The Establishment Of Standard Operating Procedure Of Immune Cells Derived From Human Peripheral Blood Cultured In Vitro And Preliminary Study On Immune Function Of Individual Differences

Dendritic cell (DC) and cytokine induced killer cell (CIK) are playing a key role in tumor immunotherapy. As the most potent antigen-presenting cells (APC), DCs recognize tumor antigen through major histocompatibility complex (MHC) restricted manner to activate antigen-specific T lymphocyte to kill the tumor cells. CIKs directly kill the tumor cells by non-MHC restricted manner. However there are scarce DCs and CIKs in human peripheral blood. So culturing and amplification DCs and CIKs be qualified and with full quantity in vitro by using the precursor cells in peripheral blood, is the premise on treatment cancer patient safely and effectively.Objective:the aim of the first part of this study is to obtain clinical grade cell product, so that ensure the safty and effectively on treatment cancer disease, standard operating procedures (SOPs) and quality control standards of process on culturing DCs and CIKs in vitro need to be set up in our laboratory. The second part is to support the concept of individual differences on immune system, and provide the experimental basis for individualized immunotherapy of cancers. We compared the absolute number, cell phenotype and cell function of DCs and CIKs cultured in vitro between two cancer patients and among five healthy adults respectively.Method:with reference to documents of "the guiding for human somatic cell therapy research and the quality control on cell preparation","Management of autologous cell immunotherapy" in China,"Chinese Pharmacopoeia", EU GMP guiding and "Implementation Guiding for GMP in China", we developed the SOPs about the whole process of preparing DC and CIK in vitro, including the collecting, separating and cryopreservation of precursor cells PBMCs, culturing and amplification of DCs and CIKs, and phenotypic and functional identification of these immune cells. Following these SOPs we also developed quality control standards from cell culturing in vitro to testing performed during culturing and after culturing. We compared the differences between individuals with tumor or not on a series of immune-related parameters of PBMCs,DC and CIK.Result:1. we have established a series of SOPs and quality control standards of clinical grade DC and CIK in GMP condition in our laboratory.2. We have found that there was no significant difference between autologous plasma and FBS or human AB serum culture condition on DC phenotype and function.3. We have found that there was no significant difference between adherent DCs and traditional non-adherent DCs on DC phenotype and function. So when harvest, it is no need to discard the adherent DC.4. The preliminary studies of individual differences have shown that there were some differences about the number, phenotype and function of immune cells between individuals.Conclusion:the successfully establishment of SOPs and the quality control standards on DC and CIK culture in vitro under GMP condition is the premise of production clinical-grade DC and CIK, it provides security guarantees for the clinical application of DC and CIK, it also meets the requirements of the regulations. The results of individual differences study suggested that there is a possible to immunotherapy individually in the future.