Share This Article:

Mixed enzymatic-explant protocol for isolation of mesenchymal stem cells from Wharton’s jelly and encapsulation in 3D culture system

Abstract Full-Text HTML XML Download Download as PDF (Size:1021KB) PP. 580-586
DOI: 10.4236/jbise.2012.510071    5,212 Downloads   9,700 Views   Citations


We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth, 6 - 9 pieces of WJ were transferred onto tissue culture flask and waited for attachment. For mixed enzymatic-explant, 1 cm3 pieces WJ were placed in enzymatic cocktail comprising 4 mg/ml Collagenase Type I and 1 mg/ml Hyaluronidase and 0.1% trypsin-EDTA. Then isolated cells were analyzed for surface cell markers such as CD73, CD31. Isolated 1.0 × 106 MSCs/ml were encapsulated in alginate hydrogel. Cells with MSCs phenotype were isolated from mixed enzymatic-explant and explant procedures within 24 - 48 hrs and 7 - 10 days, respectively. Both of procedures were shown to form clumps and colonies with dense centers. Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shaped and typical fibroblast-like shape cells. By using flow cytometery MSCs showed positive for CD73, and negative for CD31. the morphology of viable MSCs in the beads did not significantly show a different morphology pattern before and after the bead formation process. These findings are indicated that when mixed enzymatic-explant procedure is performed MSCs can be isolated faster and much higher from WJ. These finding is important in comparing with time consuming explants culture for isolation of MSCs.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Azandeh, S. , Orazizadeh, M. , Hashemitabar, M. , Khodadadi, A. , Shayesteh, A. , Nejad, D. , Gharravi, A. and Allahbakhshi, E. (2012) Mixed enzymatic-explant protocol for isolation of mesenchymal stem cells from Wharton’s jelly and encapsulation in 3D culture system. Journal of Biomedical Science and Engineering, 5, 580-586. doi: 10.4236/jbise.2012.510071.


