Cytokine production in <i>ex-vivo</i> stimulated fresh and cryopreserved T-cells

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability functionality. This study aims to investigate synthesis in ex-vivo fresh cryopreserved CD4+ CD4- T cells. Methods : PBMCs were obtained Ficoll gradient centrifugation from heparinized 6 middle-aged clinically healthy subjects. Half these (labeled “Fresh”) was further processed the other half “Cryo”) at -140°C up 3 months. Fresh-PBMCs activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin 5 hours immediately while Cryo-PBMCs identically thawing resting. Activated fixed, permeabilized intracellular staining performed using Phycoerythrin (PE)-conjugated antibodies Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), Interferon-gamma (IFN-g). All samples analyzed within 24 flow cytometry. Results: Both Fresh Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each three cytokines. A higher percentage IL-2 TNF-a a greater found produce IFN-g. significantly Cryo-lymphocytes shown both CD3+CD4+ (31.4% vs 24.9%, p=0.031) CD3+CD4- (22.7% 17.9%, subpopulations. No notable difference IFN-g between Conclusion: Cryopreservation months increases T-cells