Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

  • Monica Vuță
  • Ionela Maria Cotoi "George Emil Palade” University of Medicine, Pharmacy, Science and Technology of Târgu-Mureș
  • Ion Bogdan Mănescu
  • Doina Ramona Manu
  • Minodora Dobreanu
Keywords: Stimulated T cells, PBMC, cryopreservation, cytokine, intracellular cytokine staining



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 and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells.


PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140ᵒC for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry.


Both Fresh and Cryo CD3+CD4+/CD3+CD4- subpopulations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells.


Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

How to Cite
Vuță M, Cotoi I, Mănescu I, Manu D, Dobreanu M. Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells. amm [Internet]. 19Mar.2021 [cited 17Sep.2021];67(2). Available from:
Original article