Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

  • Parvin Salehinejad
  • Noorjahan Banu Alitheen
  • Abdul Manaf Ali
  • Abdul Rahman Omar
  • Maryam Mohit
  • Ehsan Janzamin
  • Fazel Sahraneshin Samani
  • Zahra Torshizi
  • Seyed Noureddin Nematollahi-MahaniEmail author


Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.


Enzymatic isolation Explant Umbilical cord matrix-derived cells 



This work was supported by a grant from Kerman Neuroscience Research Center, Iran. We thank Dr Alp Can and Deniz Balci from Ankara University School of Medicine for their guidance in enzymatic cell isolation from an umbilical cord.


  1. Aghaee-Afshar M.; Rezazadehkermani M.; Asadi A.; Malekpour-afshar R.; Shahesmaeili A.; Nematollahi-Mahani S. N. Potential of human umbilical cord matrix and rabbit bone marrow-derived mesenchymal stem cells in repair of surgically incised rabbit external anal sphincter. Dis Colon Rectum 52: 1753–1761; 2009.PubMedCrossRefGoogle Scholar
  2. Atala A.; Lanza P. R. Methods of tissue engineering. Academic, 2002.Google Scholar
  3. Baksh D.; Song L.; Tuan R. S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 8: 301–316; 2004.PubMedCrossRefGoogle Scholar
  4. Bobis S.; Jarocha D.; Majka M. Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol 44: 215–230; 2006.PubMedGoogle Scholar
  5. Bowman C. L.; Yohe L.; Lohr J. W. Enzymatic modulation of cell volume in C6 glioma cells. Glia 27: 22–31; 1999.PubMedCrossRefGoogle Scholar
  6. Can A.; Karahuseyinoglu S. Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells 25: 2886–2895; 2007.PubMedCrossRefGoogle Scholar
  7. Chamberlain G.; Fox J.; Ashton B.; Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 25: 2739–2749; 2007.PubMedCrossRefGoogle Scholar
  8. Conconi M. T.; Burra P.; Di Liddo R.; Calore C.; Turetta M.; Bellini S.; Bo P.; Nussdorfer G. G.; Parnigotto P. P. CD105(+) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 18: 1089–1096; 2006.PubMedGoogle Scholar
  9. Costa A.; Silvestrini R.; Del Bino G.; Motta R. Implications of disaggregation procedures on biological representation of human solid tumours. Cell Tissue Kinet 20: 171–180; 1987.PubMedGoogle Scholar
  10. Declercq H.; Van den Vreken N.; De Maeyer E.; Verbeeck R.; Schacht E.; De Ridder L.; Cornelissen M. Isolation, proliferation and differentiation of osteoblastic cells to study cell/biomaterial interactions: comparison of different isolation techniques and source. Biomaterials 25: 757–768; 2004.PubMedCrossRefGoogle Scholar
  11. Freshney R. I. Culture of animal cells; a manual of basic technique. 4th ed. Wiley, New York; 2005.CrossRefGoogle Scholar
  12. Huang P.; Lin L. M.; Wu X. Y.; Tang Q. L.; Feng X. Y.; Lin G. Y.; Lin X.; Wang H. W.; Huang T. H.; Ma L. Differentiation of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells into germ-like cells in vitro. J Cell Biochem 109: 747–754; 2010a.PubMedGoogle Scholar
  13. Huang P.; Lin W. L.; Ying Wu X.; Ling Tang Q. X. Y. F.; 1, Guang Yu Lin X. L.; 1 Hong Wu Wang; 1 Tian Hua Huang; 2 and Lian Ma. Differentiation of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells into germ-like cells in vitro. J. Cell. Biochem. 109: 747–754; 2010b.Google Scholar
  14. Ishige I.; Nagamura-Inoue T.; Honda M. J.; Harnprasopwat R.; Kido M.; Sugimoto M.; Nakauchi H.; Tojo A. Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton’s jelly explants of human umbilical cord. Int J Hematol 90: 261–269; 2009.PubMedCrossRefGoogle Scholar
  15. Kadam S. S.; Tiwari S.; Bhonde R. R. Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord. In Vitro Cell Dev Biol Anim 45: 23–27; 2009.PubMedCrossRefGoogle Scholar
