) Collagenous microbeads as a scaffold for tissue engineering with adipose-derived stem cells. Plast Reconstr Surg 120:414–424, 410.1097/1001.prs.0000267699.0000299369.a0000267698PubMedCrossRefGoogle Scholar
  24. Santos F, Andrade PZ, Abecasis MM, Gimble JM, Chase LG, Campbell AM, Boucher S, Vemuri MC, Silva CL, Cabral JM (2011) Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods 17:1201–1210. doi: 10.1089/ten.tec.2011.0255 PubMedCrossRefGoogle Scholar
  25. Sart S, Schneider Y-J, Agathos SN (2009) Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J Biotechnol 139:291–299PubMedCrossRefGoogle Scholar
  26. Sethe S, Scutt A, Stolzing A (2006) Aging of mesenchymal stem cells. Ageing Res Rev 5:91–116. doi: S1568-1637(05)00049-8 [pii]  10.1016/j.rr.2005.10.001 PubMedCrossRefGoogle Scholar
  27. Singh V (1999) Disposable bioreactor for cell culture using wave-induced agitation. Cytotechnology 30:149–158. doi: 10.1023/a:1008025016272 PubMedCrossRefGoogle Scholar
  28. Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF (2005) FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol 203:398–409PubMedCrossRefGoogle Scholar
  29. Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis CN, Papamichail M (2006) Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 24:462–471PubMedCrossRefGoogle Scholar
  30. Stewart A, Guan H, Yang K (2010) BMP-3 promotes mesenchymal stem cell proliferation through the tgf-beta/activin signaling pathway. J Cell Physiol 223:658–666. doi: 10.1002/jcp.22064 PubMedGoogle Scholar
  31. Tamama K, Fan VH, Griffith LG, Blair HC, Wells A (2006) Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells. Stem Cells 24:686–695. doi: 2005-0176 [pii]  10.1634/stemcells.2005-0176 PubMedCrossRefGoogle Scholar
  32. Timmins NE, Kiel M, Günther M, Heazlewood C, Doran MR, Brooke G, Atkinson K (2012) Closed system isolation and scalable expansion of human placental mesenchymal stem cells. Biotechnol Bioeng 109:1817–1826. doi: 10.1002/bit.24425 PubMedCrossRefGoogle Scholar
  33. Tsagias N, Koliakos I, Karagiannis V, Eleftheriadou M, Koliakos GG (2011) Isolation of mesenchymal stem cells using the total length of umbilical cord for transplantation purposes. Transfus Med 21:253–261. doi: 10.1111/j.1365-3148.2011.01076.x PubMedCrossRefGoogle Scholar
  34. Tsutsumi S, Shimazu A, Miyazaki K, Pan H, Koike C, Yoshida E, Takagishi K, Kato Y (2001) Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF. Biochem Biophys Res Commun 288:413–419PubMedCrossRefGoogle Scholar
  35. Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, Benes V, Blake J, Pfister S, Eckstein V, Ho AD (2008) Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 3:e2213. doi: 10.1371/journal.pone.0002213 PubMedCrossRefGoogle Scholar
  36. Wagner W, Bork S, Lepperdinger G, Joussen S, Ma N, Strunk D, Koch C (2010a) How to track cellular aging of mesenchymal stromal cells? Aging 2:224–230PubMedGoogle Scholar
  37. Wagner W, Ho AD, Zenke M (2010b) Different facets of aging in human mesenchymal stem cells. Tissue Eng Part B Rev 16:445–453. doi: 10.1089/ten.TEB.2009.0825 PubMedCrossRefGoogle Scholar
  38. Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, Merchav S, Luo YQ, Rao MS, Velagaleti G, Troyer D (2006) Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson’s disease. Stem Cells 24:781–792. doi: 10.1634/stemcells.2005-0330 PubMedCrossRefGoogle Scholar
  39. Yang Y, Rossi FMV, Putnins EE (2007) Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture. Biomaterials 28:3110–3120PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Sonja Kress
    • 2
  • Anne Neumann
    • 1
  • Tim Hatlapatka
    • 1
  • Antonina Lavrentieva
    • 2
  • Cornelia Kasper
    • 1
  1. 1.Department of BiotechnologyUniversity of Natural Resources and Life Sciences, Vienne (BOKU)ViennaAustria
  2. 2.Institute for Technical Chemistry, Leibniz Universiy of HannoverHannoverGermany

Personalised recommendations