Study On Efficient Preparation Of Human Umbilical Cord Mesenchymal Stem Cells By Secondary Adherence Method

Human umbilical cord derived mesenchymal stem cells(hUCMSCs).The convenience of material selection to avoid secondary injury,as well as the advantages of weak immunogenicity and strong reproductive ability,are widely recognized in the biological and medical fields,and have been applied in multi-directional clinical research,with good application prospects.In this study,an animal-derived culture system was established,using human umbilical cord mesenchymal stem cells obtained by primary and secondary adhesion,and comparing their differences,the main cell bank(MCB)and working bank(WCB)were established by detecting the hUCMSCs proliferation ability,immune phenotype,karyotype and in vitro.Establish the efficient culture method of hUCMSCs and provide the experimental basis for the safety in clinical application.Objectives: 1)A serum-free culture system was adopted to solve the existing problems and risks of traditional serum.2)Use the hUCMSCs to extract the same umbilical cord.3)Test the biological function of hUCMSCs collected by secondary wall method.Methods: 1)Separate umbilical cord Huatong adhesive and conduct tissue block adherent culture.The complete medium was replaced every 3-4 days,and the suspended tissue blocks were collected when the cells reached 65-75% confluence and seeded into a new T75 culture bottle.The original vial cells reached 85% confluence were digested and passaged with trypsin replacement.2)Observe the appearance of the primary and secondary cells;test the proliferation capacity respectively,take the detection days as the horizontal coordinate and draw the cell growth curve;perform immunophenotyping analysis by flow analyzer;detect the differentiation ability of osteoblasts and chondrocytes;conduct in vitro tumorigenicity test;conduct karyotype analysis.Results: The cells cultured in the two animal-derived culture system had the same morphology and similar doubling time;the surface antigen expression of the secondary adherent P3 cells met the hUCMSCs phenotype;both cells had osteoblastic chondrogenic differentiation ability;no live cells were observed under the microscope after 14 days of culture in agar medium;the karyotype analysis showed that the chromosome number of the secondary adherent P12 hUCMSCs cells was normal with no structural distortion.Conclusion: The animal-free culture system can greatly increase the number of cells extracted from the same umbilical cord,and establish a larger master cell bank and working cell bank,reduce the batch difference and effect difference in the process of treatment and use,and make the treatment results more stable.