  16. Kadivar M.; Khatami S.; Mortazavi Y.; Soleimani M.; Taghikhani M.; Shokrgozar M. Isolation, culture and characterization of postnatal human umbilical vein-derived mesenchmal stem cells. Daru 13: 170–176; 2005.Google Scholar
  17. Karahuseyinoglu S.; Cinar O.; Kilic E.; Kara F.; Akay G.; Demiralp D.; Tukun A.; Uckan D.; Can A. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells 25: 319–331; 2007.PubMedCrossRefGoogle Scholar
  18. Konig J. J.; van Dongen J. W.; Schroder F. H. Preferential loss of abnormal prostate carcinoma cells by collagenase treatment. Cytometry 14: 805–810; 1993.PubMedCrossRefGoogle Scholar
  19. La Rocca G.; Anzalone R.; Corrao S.; Magno F.; Loria T.; Lo Iacono M.; Di Stefano A.; Giannuzzi P.; Marasa L.; Cappello F.; Zummo G.; Farina F. Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers. Histochem Cell Biol 131: 267–282; 2009.PubMedCrossRefGoogle Scholar
  20. Latifpour M.; Nematollahi-Mahani S. N.; Deilamy M.; Azimzadeh B. S.; Eftekhar-Vaghefi S. H.; Nabipour F.; Najafipour H.; Nakhaee N.; Yaghoubi M.; Eftekhar-Vaghefi R.; Salehinejad P.; Azizi H. Improvement in cardiac function following transplantation of human umbilical cord matrix-derived mesenchymal cells. Cardiology 120: 9–18; 2011.PubMedGoogle Scholar
  21. Leeb C.; Jurga M.; McGuckin C.; Moriggl R.; Kenner L. Promising new sources for pluripotent stem cells. Stem Cell Rev 6: 15–26; 2009.CrossRefGoogle Scholar
  22. Logeart-Avramoglou D.; Anagnostou F.; Bizios R.; Petite H. Engineering bone: challenges and obstacles. J Cell Mol Med 9: 72–84; 2005.PubMedCrossRefGoogle Scholar
  23. Lorenzini S.; Gitto S.; Grandini E.; Andreone P.; Bernardi M. Stem cells for end stage liver disease: how far have we got? World J Gastroenterol 14: 4593–4599; 2008.PubMedCrossRefGoogle Scholar
  24. Lu L. L.; Liu Y. J.; Yang S. G.; Zhao Q. J.; Wang X.; Gong W.; Han Z. B.; Xu Z. S.; Lu Y. X.; Liu D.; Chen Z. Z.; Han Z. C. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica 91: 1017–1026; 2006.PubMedGoogle Scholar
  25. Majore I.; Moretti P.; Hass R.; Kasper C. Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord. Cell Communication and Signaling 7: 1–8; 2009.CrossRefGoogle Scholar
  26. Miyazaki M.; Tsunashima M.; Wahid S.; Miyano K.; Sato J. Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion. Res Exp Med (Berl) 184: 191–204; 1984.CrossRefGoogle Scholar
  27. Nematollahi-Mahani S.; Rezazadekermani M.; Latifpour M.; Salehinejad P. Biological and biochemical characteristics of human umbilical cord mesenchymal cells. Journal of Reproduction and Infertility 10: 7–15; 2008. Persian, Abstract in English.Google Scholar
  28. Nematollahi-Mahani S. N.; Rezazadeh-kermani M.; Mehrabani M.; Nakhaee N. Cytotoxic effects of Teucrium polium on some established cell lines. Pharmaceutical Biology 45: 295–298; 2007.CrossRefGoogle Scholar
  29. Oyama Y.; Hori N.; Allen C. N.; Carpenter D. O. Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia californica. Cell Mol Neurobiol 10: 193–205; 1990.PubMedCrossRefGoogle Scholar
  30. Petsa A.; Gargani S.; Felesakis A.; Grigoriadis N.; Grigoriadis I. Effectiveness of protocol for the isolation of Wharton’s Jelly stem cells in large-scale applications. In Vitro Cell. Dev. Biol. Anim. 2009.Google Scholar
  31. Phinney D. G.; Prockop D. J. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 25: 2896–2902; 2007.PubMedCrossRefGoogle Scholar
  32. Qiao C.; Xu W.; Zhu W.; Hu J.; Qian H.; Yin Q.; Jiang R.; Yan Y.; Mao F.; Yang H.; Wang X.; Chen Y. Human mesenchymal stem cells isolated from the umbilical cord. Cell Biol Int 32: 8–15; 2008.PubMedCrossRefGoogle Scholar
