1. Introduction
Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the mesoderm, with the potential for self-renewal and multi-directional differentiation. Research has shown that MSCs are widely present in various tissues of adults and fetuses, such as bone marrow [1], peripheral blood [2], adipose tissue [3], amniotic membrane and umbilical cord [4], placenta [5], dental pulp [6], skin, etc. [7].
2. Technical process [8-9]
2.1 Isolation and culture of umbilical cord mesenchymal stem cells
(1) Aseptic collection of 15-20 cm fetal umbilical cord from healthy parturients undergoing cesarean section, packed in a sealed container pre placed with PBS (containing 2% penicillin+2% streptomycin) and brought back to the laboratory.
(2) In the laboratory ultra clean workbench, rinse repeatedly to remove blood clots on the surface of umbilical cord tissue, and use hemostatic forceps
(3) Clamp both ends of the umbilical cord and soak them in 75% medical grade alcohol for about 1 minute.
(4) Then transfer to PBS to remove residual alcohol on the surface of the umbilical cord, cut the umbilical cord into 2-3 cm segments, and place them in PBS to remove blood cells from the umbilical cord arteries and veins.
(5) Subsequently, the umbilical vein, umbilical artery, and outer membrane of the umbilical cord were cut open using tissue scissors, and the Wharton's gel tissue was separated. After repeated washing with PBS, the Wharton's gel was cut into 2-3 mm tissue blocks and evenly spread in a 60 mm culture dish.
(6) Add a small amount of culture medium to keep the tissue block moist. Place the culture dish in a CO2 incubator and let it stand for 12 hours. Gently add a small amount of culture medium along the side wall of the dish and continue to culture. Change the medium every 5 days.
2.2 Determination of Cell Proliferation Ability
(1) Measure the cell proliferation ability of cultured 4th generation human umbilical cord mesenchymal stem cells, digest them with 0.25% trypsin, centrifuge and remove the supernatant.
(2) Add DMEM culture medium containing 10% fetal bovine serum by volume to resuspend the cells, adjust the cell concentration to 1x104 cells/ml, and inoculate them into a 24 well cell culture plate with 1 ml of cell suspension per well.
(3) Place the culture plate in a CO2 incubator and replace the culture medium every 3 days. On days 2, 4, 6, 8, 10, and 12, 3 wells of cells were collected and digested, and then counted using a cell counter.
(4) Plot the growth curve of 4th generation human umbilical cord mesenchymal stem cells with culture time as the horizontal axis and cell count as the vertical axis.
2.3 Cell surface antigen detection
(1) Measure the cell proliferation ability of cultured 4th generation human umbilical cord mesenchymal stem cells, digest them with 0.25% trypsin, centrifuge and remove the supernatant.
(2) Add DMEM culture medium containing 10% fetal bovine serum by volume to resuspend the cells, adjust the cell concentration to 1x105 cells/ml, fix in 70% cold ethanol by volume, overnight at 4 ℃, and use for flow cytometry to determine cell surface markers.
(3) The cells reacted with mouse anti human FITC-CD90, PE-CCD86, FITC-CD45, PE-CD19, FITC-CD105, PE-HDR, FITC-HLA-ABC, and PE-CD34 antibodies respectively, and were placed in a dark room at 4 ℃ for 30 minutes. Using mouse anti human PE/FITC-IgG1 as a parallel control. The data was processed using Cell Quest software.
2.4 Testing of Multiple Differentiation Abilities
(1) Extract cultured 4th generation human umbilical cord mesenchymal stem cells and induce identification of osteogenic, adipogenic, and chondrogenic directions.
3. Progress in directed differentiation research [10]
At present, there are more than 18 types of cells recorded in domestic and foreign literature that have differentiated from human umbilical cord derived mesenchymal stem cells. In the past 5 years of literature reports, some studies have improved the previous induction methods; Another part of the research has opened up a new direction for the directed differentiation of human umbilical cord derived mesenchymal stem cells; Some studies have preliminarily explored the signaling pathways and related molecular mechanisms involved in the differentiation process.
4. References
【1】Gardner OF, Alini M, Stoddart MJ. Mesenchymal Stem Cells Derived from Human Bone Marrow. Methods Mol Biol. 2015;1340:41-52.
【2】Chong PP, Selvaratnam L, Abbas AA, Kamarul T. Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells. J Orthop Res. 2012 Apr;30(4):634-42
【3】Doornaert M, De Maere E, Colle J, Declercq H, Taminau J, Lemeire K, Berx G, Blondeel P. Xenogen-free isolation and culture of human adipose mesenchymal stem cells. Stem Cell Res. 2019 Oct;40:101532.
【4】Hendrijantini N, Hartono P. Phenotype Characteristics and Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells Derived from Amnion Membrane (HAMSCs) and Umbilical Cord (HUC-MSCs). Acta Inform Med. 2019 Jun;27(2):72-77.
【5】Thaweesapphithak S, Tantrawatpan C, Kheolamai P, Tantikanlayaporn D, Manochantr S. The Biological Characteristics of Placenta Derived Mesenchymal Stem Cells Cultured in Human Serum. J Med Assoc Thai. 2016 Jul;99 Suppl 4:S75-83. PMID: 29917345.
【6】Tsai AI, Hong HH, Lin WR, Fu JF, Chang CC, Wang IK, Huang WH, Weng CH, Hsu CW, Yen TH. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp. Biomed Res Int. 2017;2017:2851906.
【7】Mushahary D, Spittler A, Kasper C, Weber V, Charwat V. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A. 2018 Jan;93(1):19-31.
【8】Chen Feng, Yang Min, Li Yanjie, etc Isolation, culture, and multipotent differentiation potential of human umbilical cord mesenchymal stem cells [J] Journal of Biology, 2021
【9】 Wang Fei, Zhou Hong, Guo Yucheng, et al. Study on the Culture Method of Primary Human Umbilical Cord Mesenchymal Stem Cells [J] Chinese Journal of Tissue Engineering, 2014, 18 (19): 6
【10】 Shi Qin, Yang Xiaoqing, Zhang Yuquan, etc Directional differentiation of human umbilical cord Huatong collagen mesenchymal stem cells: current status and prospects [J] China Tissue Engineering Research, 2018
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