Construction Of Quality Control Standards For Human Mesenchymal Stem Cells

Human-derived Mesenchymal stem cell(MSC)is a kind of pluripotential stem cell,which with wide availability of raw materials.MSCs could be widely used in the construction of tissue engineering and regenerative medicine.Here comes up with higher requirements of quality identification and testing system through the procedures of large-scale preparations and low temperature storage processes,for ensuring the safety of MSCs and the excepted use of them.The generalduty quality identification and testing system is absent for MSCs culture process,no matter domestic or overseas.This task is based on Shenzhen China National Geneback,here we forced on pudding control system establish for MSCs from different resources,including UC-MSCs?DP-MSCs?DPMSCs?A-MSCs.We derived UC-MSCs?DP-MSCs?DP-MSCs?A-MSCs from umbilical cord?placenta?milk teeth and adipose,respectively.Primary cell we dissociate by tissue adhering method UC-MSCs,enzyme digestion method(including DP-MSCs?DP-MSCs?AMSCs),and then culture to P2 after freeing in vitro.During these process,we investigated cell morphology and living cell counts.In order to ensure securing of these cells,which maybe clinical application in the future,we did some common infectious disease screening,and mycoplasma?bacterial?fungi and endotoxin tests during all cell culture ?passage ?freezing,meanwhile,we checked contamination between cells from different tendency by STR test.purity is important for cell functions stability,here we applied FACS technique to test purity of cells prepared.In addition,we detected them function by three-line differentiation.In vitro,and verified by RT-PCR to indentify RUNX2 and OCN?SOX9?COL2?PPAR??LPL,which are specially expressed in osteoblasts,chondrocytes,adipocytes respectively.The process quality control system for MSC we preliminarily established here laid a foundation for future development and application.The quality control system of MSCs showed the cell morphology was uniform,and the prefrozen cell viability was greater than 90%.The donor was proved to be qualified and the MSCs were no contamination of exogenous elements including bacteria,anaerobic bacteria,fungi and mycoplasma.The endotoxin was less than 0.5 EU/ml.There's no cell cross contamination according to the STR assay.The MSC surface expression was greater than 95%(CD90,CD73,CD105,CD29,CD44)and negative the were less than 2%(CD45?CD34?CD19?CD11b?HLA-DR)in more than 95%.Meanwhile,the MSCs can differentiate into osteoblasts,adipocytes,chondrocytes with Oil Red O,Alcian Blue and Alizarin Red Staining.The differentiated cells were also found to higher expression of mRNA specific for adipogenesis(PPAR?,LPL),chondrogenesis(SOX9,COL2)and osteogenesis(RUNX2,OCN)than the control group(P <0.01)?The results all above indicate that the qualities of MSCs preparation could be effectively controlled by the quality identification and testing system in the processes of cell preparation and storage for MSCs of separate sources.The system that is built in this article guarantee the safety and efficacy of the products which could be adapted in clinical therapeutic application.And this system could be a sound basis for the construction of quality appraisal system in Genebank,the preparation of clinical-grade cells,and the long-term stability of cell storage quality,in the meantime,it also provides a reference to found a quality identification system for other cell types.