Cell and Tissue Banking

, Volume 17, Issue 2, pp 289–302 | Cite as

Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

  • Phuc Van PhamEmail author
  • Nhat Chau Truong
  • Phuong Thi-Bich Le
  • Tung Dang-Xuan Tran
  • Ngoc Bich Vu
  • Khanh Hong-Thien Bui
  • Ngoc Kim Phan
Original Paper


Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC–MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm2) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications.


Activated platelet rich plasma Clinical application of mesenchymal stem cells Umbilical cord Umbilical cord derived mesenchymal stem cells Good manufacturing practice UC–MSCs 



This research was funded by Ministry of Science and Technology via project Grant No. DTDL.2012-G/23.

Authors’ contributions

PVP conceived the study, performed PRP preparation, evaluated the effects of PRP on mesenchymal stem cell proliferation. NBV primarily cultured mesenchymal stem cells from mononuclear cells; TDXT, PTBL collected umbilical cord blood, isolated mononuclear cells from umbilical cord blood; KHTB carried out the differentiation assays; NTC, NKP evaluated the, karyotype, and tumorigenecity of MSCs in mice model. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no competing interests.


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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Phuc Van Pham
    • 1
    Email author
  • Nhat Chau Truong
    • 1
  • Phuong Thi-Bich Le
    • 2
  • Tung Dang-Xuan Tran
    • 2
  • Ngoc Bich Vu
    • 1
  • Khanh Hong-Thien Bui
    • 3
  • Ngoc Kim Phan
    • 1
  1. 1.Laboratory of Stem Cell Research and ApplicationUniversity of Science, Vietnam National UniversityHo Chi Minh CityVietnam
  2. 2.Van Hanh Stem Cell UnitVan Hanh HospitalHo Chi Minh CityVietnam
  3. 3.University Medical Center, University of Medicine and PharmacyHo Chi Minh CityVietnam

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