, Volume 65, Issue 5, pp 819–827 | Cite as

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

  • Yan-Fu Han
  • Ran Tao
  • Tian-Jun Sun
  • Jia-Ke ChaiEmail author
  • Guang Xu
  • Jing Liu
Brief Report


Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.


Mesenchymal stem cell Umbilical cord Culture Isolation Modified enzyme digestion 



This study was supported by grants from the National Health Public Welfare Special Scientific Research Foundation of China (200802066), China Postdoctoral Science Foundation special fund project (201104777), Capital Medical University basic-clinical medical research cooperation project (12JL81), National Natural Science Foundation of China (81101423), and Military Medical Science and Technology Research Project of “Twelfth Five-Year Plan” of China (CWS11J111).

Supplementary material

10616_2012_9528_MOESM1_ESM.doc (36 kb)
Supplementary material 1 (DOC 36 kb)


  1. Baksh D, Yao R, Tuan RS (2007) Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells 25:1384–1392CrossRefGoogle Scholar
  2. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I (2004) Study of telomere length reveals rapid aging of human marrow stromal cells, following in vitro expansion. Stem Cells 22:675–682CrossRefGoogle Scholar
  3. Can A, Karahuseyinoglu S (2007) Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells 25:2886–2895CrossRefGoogle Scholar
  4. Chamberlain G, Fox J, Ashton B, Middleton J (2007) Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 25:2739–2749CrossRefGoogle Scholar
  5. Conconi MT, Burra P, Di Liddo R, Calore C, Turetta M, Bellini S, Bo P, Nussdorfer GG, Parnigotto PP (2006) CD105 (+) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 18:1089–1096Google Scholar
  6. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop DJ, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317CrossRefGoogle Scholar
  7. Han Y, Chai J, Sun T, Li D, Tao R (2011) Differentiation of human umbilical cord mesenchymal stem cells into fibroblasts in vitro. Biochem Biophys Res Commun 413:561–565CrossRefGoogle Scholar
  8. Karahuseyinoglu S, Cinar O, Kilic E, Kara F, Akay GG, Demiralp DO, Tukun A, Uckan D, Can A (2007) Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells 25:319–331CrossRefGoogle Scholar
  9. Kobayashi K, Kubota T, Aso T (1998) Study on myofibroblast diferentiation in the stromal cells of Wharton’s jelly: expression and localization of alpha-smooth muscle actin. Early Hum Dev 51:223–233CrossRefGoogle Scholar
  10. Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, Gronthos S (2005) Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood 105:3793–3801CrossRefGoogle Scholar
  11. Lavik E, Langer R (2004) Tissue engineering: current state and perspectives. Appl Microbiol Biotechnol 65:1–8CrossRefGoogle Scholar
  12. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675CrossRefGoogle Scholar
  13. Ma L, Feng XY, Cui BL, Law F, Jiang XW, Yang LY, Xie QD, Huang TH (2005) Human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J 118:1987–1993Google Scholar
  14. Meyer FA, Laver-Rudich Z, Tanenbaum R (1983) Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton’s jelly. Biochim Biophys Acta 755:376–387CrossRefGoogle Scholar
  15. Oyama Y, Hori N, Allen CN, Carpenter DO (1990) Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia califonica. Cell Mol Neurobiol 10:193–205CrossRefGoogle Scholar
  16. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefGoogle Scholar
  17. Qiao C, Xu W, Zhu W, Hu J, Qian H, Yin Q, Jiang R, Yan Y, Mao F, Yang H, Wang X, Chen Y (2008) Human mesenchymal stem cells isolated from the umbilical cord. Cell Biol Int 32:8–15CrossRefGoogle Scholar
  18. Schneider RK, Püllen A, Kramann R, Bornemann J, Knüchel R, Neuss S, Perez-Bouza A (2010) Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents. Differentiation 79:182–193CrossRefGoogle Scholar
  19. Seshareddy K, Troyer D, Weiss ML (2008) Method to isolate mesenchymal-like cells from Wharton’s Jelly of umbilical cord. Methods Cell Biol 86:101–119CrossRefGoogle Scholar
  20. Thomson JA, Odorico JS (2000) Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol 18:53–57CrossRefGoogle Scholar
  21. Tong CK, Vellasamy S, Tan BC, Abdullah M, Vidyadaran S, Seow HF, Ramasamy R (2011) Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method. Cell Biol Int 35:221–226CrossRefGoogle Scholar
  22. Vizza E, Correr S, Goranova V, Heyn R, Muglia U, Papagianni V (1995) The collagen fibrils arrangement in the Wharton’s jelly of full-term human umbilical cord. Ital J Anat Embryol 100:495–501Google Scholar
  23. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, Fu YS, Lai MC, Chen CC (2004) Mesenchymal stem cells in the Wharton’s Jelly of the human umbilical cord. Stem Cells 22:1330–1337CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Yan-Fu Han
    • 1
    • 2
  • Ran Tao
    • 3
  • Tian-Jun Sun
    • 2
  • Jia-Ke Chai
    • 2
    • 4
    Email author
  • Guang Xu
    • 1
  • Jing Liu
    • 1
  1. 1.Department of Plastic Surgery, Affiliated Beijing Shijitan HospitalCapital Medical UniversityBeijingPeople’s Republic of China
  2. 2.Department of Burn and Plastic Surgery, Burns InstituteThe First Affiliated Hospital of PLA General HospitalBeijingPeople’s Republic of China
  3. 3.Department of Plastic SurgeryPLA General HospitalBeijingPeople’s Republic of China
  4. 4.Department of Burn and Plastic Surgery, Burns InstituteThe First Affiliated Hospital of PLA General HospitalHaidian, BeijingChina

Personalised recommendations