| Amino acids are important part of the serum-free medium,and the types and concentrations of amino acid are of great significance to the proliferation and activation of immune cells.Therefore,in this article Natural Killer Cells(NK-92)were regarded as the research object to study the relationship between NK-92 cells expansion and amino acid metabolism in vitro to designe and optimize serum-free medium.Firstly,the amino acid metabolism characteristics of NK-92 cells during the cultivation process were analyzed in the self-designed serum-free medium SFM-MIN with simple and clear composition.According to the specific consumption rate of amino acids,glutamine,serine,leucine and arginine were determined as the key amino acid affecting the expansion of NK-92 cells in vitro,and the optimal concentrations of the four amino acids were determined through orthogonal experiments which were 13.00,0.40,1.15 and 0.70 mmol/1.The new medium which was adjusted the concentration of the four amino acids to the optimal concentration was named SFM-OPT,and the commercial serum-free medium was used as a control to evaluate the expansion effect of SFM-OPT on NK-92 cells.The results showed that after 10 days of cultivation in vitro,the total cell expansion in SFM-OPT and the proportion of CD3-CD56+cells were 164.06±35.94 folds and(88.70±1.47)%,which were significantly higher than 54.25±12.14 folds and(69.83±3.11)%in the control group(p<0.05)while the cytotoxicity to K562 cells of the expanded NK-92 cells in the two serum-free mediums was comparable.It was found that the SFM-OPT medium with optimized amino acid concentration can promote the expansion of NK-92 cells effectively without affecting its function in vitro.Further,the concentration of glutamine in SFM-OPT had been change to investigate the effect of glutamine on the expansion of NK-92 cells.It was found that after increasing the glutamine concentration to 13.00 mmol/1 in SFM-OPT and culturing for 10 days,the expansion folds of NK-92 cells and the proportion of CD3-CD56+cells were 161.87±21.02 folds and(91.23±1.33)%respectively,which were significantly higher than those of the control group 55.55±7.00 folds and(73.23±0.85)%(p<0.05);the cytotoxicity to K562 cells of expanded NK-92 cells,the expression of CD 107a,perforin and granzyme B were also significantly higher than that of the control group(p<0.05);Compared to the control group,the apoptosis ratio and intracellular ROS level in NK-92 cells were significantly reduced(p<0.05);And the intracellular GSH/GSSG and NADPH/NADP+ were significantly increased(p<0.05).Thereby,it was illustrated that increasing the concentration of glutamine could maintain the intracellular redox state better and reduce the intracellular ROS level to promote the expansion of NK-92 cells and enhance their cytotoxicity in vitro.In view of the fact that NK-92 cells were a kind of high energy-consuming metabolic supporting cells,the effects of sodium pyruvate and glutamine on the expansion of NK-92 cells in vitro were investigated.The optimal concentrations were determined to be 2.50 and 13.00 mmol/1 by orthogonal experiments based on SFM-MIN.And the commercial serum and serum-free media were selected to increase the concentration of sodium pyruvate and glutamine to the optimal levels to verify the results.It was showed that after 15 days of culture,in the serum culture system,the total cell expansion folds of NK-92 cells were up to 3712.85±225.74 folds,which was significantly higher than 2255.93±243.00 folds in the control group(p<0.05)and the proportion of CD3-CD56+ cells had no significant difference in the two groups of cultures modes.But the average killing activity of NK-92 cells were increased from 57.23%to 63.53%.In serum-free medium of T009,the expansion folds of NK-92 cells and the proportion of CD3-CD56+ cells were up to 3193.59±199.99 folds and(90.37±0.90)%,which were significantly higher than 1917.16±242.87 folds and(82.68±2.28)%(p<0.05)in the control group.and the cytotoxicity of NK-92 cells were also increased from 63.53%to 71.22%.It could be summarized that increasing the concentration of sodium pyruvate and glutamine can promote the expansion of NK-92 cells and enhance their killing activity. |
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