<abstract xmlns="http://www.w3.org/1999/xhtml">

<p><bold>Objective:</bold> 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. <bold>Methods</bold>: 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 -140°C 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. <bold>Results:</bold> Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations 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. <bold>Conclusion:</bold> Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.</p>
</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 and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: 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 -140°C 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. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations 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. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

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

George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures

Acta Marisiensis - Seria Medica

This work is licensed under the Creative Commons Attribution 4.0 International License.

Cytokine production in  stimulated fresh and cryopreserved T-cells

Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be...

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

Publié en ligne: 12 juil. 2021

Pages: 95 - 101

Reçu: 22 févr. 2021

Accepté: 15 mars 2021

DOI: https://doi.org/10.2478/amma-2021-0012

Mots clés
stimulated T cells, PBMC, cryopreservation, cytokine, intracellular cytokine staining

© 2021 Monica Vuță et al., published by Sciendo

This work is licensed under the Creative Commons Attribution 4.0 International License.

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

Publié en ligne: 12 juil. 2021

Pages: 95 - 101

Reçu: 22 févr. 2021

Accepté: 15 mars 2021

DOI: https://doi.org/10.2478/amma-2021-0012

Mots clésstimulated T cells, PBMC, cryopreservation, cytokine, intracellular cytokine staining

© 2021 Monica Vuță et al., published by Sciendo

This work is licensed under the Creative Commons Attribution 4.0 International License.

Mots clés
stimulated T cells, PBMC, cryopreservation, cytokine, intracellular cytokine staining