Preliminary Study On Suspension Culture Of Effector Cells And Optimization Of Serum-free Medium Based On Bioreactor

In vitro expansion is one of the effective ways to solve the problem of insufficient cell dose for treatment,impacts of which mainly come from the medium and the culture device.Therefore,in vitro expansion of cytokine-induced killer cells(CIK)and hematopoietic stem cells(HSC)were optimized from two perspectives of culture medium and culture device in this paper.In this paper,the effect of monosaccharide species such as glucose,mannose and glucuronic acid and their combinations on the in vitro expansion of CIK cells were determined,and the optimal additive amount was further determined based on orthogonal experiments.The results indicated that when 2.2 mmol/L mannose and 0.10 mmol/L glucuronic acid are used in combination,the expansion fold of total cells was up to 93.2825.73 after 14 days of CIK cell culture in vitro,which was significantly higher than that of the control group(45.56±4.38)(p<0.05).The percentage of CD3+,CD3+CD56+,CD3+CD8+cells in culture was comparable to that of the control group(p>0.05).The expansion fold of CD3+,CD3+CD56+,CD3+CD8+cells was 269.40±59.98,473.47±112.37,453.38±91.43,which were significantly higher than those of the control group(127.12±21.86,177.12±23.79,226.93±21.66)(p<0.05).Therefore,the combined application of mannose and glucuronic acid is able to effectively promote the expansion of CIK cells.The percentage of Gzm B++cells in CD3+and CD3+CD8+cells were(61.50±17.11)%and(70.23±18.05)%,and the percentage of Perforin+cells in CD3+ and CD3+CD56+cells were(77.00±7.50)%and(69.63±6.90)%,which both were significantly higher than those of the control group(p<0.05).After 14 days of expansion,the killing activity of CIK cells against K562 cells was(35.77±10.79)%,which was also significantly higher than that of the control group(22.27±6.16)%(p<0.05),indicating that the combined application of mannose and glucuronic acid could enhance tumor killing activity of expanded CIK cells.Hematopoietic stem cells are mainly enriched in CD34+cells.In this paper,we attempted to establish a suspension culture system suitable for CD34+ cells in vitro.KG-1a cells were used as the model cell to investigate the effect of dynamic culture device and stirring mode on the in vitro expansion of KG-la cell based on the preliminary evaluation of the feasibility of CD34+ cell suspension culture.The results indicated that the use of pendulum stirring paddle and the stirring speed of 15 rpm in the spinner flask culture system were more favorable for KG-la cell expansion,and then CD34+cells were used for verification.Therefore,in vitro suspension culture system of CD34+ cells was initially constructed.Based on the above results,further attempts were made to explore the possibility of culturing CIK cells using the MINIFOR bioreactor.The above results provide technical support for the optimization of in vitro expansion techniques for CIK cells and CD34+cells.