[1] Watt, F.M., and B.L.M. Hogan. (2000) Out of Eden: Stem cells and their niches. Science, 287:1427-143... DOI: 10.1126/science.287.5457.1427
[2] Zhang, X.Y., J.S. Li, J. Nie, K. Jiang, Z.K. Zhen, J.J. Wang, and L. Shen. (2010) Differentiation Character of Adult Mesenchymal Stem Cells and Transfection of MSCs with Lentiviral Vectors. J Huazhong U Sci-Med, 30, 687-693 DOI: 10.1007/s11596-010-0641-z
[3] Troyer, D.L., and M.L. Weiss. (2008) Wharton's jelly-derived cells are a primitive stromal cell population. Stem Cells, 26, 591-599. DOI:10.1634/stemcells.2007-0439
[4] Sarugaser, R., D. Lickorish, D. Baksh, M.M. Hosseini, and J.E. Davies. (2005) Human umbilical cord perivascular (HUCPV) cells: A source of mesenchymal progenitors. Stem Cells, 23, 220-229. DOI:10.1634/stemcells.2004-0166
[5] Ruetze, M., S. Gallinat, I.J. Lim, E. Chow, T.T. Phan, F. Staeb, H. Wenck, W. Deppert, and A. Knott. (2008) Common features of umbilical cord epithelial cells and epidermal keratinocytes. J Dermatol Sci, 50, 227-231. DOI:10.1016/j.jdermsci.2007.12.006
[6] Ilancheran, S., A. Michalska, G. Peh, E.M. Wallace, M. Pera, and U. Manuelpillai. (2007) Stem cells derived from human fetal membranes display multilineage differentiation potential. Biol Reprod, 77, 577-588. DOI: 10.1095/biolreprod.106.055244
[7] Weiss, M.L., S. Medicetty, A.R. Bledsoe, R.S. Rachakatla, M. Choi, S. Merchav, Y.Q. Luo, M.S. Rao, G. Velagaleti, and D. Troyer. (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
[8] Fong, C.Y., M. Richards, N. Manasi, A. Biswas, and A. Bongso. (2007) Comparative growth behaviour and characterization of stem cells from human Wharton's jelly. Reprod Biomed Online, 15, 708-718. DOI: 10.1016/S1472-6483(10)60539-1
[9] Chang, P.L. (1999) Encapsulation for somatic gene therapy. Ann Ny Acad Sci, 875, 146-158. DOI:10.1111/j.1749-6632.1999.tb08500.x
[10] Penolazzi, L., E. Tavanti, R. Vecchiatini, E. Lambertini, F. Vesce, R .Gambari, S. Mazzitelli, F. Mancuso, G. Luca, C. Nastruzzi, and R. Piva. (2010) Encapsulation of mesenchymal stem cells from Wharton’s jelly in alginate microbeads. Tissue Eng Part C-Me, 16, 141-155. DOI:10.1089/ten.tec.2008.0582.
[11] Park, J.S., D.G. Woo, H.N. Yang, H.J. Lim, K.M. Park, K. Na, and K.H. Park. (2010) Chondrogenesis of human mesenchymal stem cells encapsulated in a hydrogel construct: Neocartilage formation in animal models as both mice and rabbits. J Biomed Mater Res A, 92, 988- 996. DOI: 10.1002/jbm.a.32341
[12] Fong, C.Y., A. Subramanian, A. Biswas, K. Gauthaman, P. Srikanth, M.P. Hande, and A. Bongso. (2010) Derivation efficiency, cell proliferation, freeze-thaw survival, stem-cell properties and differentiation of human Wharton's jelly stem cells. Reprod Biomed Online 21, 391-401. DOI:10.1016/j.rbmo.2010.04.010
[13] Salehinejad, P., N.B. Alitheen, A.M. Ali, A.R. Omar, M. Mohit, E. Janzamin, F.S. Samani, Z. Torshizi ,and S.N. Nematollahi-Mahani. (2012) Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly. In Vitro Cell Dev-An 48, 75-83. DOI: 10.1007/s11626-011-9480-x
[14] Montanucci, P., G. Basta, T. Pescara, I. Pennoni, F. Di Giovanni ,and R. Calafiore. (2011) New simple and rapid method for purification of mesenchymal stem cells from the human umbilical cord Wharton jelly. Tissue Eng Part A 17, 2651-2661. DOI:10.1089/ten.tea.2010.0587
[15] Tsagias ,N., I. Koliakos, V. Karagiannis, M. Eleftheriadou, and G.G. Koliakos. (2011) Isolation of mesenchymal stem cells using the total length of umbilical cord for transplantation purposes. Transfusion Med 21, 253-261. DOI: 10.1111/j.1365-3148.2011.01076.x
[16] Lu, L.L., Y.J. Liu, S.G. Yang, Q.J. Zhao, X. Wang ,W. Gong, Z.B. Han, Z.S. Xu, Y.X. Lu, D. Liu, Z.Z. Chen, and Z.C. Han. (2006) Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica 91, 1017-1026.
[17] Karahuseyinoglu ,S., O. Cinar, E. Kilic, F. Kara, G.G. Akay, D.O. Demiralp, A. Tukun, D. Uckan, and A. Can. (2007) Biology of stem cells in human umbilical cord stroma: In situ and in vitro surveys. Stem Cells 25, 319-331. DOI: 10.1634/stemcells.2006-0286
[18] Meyer, T., V. Lechner, K. Ulrichs, J. Dietl, B. Hocht, and A. Thiede. (2006) Isolation and characterization of umbilical cord mesenchymal stem cells: Potent cells for cell-based therapies in pediatric surgery? Cytotherapy 8, 39-39. DOI: 10.1007/s10353-008-0417-x
[19] Manca, M.F. ,I. Zwart, J. Beo, R. Palasingham, L.S. Jen, R. Navarrete, J. Girdlestone, and C.V. Navarrete. (2008) Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood. Cytotherapy 10, 54-68 DOI:10.1080/14653240701732763
[20] Han, Y.F., J.K. Chai, T.J. Sun, D.J. Li, and R. Tao. (2011) Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro. Biochem Bioph Res Co 413, 561-565. DOI: org/10.1016/j.bbrc.2011.09.001
[21] Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F.C. Marini, D.S. Krause, R.J. Deans, A. Keating, D.J. Prockop, and E.M. Horwitz. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8, 315-317. DOI:10.1080/14653240600855905
[22] Wang, H.S., S.C. Hung, S.T. Peng, C.C. Huang, H.M. Wei, Y.J. Guo, Y.S. Fu, M.C. Lai, and C.C. Chen. (2004) Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells 22, 1330-1337. DOI:10.1634/stemcells.2004-0013.

comments powered by Disqus

Copyright © 2018 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.