| Objective:In recent years,a large number of studies have demonstrated that small extracellular vesicles?sEVs?derived from human induced pluripotent stem cells?hiPSCs??hiPSCs-sEVs?play a therapeutic role in various diseases.However,there are no clear reports on the culture conditions and cell status of sEVs-derived donor cells.Therefore,the purpose of this study is to explore the culture conditions of hiPSCs in the extraction of hiPSCs-sEVs and whether they can maintain the normal state of the cells to ensure the extraction of biologically active sEVs while reducing the interference of foreign substances on sEVs.1.Determine the composition of the conditioned medium required to collect the culture supernatant according to the growth and differentiation status of the hiPSCs;2.Further determining the collection time of the culture supernatant;3.Modifying and optimizing the sEVs extraction methods;4.Verifying the physical characteristics of the extracted sEVs by detecting the shape and size;5.Verify the expression of surface marker proteins in sEVs;6.The biological activity of the extracted sEVs was demonstrated using HepG2 cells.Methods:1.The Preparation of the extracellular vesicle depleted knockout serum replacement?ED-KSR?by continuous 16h ultracentrifugation;2.Preparing the conditioned medium?CM?containing the different concentrations?0%?0.25%?0.5%?2%?5%?20%?;3.Removed and replaced with different concentrations of CM when hiPSCs were grown to a certain density,after 24 h,the cell morphology was observed by phase contrast microscopy,cell survival rate was determined by flow cytometry,and the protein concentration and band distributions of sEVs extracted from different concentrations of CM were determined by BCA and SDS-PAGE method respectively,and then determined the optimal concentration of ED-KSR.4.The culture supernatants were collected in 0.5%CM for 1 d,3 d,and 5 d respectively,then the cell viability of hiPSCs was measured by flow cytometry,alkaline phosphatase?ALP?staining,immunofluorescence staining of pluripotency markers OCT4,SEEA4 and SOX2,qRT-PCR analysis of pluripotency markers NANOG,OCT4 and SOX2 to detect pluripotency of hiPSCs and determine the suitable time to collect culture supernatants;5.Extract sEVs using modified ultracentrifugation?M-UC?and Exo-Quick kit?EQ?,and use transmission electron microscopy?TEM?,nanoparticle tracking analysis?NTA?and Western blot?WB?to verify the extracted sEVs;6.PKH67 labeled sEVs,and identified whether it can be taken up by the recipient cell HepG2;7.Different concentrations of sEVs were added to HepG2 cells to observe its effect on it and identify the biological activity of sEVs.Results:After replacing CM prepared with ED-KSR at 0%,0.25%,0.5%,2%,5%and 20%for 24 hours,the morphology of two hiPSCs was observed under phase contrast microscope,and found that the cell morphology of 0.5%,2%,5%and 20%CM did not change significantly,and all of them showed characteristic undifferentiated state compared with the cells cultured in stem cell culture medium.The data analysis of flow cytometry showed that the difference between the groups was not statistically significant?P>0.05?,however,the morphology of hiPSCs in 0%and 0.25%CM has changed slightly,and differentiation began to appear,the cell survival rate was also significantly lower than that of the control group?P<0.05?;The BCA results showed that the protein concentrations of sEVs derived from two hiPSCs collected in 0.5%,2%,5%and 20%CM were 4.36±0.53,7.5±2.0,10.2±1.2,14.99±1.12;4.36±0.54,7.14±0.68,10.67±1.84,13.63±0.06 respectively,and the difference between the groups was significant?P<0.05?,SDS-PAGE also showed that the protein in KSR had the least interference with 0.5%CM extracted sEVs,and the above results indicated that 0.5%ED-KSR was the suitable concentration;Subsequently,after continuously collecting the culture supernatant in 0.5%CM for 5days,the morphology of the cells was not significantly changed,and the results of flow cytometry showed that the survival rate of hiPSCs decreased slightly,but the data analysis showed that the difference was not statistically significant?P>0.05?;then alkaline phosphatase staining,OCT4,SEEA4 and SOX2 immunofluorescence,NANOG,OCT4 and SOX2 RT-PCR analysis showed that hiPSCs still expressed hiPSCs pluripotency markers after 5 days of collection,showed an undifferentiated state.Modified ultracentrifugation?M-UC?and Exo-QuickTM-TC kit?EQ?were used to extract sEVs,and TEM result showed that the extracted sEVs were all 50-200 nm,double-layered and oval or round cellular vesicles;the NTA results showed that the sEVs extracted by the two methods were respectively 186.6±2.6 nm,167.5±2.0 nm;western blotting showed that two hiPSCs-derived sEVs all expressed marker proteins CD63,TSG101 and HSP70,and did not express calreticulin.In addition,the PKH67staining experiments showed that the sEVs could be endocytosed by HepG2 cells and aggregated in a granular form in the cytoplasm,and a certain concentration of sEVs can promote cell proliferation.Conclusion:Under the condition of maintaining the pluripotency and survival rate of hiPSCs,0.5%ED-KSR is the optimal concentration of conditioned medium used to collect the supernatant of hiPSCs,and can be collected continuously for 5 days;Both M-UC and EQ methods can extract sEVs with biological activities that promote receptor cell proliferation,providing a basis for subsequent functional studies. |
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