Isolation And Identification Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells And Its Effect On Osteosarcoma Saos-2 Cells

Objective:In this study,umbilical cord mesenchymal stem cells(hUC-MSCs)were isolated from human umbilical cord tissue by tissue adherence method,exosomes were separated and identified by precipitation method,so as to explore the extraction and identification method of human umbilical cord mesenchymal stem cell derived exosomes(hUC-MSCs-exo),laying a foundation for future studies.The effect of hUC-MSCs-exo on the growth of osteosarcoma Saos-2 cells was further investigated.Methods:With the approval of the ethics committee of the hospital,fresh qualified human umbilical cord tissue samples were obtained from the delivery room.hUC-MSCs cells were obtained by tissue adherence method.Mesenchymal stem cells(MSCs)were cultured,subcultured,cryopreserved and resuscitated.hUC-MSCs-exo was separated and identified by precipitation method.Flow cytometry was used to detect exosome surface marker proteins CD63 and CD81.The effect of hUC-MSCs-exo suspension on osteosarcoma Saos-2 cells was further investigated.Saos-2 cells were stained with DAPI fluorescent nuclei,and exosomes were labeled with PKH26 fluorescence.The labeled hUC-MSCs-exo solution was added to the culture medium of osteosarcoma Saos-2 cells for 24 h,and fluorescence microscope was used to observe whether the exosomes could be absorbed by osteosarcoma Saos-2cells.hUC-MSCs-exo suspensions of different concentrations were collected and prepared,and the experiment was divided into four groups:(a)the exosomes group:200,400,and 800 cells were added by dropping exosomes suspension on osteosarcoma saos-2 cells.(b)autophagy inhibition group: when dropping 200 living g/ml exosome suspension,add autophagy inhibitor 3-MA;(c)control group: the same volume of serum-free medium was added,and the remaining conditions were the same as above.The proliferation,migration and invasion capacity of osteosarcoma Saos-2 cells were detected by cell counting,CCK-8 assay,and Transwell assay.Thechange of Saos-2 autophagy level was detected by MDC autophagy staining kit,and the expression of Beclin1,a marker protein of autophagy,was detected by Western blot.Results:hUC-MSCs cells were successfully isolated by tissue mass adherent method,and the supernatant precipitation of hUC-MSCs was successfully isolated by precipitation method.Flow cytometry was used to identify the supernatant precipitation,and the results showed that the extracted supernatant precipitation enriched CD81 and CD63 proteins,indicating a high concentration of hUC-MSCs-exo in the extracted supernatant precipitation.After PKH26-labeled exosomes were co-cultured with tumor cells for 24 h,fluorescence microscope observation confirmed that Saos-2 cells had an uptake effect on exosomes.Cell counting and CCK-8 experiments showed that compared with the control group,the MSCs-exo group could promote the proliferation of osteosarcoma Saos-2 cells,and the proliferation ability of Saos-2 cells was enhanced with the increase of the concentration of exosome suspension.However,the number of cells and OD values in the exo+3-MA group were significantly lower than those in the exosome group.Transwells assay detected the cell migration and invasion of osteosarcoma in each group.Microscopically,the number of transmembrane penetrating cells in the exosome group was significantly higher than that in the control group,and with the increase of exosome concentration,the migration and invasion capacity of osteosarcoma cells were significantly enhanced.In the exo+3-MA group,the migration and invasion capacity of Sao-2 cells were significantly reduced compared with the exosome group.MDC staining was used to re-examine the autophagy situation,and it was found that the number of fluorescent spots increased significantly with the concentration after exosome treatment.Western blot analysis showed that the gray level of Beclin1 increased significantly with the increase of MSCs-exo concentration.Conclusion:(1)In this experiment,hUC-MSCs were obtained by tissue mass adherent culture method,and the supernatant of hUC-MSCs was separated by precipitation method,which was later identified as hUC-MSCs-exo by flow cytometry,indicatingthat our team successfully mastered the method to extract exosomes with appropriate concentration.(2)hUC-MSCs-exo can activate autophagy on osteosarcoma Saos-2 cells,promoting the growth and invasion of the cells,and its ability to act is enhanced with the increase of exosome fluid concentration.