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The effects of cryopreservation on the expression of canine regulatory T‐cell markers
Author(s) -
Tarpataki Noemi,
Wawrzyniak Marcin,
Akdis Cezmi A.,
Rückert Beate,
Meli Marina L.,
Fischer Nina M.,
Favrot Claude,
Rostaher Ana
Publication year - 2017
Publication title -
veterinary dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.744
H-Index - 60
eISSN - 1365-3164
pISSN - 0959-4493
DOI - 10.1111/vde.12438
Subject(s) - cryopreservation , flow cytometry , andrology , peripheral blood mononuclear cell , biology , cell , immune system , immunology , phenotype , microbiology and biotechnology , medicine , embryo , in vitro , gene , genetics
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 CD 4 + CD 25 + , CD 4 + FOXP 3 + and CD 4 + CD 25 + FOXP 3 + 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 CD 4 + CD 25 + , CD 4 + FOXP 3 + and CD 4 + CD 25 + FOXP 3 + 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.