| Objectives:(1)Isolation and culture of human umbilical cord mesenchymal stem cells through optimized tissue adherence method,normalization and standardization preparation process was determined,and cell bank was established,so as to provide reliable cell sources for basic research and clinical research.Quality verification was carried out around the whole process of cell bank establishment,and a quality evaluation system corresponding to "four categories" of quality attributes was preliminarily constructed,namely,basic cell biological attributes,microbiological safety,biological safety and biological effectiveness.(2)The gradient descending serum acclimation culture was carried out on the working bank cells to study the changes in the expression levels of VEGF,PDGF and bFGF in cells and paracrine substances under "starvation" condition,and explore the influencing factors of cytokines secreted by stem cells.Methods:(1)Establishment and verification of human umbilical cord mesenchymal stem cell bank:Umbilical cord mesenchymal stem cells were isolated and cultured by tissue adherence method by collecting full-term neonatal umbilical cords,and then continuously expanded to P2 generation cryopreservation all the cells to establish a seed cell bank.The resuscitation seed bank cells were continuously expanded to P5 generation cryopreservation all the cells to establish a working cell bank.The resuscitation working bank cells were expanded to P10 generation for higher generation cell safety detection.Cell morphology,numbers,viability,surface markers,cycle,STR authentication and growth curve of P2,P5,P10 generation were detected during the preparation process.Each generation of P0-P10 cells was examined for sterility,and P2,P5,P10 cells were tested for microbiological safety of mycoplasma,endotoxin and specific human virus.The tumorigenicity of P10 generation was examined for the biological safety of cells.P5,P10 have the ability to differentiate into osteoblasts,lipoblasts and chondroblasts.(2)Study on the expression levels of VEGF,PDGF and bFGF in human umbilical cord mesenchymal stem cells:the first generation of resuscitated cells were cultured in 15%Fetal Bovine Serum medium;the second generation cells were cultured in 7.5%Fetal Bovine Serum medium;the third generation cells were cultured in 3.75%Fetal Bovine Serum medium.In the process of each generation culture,the old culture medium was replaced by the basic medium DMEM/F12 containing no serum to carry out gradient descending serum culture,and the serum was close to zero in the third generation after resuscitation.The expression levels of VEGF,PDGF and bFGF in cells or cell suspensions were detected by qPCR.By resuscitation of different generations of cells(P3,P8,P13)and culture under the same conditions,the expression levels of VEGF,PDGF and bFGF in different generations of cells were compared.Results:(1)The cell bank of human umbilical cord mesenchymal stem cells was established by this process.The cells were long fusiform and uniform in morphology,and the viability rate was higher than 95%before cryopreservation.The results of growth curve of P5 and P10 generation were consistent with the increasing trend of culture days before cell passage.The cell cycle test results showed that 80%of cells in P2,P5 and P10 generation were in G1 phase,with strong proliferation and division ability.The number of cells in S and G2 phase increased with the increase of generation,and the proliferation ability of cells decreased.Flow cytometry(FCM)to detect cell surface markers results accord with standard of the international association of cellular therapy(ISCT),positive surface markers CD73,CD90,CD 105,CD44,CD 166,the positive rate of no less than 95%,the negative surface markers CD11b or CD 14,CD 19,CD34,CD45 and HLA-DR,the positive rate is not higher than 2%;STR authentication showed that P2 and P5 generation cells were derived from the same individual,and there was no cross-contamination from other cells.The donor was from a healthy population,and the cell test results showed that there was no contamination of specific person viruses(HBV,HCV,HIV,HPV),bacteria,fungi,mycoplasma,or endotoxin.No tumorigenicity risk was detected in P10 generation cells by soft AGAR clonal formation assay.The effectiveness test results showed that the cells of P5 and P10 generation had the ability to differentiate into osteoblasts,lipoblasts and chondroblasts.The ability of osteoblast differentiation and chondroblast differentiation of P10 generation was weaker than that of P5 generation(P<0.05),and there was no significant difference in the ability of adipogenic differentiation in P5 and P10 generations,and it did not decrease with the increase of generations.(2)After "starvation" treatment,VEGF expression levels in 5.625%-cells and 5%FBS-suspension were significantly different from those in other groups;the expression levels of VEGF,PDGF and bFGF in P3,P8 and P13 all decreased with the increase of generation.The expression levels of PDGF were significantly different among all generations(P<0.01),and the expression levels of bFGF in P3 were significantly different from those in all higher generations(P<0.001).Conclusions:In this study,a simple,efficient and standardized preparation process was determined by optimizing the tissue adherence method,and the established cell bank provided a reliable source of cells for subsequent studies.Moreover,the preliminary quality evaluation system and standard operating procedures are suitable for the quality control of mesenchymal stem cells,ensuring the safety and effectiveness of cells,and laying a foundation for the establishment of large-scale cell bank and third-party testing platform for enterprises.The expression level of VEGF in 5%FBS-cell suspension was significantly different from that in 15%FBS-cell group and other groups.Whether the reason for the high expression was related to paracrine substances needs further analysis.The expression levels of VEGF,PDGF and bFGF decreased with the increase of cell generation,and low-generation cells should be selected to prepare conditional media using paracrine substances. |
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