| Adipose stem cells are a kind of stem cells with multidirectional differentiation potential isolated from adipose tissue in recent years.They have a wide range of sources and large reserves.Under certain induction conditions,adipose stem cells can differentiate into osteoblasts,cartilage,fat and other cells.In addition,adipose-derived stem cells are easy to obtain,suitable for large-scale culture,and have little damage to the collective,so they have gradually become one of the new research hotspots in recent years.Adipose stem cells are important seed cells in tissue engineering,especially in repairing bone or cartilage tissue damage.However,microbial contamination or genotype changes sometimes occur during long-term culture.In order to obtain mesenchymal stem cells from convenient sources,carry out effective stem cell transplantation and cell bank establishment,cryopreservation is often used to maintain the biological activity of stem cells for a long time in order to achieve the purpose of on demand.Studies have shown that dimethyl sulfoxide(DMSO),trehalose and L-Carnitine have better cryoprotective effects on cells.In order to establish and optimize cryopreservation technology of adipose-derived stem cells,dimethyl sulfoxide,trehalose and L-carnitine were used as cryopreservation agents in this experiment.The cell viability and cell cycle of resuscitated adipose-derived stem cells were detected and analyzed,and the results were reversed.1.Cell viability test results showed that the recovery effect of the control group cells after cryopreservation was better than that of the experimental group,and the cryopreservation effect of different cryopreservation agents was also different.The control group(5% DMSO + 10% FBS)showed the best cryopreservation effect.After 1,3 and 7 days of liquid nitrogen cryopreservation,the cell viability was 0.413,0.267 and 0.218 24 hours after resuscitation,and 0.532,0.366 and 0.256 48 hours after resuscitation,respectively.The cell viability of the control group(5% DMSO),3(5% T + 10% FBS)and 5(5% L + 10% FBS)was higher than that of the experimental group(5% L + 10% FBS).2.Cell cycle test results showed that after cryopreservation and resuscitation,the cells in G1 phase accounted for the largest proportion and those in G2 phase accounted for the smallest proportion.After further analysis of S-phase cells,it was found that after short-term cryopreservation(1-3 days),the proportion of S-phase cells in No.2 group was the largest,27.38% and 26.34% respectively,which was significantly higher than that in control group(11.15% and 17.24%).When the freezing time was prolonged to 7 days,the proportion of cells in S phase was the largest in the control group,which was 3.35%.The effect of the experimental group 5 was similar to that of the control group,but slightly lower than that of the control group,which was 3%.3.Mesenchymal stem cells can be induced into osteoblasts by adding induction solution in the culture medium.Osteoblasts can be induced to become red by oil red O staining.The deeper or more red,the more cells are induced.The frozen storage agent was 5% DMSO + 10% FBS for 1 day,followed by 5% DMSO + 10% FBS for 5 days,and the lure agent was 5% L + 10% FBS for the least.Deposit for 1 days.4.The results of transcriptome sequencing and bioinformatics analysis showed that the gene expression profile of adipose-derived stem cells changed after cryopreservation,which indicated that cryopreservation did affect gene expression of cells.In this study,we compared the effects of different cryopreservation agents on cell viability and cell cycle by detecting the biological characteristics of cells after resuscitation under different cryopreservation agents,and analyzed the effects of 5% DMSO + 10% FBS cryopreservation agents on the expression profile of adipose stem cells through transcriptome data analysis,which laid a foundation for making full use of stem cells for artificial tissue construction,cell transplantation and other aspects of research.A certain basis. |
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