Application of leukocyte filters to obtain a preparation of cytokine-induced killer cells

The ability of cytokine-induced killer (CIK) cells to recognize and lyse tumor cells has been shown in preclinical in vitro studies. The database ClinicalTrials.gov currently registers over 600 completed clinical studies on cellular immunotherapy including more than 30 trials with CIK cells. There are many ways to obtain the lymphocyte-based cell products. In our opinion, their common drawback is the need for additional blood samples to obtain cell preparation in large volumes, thus requiring the development of new approaches in the field. We have proposed an idea of lymphocyte extraction using leukocyte filters with subsequent in vitro activation of these cells. The purpose of our study was to test the opportunity of using leukocyte filters as a source of lymphocytes to obtain large doses of CIK. The study included data from 30 donors. The article describes in detail the procedure for extracting lymphocytes from leukocyte filters. The isolated lymphocytes were cultured in plastic bottles in the CO₂-incubator for 10-14 days in a medium based on RPMI-1640 supplied with IL-2 and IL-15. The level of cell activation was assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The subpopulation profile of T, NK, TNK, B lymphocytes (CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD56) and surface activation markers (CD38 and HLA-DR) before and after cell activation were assessed. We have assessed the possibility of using leukocyte filters as a tool of cell separation from leukocyte-rich plasma. The functional activity of extracted lymphocytes and cytokine contents of supernatants were studied after cultivation in a complete nutrient medium. The supernatant peptide complex was shown to contain high doses of signaling proteins, i.e., IL-2, IL-6 IL-10, IFNγ, TNFα and retained its functional activity after long-term storage at low temperatures. The resulting cell product is sterile and consists predominantly of T, TNK and NK cells with a high level of viability and expressed activation markers (HLA-DR, CD38). Each single filter procedure allowed getting of up to 350 million activated lymphocytes and supernatant aliquots. This preparation may be used both for basic research and for administering autologous immune therapy to cancer patients in order to restore the patients’ own antitumor immunity potential. This amount of cellular and cell-free products is sufficient to conduct more courses of immune therapy without additional blood sampling.

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