| Adoptive cellular immunotherapy is a new promising clinical treatment strategy to eliminate cancer cells.Cytokine induced killer(CIK)is one of frequently-used immune effector cells.CIK cell-based therapy has acquired very positive results in clinical treatment of various blood and solid tumors.The clinical therapeutic effect of CIK cell-based therapy is closely related to the quantity and quality of infused CIK cells.Thus,because of the limited initial numbers of CIK cells in human cord and peripheral blood,how to get enough CIK cells with high antitumor activity in a short time becomes one of the key steps to achieve the desired effect.The conventional ex vivo expansion of CIK cells was carried out mainly in static culture with gas-permeable culture bags.However,because of the poor mixing effect in static cultures,it would result in the nutrient/metabolites concentration gradient and is hard to control,which not only lead to unstable cell product quantity and quality,but also lead to hardly repeated expansion process.In this paper,with the object to realize the efficient ex vivo expansion of CIK cells,a dynamic suspension culture process for ex vivo expansion of CIK cells was established,looking forward to solve the environmental heterogeneity and increase the mass transfer to improve the utilization efficiency of nutrients,which will also provide technical support for the large-scale,efficient,bioreactor based-ex vivo expansion CIK cells and other immune effector cells in the future.In this work,using cord blood mononuclear cells(CBMNC)as initial cell,the effects of static and dynamic cultures on ex vivo expansion of CIK cells were investigated in a serum-containing culture system.The results showed that cell viability and the percentages of subset cells was similar in two culture modes.On day 14,the percentages of CD3+cells increased from 23.42±9.43%to above 90%,the expansion folds of total cells in dynamic cultures achieved 69.36±30.36 folds,significantly higher than the 9.24±1.12 folds in static cultures(P<0.05).The main effector cells--CD3+CD56+ cells could expand 2579.47±2242.43 folds in dynamic cultures,obviously higher than the 505.80±510.21 folds in static cultures(P<0.05).Further analysis of the expression of three activation antigens CD69,CD25 and CD71,which were closely related to cell proliferation,in CD3+cell populations found that the percentages of CD3+CD69+ cells,CD3+CD25+ cells and CD3+CD71+ cells gated on CD3+ cell populations in dynamic cultures were all significantly higher than those in static cultures(P<0.05),thus,it was speculated that dynamic cultures prompt more CD3+ cells into the activation state,thereby enhance cell expansion efficiency.Moreover,the cytotoxic capacity on tumor cells,the expression of surface CD 107a and the intracellular perforin(PFN)and granzyme B(Gzm B),the secretion of TNF-? and IFN-? were investigated,the results showed that these indexes which were related to cell cytotoxicity of expanded CIK cells were all similar in two culture modes.Collectively,these results demonstrated that dynamic cultures could improve the expansion efficiency while maintaining comparable cell physiological functions.On this basis,the growth kinetics parameters and metabolic characteristics of cells expanded in static and dynamic cultures were analyzed and compared.The results showed that compared to static cultures,not only the maximal cell growth rate and the specific consumption rate of glucose,but also the yield coefficient of total cells,CD3+ cells and CD3+CD56+ cells to glucose were obviously higher in dynamic cultures,which demonstrating dynamic cultures improve the glucose metabolic rate of cells,the absorbed glucose was more used to support the rapid growth of the cells.Further,although the specific lactate production rate of cells in dynamic cultures was higher,the yield coefficient of lactate to glucose in cells of two cultures were both between 1.5 and 2,which indicating that the absorbed glucose was mostly converted to lactate.In order to further understanding the reason why cells in dynamic cultures accelerated glucose metabolic rate,from the perspective of glucose transport and metabolic enzyme activity in key metabolic pathway,the effect of culture modes on the regulation of glucose metabolism of CIK cells were analyzed.The results showed that compared to static cultures,the surface glucose transporter GLUT1 expression on expanded CIK cells in dynamic cultures were obviously enhanced,which pointed out that the glucose uptake ability was boosted;in addition,the activities of two key enzymes in glycolysis and pentose phosphate pathway,PFK and G6PDH in cells of dynamic cultures were improved by 60%and 62%,respectively.This results was in accordance with the results