  33. Rao M. S.; Mattson M. P. Stem cells and aging: expanding the possibilities. Mech Ageing Dev 122: 713–734; 2001.PubMedCrossRefGoogle Scholar
  34. Romanov Y. A.; Svintsitskaya V. A.; Smirnov V. N. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 21: 105–110; 2003.PubMedCrossRefGoogle Scholar
  35. Saward L.; Zahradka P. Coronary artery smooth muscle in culture: migration of heterogeneous cell populations from vessel wall. Mol Cell Biochem 176: 53–59; 1997.PubMedCrossRefGoogle Scholar
  36. Schugar R. C.; Chirieleison S. M.; Wescoe K. E.; Schmidt B. T.; Askew Y.; Nance J. J.; Evron J. M.; Peault B.; Deasy B. M. High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue. J Biomed Biotechnol 2009: 789526; 2009.PubMedCrossRefGoogle Scholar
  37. Semenov O. V.; Koestenbauer S.; Riegel M.; Zech N.; Zimmermann R.; Zisch A. H.; Malek A. Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation. Am J Obstet Gynecol 20: 193 e191–193 e113; 2009.Google Scholar
  38. Sotiropoulou P. A.; Perez S. A.; Salagianni M.; Baxevanis C. N.; Papamichail M. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 24: 462–471; 2006.PubMedCrossRefGoogle Scholar
  39. Stricklin G. P.; Bauer E. A.; Jeffrey J. J.; Eisen A. Z. Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry 16: 1607–1615; 1977.PubMedCrossRefGoogle Scholar
  40. Struys T.; Moreels M.; Martens W.; Donders R.; Wolfs E.; Lambrichts I. Ultrastructural and immunocytochemical analysis of multilineage differentiated human dental pulp- and umbilical cord-derived mesenchymal stem cells. Cells Tissues Organs 193: 366–378; 2011.PubMedCrossRefGoogle Scholar
  41. Vawda R. Characterisation and neurogenic potential of stem cells from the human umbilical cord matrix. Imperial College London. 2008.Google Scholar
  42. Wang H. S.; Hung S. C.; Peng S. T.; Huang C. C.; Wei H. M.; Guo Y. J.; Fu Y. S.; Lai M. C.; Chen C. C. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 22: 1330–1337; 2004.PubMedCrossRefGoogle Scholar
  43. Weiss M. L.; Medicetty S.; Bledsoe A. R.; Rachakatla R. S.; Choi M.; Merchav S.; Luo Y.; Rao M. S.; Velagaleti G.; Troyer D. 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; 2006.PubMedCrossRefGoogle Scholar
  44. Williams S. K.; McKenney S.; Jarrell B. E. Collagenase lot selection and purification for adipose tissue digestion. Cell Transplant 4: 281–289; 1995.PubMedCrossRefGoogle Scholar
  45. Zhang Y.; Li C. D.; Jiang X. X.; Li H. L.; Tang P. H.; Mao N. Comparison of mesenchymal stem cells from human placenta and bone marrow. Chin Med J (Engl) 117: 882–887; 2004.Google Scholar

Copyright information

© The Society for In Vitro Biology 2012

Authors and Affiliations

  • Parvin Salehinejad
    • 1
  • Noorjahan Banu Alitheen
    • 2
  • Abdul Manaf Ali
    • 2
  • Abdul Rahman Omar
    • 1
  • Maryam Mohit
    • 5
  • Ehsan Janzamin
    • 4
  • Fazel Sahraneshin Samani
    • 4
  • Zahra Torshizi
    • 6
  • Seyed Noureddin Nematollahi-Mahani
    • 3
    Email author
  1. 1.Institute of BioscienceUniversity Putra MalaysiaKuala LumpurMalaysia
  2. 2.Faculty of Molecular and BiotechnologyUniversity Putra MalaysiaKuala LumpurMalaysia
  3. 3.Kerman Neuroscience Research CenterKerman University of Medical SciencesKermanIran
  4. 4.Department of Stem CellsRoyan InstituteTehranIran
  5. 5.Department of PathologyKerman University of Medical SciencesKermanIran
  6. 6.Afzalipour School of MedicineKerman University of Medical SciencesKermanIran

Personalised recommendations