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Improved primary cultivation of human umbilical cord mesenchymal stem cells |
LI Xiao-Zhan, Ma-Hong, Xu-Hui, Li-Shu, Li-Yu-Mei |
(Department of Dermatology, the Affiliated Hospital of Jiangsu University, Zhenjiang Jiangsu 212001,China) |
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Abstract Objective: To improve the tissue mass primary culture protocol of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs),so as to explore their morphology, cell surface markers,osteogenic and adipogenic differentiation potential and observe biological properties. Methods: Human umbilical cords were obtained from healthy full-term delivered newborns. After stripping off blood vessels,the remaining connective tissues were cut into small fragments,and primary hUC-MSCs were separated by culture of traditional and improved tissue explants methods. The hUC-MSCs were tested with MTT method and the growth curves of them were drawn. Cell surface markers were detected by flow cytometry. After osteogenic and adipogenic differentiation,the potentiality of their differentiation was detected by chemistry staining. Results: After 3-4 days of incubation, flat or spindle-shaped fibroblast-like primary cells were presented,and cells reached 80% confluence after 5-6 days of incubation. Culture period was shortened by more than half. Flowing water-like or spiral growth morphology was presented after cell passage. MTT revealed high capability of proliferation. Flow cytometry analysis showed CD90(+), CD105(+), CD73(+), CD166(+), CD44(+), CD29(+), positive rate of 95% or more ;CD45(-),HLA-DR(-), negative rate of not more than 2%.Its differentiation potential was proved by osteogenic and adipogenic differentiation,which was proved by chemistry staining. Conclusion: An improved primary culture method of hUC-MSCs has been developed. Primary cells can be obtained from improved tissue explants method combined enzyme digestion technique. Primary cell culture time was shortened,and the efficiency of cell culture was improved. Cells isolated by the new method from the human umbilical cords had the following biological characteristics:adherent growth, high proliferation, mesenchymal stem cell phenotype,and multipotent differentiation. They could be used as the source of seed cells in scientific research and clinical applications.
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Received: 14 June 2014
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\[1\]Zhang L, Zhang HT, Hong SQ, et al. Cografted Wharton's jelly cells-derived neurospheres and BDNF promote functional recovery after rat spinal cord transection\[J\]. Neurochem Res, 2009, 34(11): 2030-2039.[2]Huang P,Lin LM,Wu XY,et al.Differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells into germ-like cells in vitro\[J\]. J Cell Biochem ,2010,109(4):747-754.[3]Eggenhofer E,Steinmann JF,Renner P,et al.Mesenchymal stem cells together with mycophenolate mofetil inhibit antigen presenting cell and T cell infiltration into allogeneic heart grafts\[J\].Transpl Immunol,2011,24(3):157-163.[4]Li L,Tian H,Yue W,et al.Human mesenchymal stem cells play a dual role on tumor cell growth in vitro and in vivo\[J\].J Cell Physiol,2011,226(7):1860-1867.[5]Friedenstein AJ,Piatetzky-Shapiro II,Petrakova KV.Osteogenesis in transplants of bone marrow cells\[J\].J Embryol Exp Morphol,1966,16(3):581-390.[6]Troyer DL,Weiss ML.Concise review: Wharton's jelly-derived cells are a primitive stromal cell population\[J\].Stem Cells,2008,26(3):591-599.[7]Zeddou M,Briquet A,Relic B,et al.The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood\[J\].Cell Biol Int,2010,34(7):693-701.[8]朱少芳, 何援利, 付霞霏. 人脐带间充质干细胞的生物学特性和超微结构\[J\]. 中国医学科学院学报, 2011, 33(4): 382-386.[9]Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord\[J\]. Stem Cells,2003, 21(1): 105-110.[10]Rubio R,Garcia-Castro J,Gutierrez-Aranda I,et al.Deficiency in p53 but not retinoblastoma induces the transformation of mesenchymal stem cells in vitro and initiates leiomyosarcoma in vivo\[J\].Cancer Res,2010,70(10):4185-4194.[11]Tong CK,Vellasamy S,Tan BC,et al.Generation of mesenchymal stem cell from human umbilical cord t3 using a combination enzymatic and mechanical disassociation method\[J\].Cell Biol Int,2011,35(3):221-226.[12]Si YL,Zhao YL,Hao HJ,et al.MSCs: Biological characteristics,clinical applications and their outstanding concerns\[J\].Ageing Res Rev,2011,10(1):93-103.[13]Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement\[J\].Cytotherapy,2006,8(4):315-317. |
[1] |
WANG Jing-wen1,2, YANG Yong2, ZHANG Zhi-jian1, CHEN Bo2, SU Chun-xiang2, HU Changlong2, CAI Hui-min1, LI Qiao-yu2. Induction and differentiation of human glioma cell line U251 into the adipocytes[J]. Journal of Jiangsu University(Medicine Edition), 2016, 26(1): 1-4. |
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