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The effects of cryopreservation on the expression of canine regulatory T-cell markers


Tarpataki, N; Wawrzyniak, M; Akdis, C A; Rückert, B; Meli, Marina L; Fischer, Nina M; Favrot, Claude; Rostaher, Ana (2017). The effects of cryopreservation on the expression of canine regulatory T-cell markers. Veterinary Dermatology, 28(4):396-e93.

Abstract

BACKGROUND Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. HYPOTHESIS/OBJECTIVES The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. ANIMALS Nine privately owned dogs. METHODS Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4(+) CD25(+) , CD4(+) FOXP3(+) and CD4(+) CD25(+) FOXP3(+) cell populations were calculated. RESULTS A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4(+) CD25(+) , CD4(+) FOXP3(+) and CD4(+) CD25(+) FOXP3(+) cell populations, respectively, was observed. CONCLUSIONS This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.

Abstract

BACKGROUND Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. HYPOTHESIS/OBJECTIVES The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. ANIMALS Nine privately owned dogs. METHODS Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4(+) CD25(+) , CD4(+) FOXP3(+) and CD4(+) CD25(+) FOXP3(+) cell populations were calculated. RESULTS A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4(+) CD25(+) , CD4(+) FOXP3(+) and CD4(+) CD25(+) FOXP3(+) cell populations, respectively, was observed. CONCLUSIONS This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Swiss Institute of Allergy and Asthma Research
05 Vetsuisse Faculty > Institute of Veterinary Pathology
05 Vetsuisse Faculty > Veterinary Clinic > Department of Farm Animals
05 Vetsuisse Faculty > Veterinary Clinic > Department of Small Animals
Dewey Decimal Classification:570 Life sciences; biology
630 Agriculture
Language:English
Date:19 March 2017
Deposited On:20 Apr 2017 14:46
Last Modified:19 Feb 2018 07:47
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:0959-4493
OA Status:Closed
Publisher DOI:https://doi.org/10.1111/vde.12438
PubMed ID:28317209

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