Study On Optimization Of Culture Technology Of Peripheral Blood Mononuclear Cells In Vitro Based On Novel Fluorescence Analyzer

Chimeric antigen receptor T-cell immunotherapy(CAR-T)is a new type of precise targeted immunotherapy,which has been successfully used for tumor treatments.Its T cells are mainly derived from human peripheral blood mononuclear cells(PBMCs).Therefore,in the process of preparing CAR-T cells,it is necessary to isolate PBMCs from the patient's blood,and then categorize and enrich T cells,later activate and genetically modify them to construct CAR-T cells.In the process of isolation and in vitro culture of PBMCs,critical quality attribute(CQA)parameters such as cell density,cell viability and cell apoptosis ratio are required to be strictly monitored.To this end,the current PBMCs cultivation process has long detection time and low counting accuracy.Herein,this paper work is based on the novel(CountStar(?)Rigel,CSR)fluorescence analyzer to study the rapid detection method of the quality parameters of PBMCs cell culture process,including cell density,cell viability,cell apoptosis and other parameters.At the same time,the optimization of PBMCs in vitro static culture conditions was studied in the T flask,including cytokines,serum concentration,feeding strategy,etc.,and then the cell culture process of PBMCs was studied in the spinner flask.Finally,the inhibitory effect of PBMCs stimulated by cytokines on HeLa cells was studied.The results are listed as follows:(1)The feasibility study of CSR fluorescence analyzer to detect the cell density,cell viability and apoptosis of PBMCs and Jurkat.By comparing with the optical microscope(OM)analysis,the CSR fluorescence analyzer performs t-test,Passing-Bablok regression analysis and Bland-Altman consistency analysis on PBMCs and Jurkat cell density and cell viability data.Moreover,in the detection of PBMCs and Jurkat cell density and cell viability,the results of CSR and OM have a highly reliable correlation,linearity and consistency.So,the detection methods of CSR fluorescence analyzer and OM can replace each other.Compared with optical microscope,the detection process of CSR fluorescence analyzer is more rapid,simple and standardized.By comparing with the flow cytometer(FCM)analysis,the CSR fluorescence analyzer performs t-test,Passing-Bablok regression analysis and Bland-Altman consistency analysis on the apoptosis data of PBMCs and Jurkat cells.The results showed that,in the detection of apoptosis of PBMCs and Jurkat cells,only in the detection of drug-induced Jurkat viable cells(Drug-Viable),CSR fluorescence analyzer and FCM have correlation,linearity and consistency,while heat-induced PBMC unosort cells(Heat-Unsort),drug-induced Jurkat early apoptosis(Drug-Early),drug-induced Jurkat late apoptosis(Drug-Late),and drug-induced Jurkat unsorted cells(Drug-Unsort),etc.The correlation,linearity and consistency of the results obtained by CSR fluorescence analyzer and FCM detection are not very high.Therefore,it is concluded that CSR fluorescence analyzer can not replace flow cytometry to detect PBMCs and Jurkat cell apoptosis.However,the follow-up exploration and improvement of the CSR fluorescence analyzer to detect cell apoptosis provide a reference for the preliminary results.(2)Optimization of cell culture of PBMCs in vitro.In the process of static culture of PBMCs in T flasks,timely supplementation of interleukin-2(IL-2)can further stimulate the proliferation of CD3+CD8+ T cells.The effect of adding ABS serum concentration on the proliferation and growth of PBMCs was studied.The results showed that there was no significant difference in the proliferation of PBMCs with the addition of 2.0%-5.0%ABS serum concentration(p<0.05).The choice of feeding strategy,this experiment comparatively studied strategy 1(Batch),strategy 2(Half-exchange medium)and strategy 3(Fed-batch).Compared with the other two groups of feeding strategies,adopting strategy 3(Fed-batch)feeding strategy is conducive to the proliferation of PBMCs.The final cell density,cell viability,and specific growth rate of the flow-feeding strategy were significantly higher(p<0.05)than strategy 1(Batch)and strategy 2(Half-exchange medium).The main metabolites glucose,lactic acid,glutamine,ammoniumion and other parameters were analyzed,and it was found that the feeding strategy can effectively control the lactic acid and ammonium ion in the medium,in order to maintain the concentration of glucose and glutamine in the culture medium,it was beneficial to the proliferation and growth of PBMCs.The PBMCs were further cultured in spinner flask,and the results were comparable to the average specific growth rate,cell expansion ratio and T cell expression of PBMCs cultured in T flasks(p>0.05).This indicates that dynamic PBMCs culture can be carried out in a spinner flask,which provides preliminary guidance for the further scale-up and cultivation of PBMCs in larger-scale bioreactors.(3)The inhibitory effect of expanded PBMCs on HeLa cells stimulated by cytokines was studied.The co-culture system was used to co culture PBMCs and HeLa cells stimulated by cytokines:PBMCs stimulated by cytokines significantly inhibited HeLa cell proliferation and promoted its apoptosis(p<0.05).The kill rate of IL-2 group was significantly higher than that of control group(p<0.05).These results indicate that PBMCs stimulated by cytokines have strong inhibitory effect on HeLa cells in co culture system.