of the specific glucose consumption rates.Moreover,the relative activities of G6PDH/PFK were higher in cells of dynamic cultures,demonstrating that cells in dynamic cultures elevated glucose metabolic flux through pentose phosphate pathway,one of the main physiological function of pentose-phosphate pathway is to provide raw materials for synthetic biology,thus,this results was consistence with the results of the cell growth rate and yield coefficient of cells to glucose.In addition to glucose,glutamine is another important carbon and energy source for cell proliferation,thus,the glutamine metabolism of CIK cells expanded in two culture modes were also compared.The results found that compared with static cultures,cells in dynamic cultures possessed higher glutamine consumption rate,ammonia production rate and yield coefficient of total cells,CD3+cells and CD3+CD56+cells to glutamine,indicating that dynamic cultures enhanced the glutamine metabolic rate of cells,the absorbed glutamine was more used to support the rapid growth of cells.Further,it was found that the expression of glutamine transporters ASCT2,SNAT1,SNAT2 and CD98/LAT1 in cells of dynamic cultures were all up-regulated,demonstrating that the glutamine uptake ability of cells in dynamic cultures was enhanced.Meanwhile,the GLS and GDH activities in cells of dynamic cultures were found 20%and 23%higher than those in cells of static cultures,resp ectively,suggesting that dynamic cultures may accelerate the intracellular catabolism rate of glutamine,strengthen the supply of TCA cycle,improve the TCA cycle and ATP generation efficiency,so as to achieve the effect of promoting rapid cell growth.In order to confirm this inference,the ATP generation ability of CIK cells in static and dynamic cultures were investigated.Since glycolysis and oxidative phosphorylation are the main pathways for ATP synthesis in cells,and ECAR and OCR are effective indicators reflecting the ability of intracellular glycolysis and oxidative phosphorylation,the ECAR and OCR of cells under the two culture modes were detected in this paper.The results showed that compared to cells in static cultures,the basal ECAR and OCR of cells in dynamic cultures were enhanced by 33%and 301%,respectively,demonstrating that cells in dynamic cultures possessed higher glycolysis and oxidative phosphorylation ability,these results were in accordance with the higher glucose and glutamine consumption ability.By calculating the OCR/ECAR ratio,it was found that the OCR/ECAR ratio of cells in dynamic cultures was 3.36 times higher than those of cells in static cultures,indicating that the oxidative phosphorylation metabolism in cells of dynamic cultures was more vigorous.Since the efficiency of ATP generation by oxidative phosphorylation is much higher than that of glycolysis,the above results also indicated that ATP synthesis by cells in dynamic cultures was more efficient.To sum up,dynamic culture improved the nutrients uptake ability of cells,shifted the cell metabolism pattern to the direction of cell anabolism,which is more conducive to support the rapid and efficient cell expansion.Considering that peripheral blood derived CIK cells(PB-CIK cells)are more widely used in clinical trials and have different growth characteristics from cord blood derived CIK cells(CB-CIK cells).the dynamic cultures of PB-CIK were also conducted in this paper.The results showed that dynamic cultures significantly increased the proportion of CD3+CD8+cells,and other effector cells was similar to those of static cultures.After 14 days of culture,the proportion of CD3+ cells and CD3+CD56+ cells in the total cells in dynamic cultures reached 92.16±2.25%and 19.34±3.07%,respectively.Further,on day 14,the expansion folds of total cells and CD3+CD56+ cells reached 104.36±8.98 folds and 522.33±47.98 folds in dynamic culture,which were both higher than those in static culture(15.07±1.46 folds and 82.00±27.73 folds).In addition,dynamic cultures enhanced the cytotoxic capacity of PB-CIK cells on K562 cells by increasing the proportion of PFN+ cells and Gzm B+cells,indicating that dynamic cultures enhanced the physiological function of expanded PB-CIK cells.Therefore,dynamic cultures can achieve efficient ex vivo of CIK cells from different sources.In this paper,the CIK cell growth and metabolism characteristics and difference in static and dynamic cultures were studied in detail,the mechanism which may lead to cell growth and metabolism difference was deeply discussed.These results would provide great guiding significance for the subsequent establishment of bioreactor-based,large-scale ex vivo expansion process of immune effector cells and for the design of serum free medium. |
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