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Volume 1 Supplement 1

Abstracts of the 28th Annual Scientific Meeting of the Society for Immunotherapy of Cancer (SITC)

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Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT

ELISPOT assays are routinely used to measure immune responses of T cells in fresh and frozen splenocytes preparations; however, standardized methods for murine ELISPOT are not widely available. Freezing cells can significantly impact the function of T cells. The aim of this study was to optimize cryopreservation protocols to retain antigen-specific T cell function at similar levels as freshly isolated T cells using murine splenocytes. We examined four factors that might have an impact on cell viability and function: freezing medium, resting cells prior to freezing, temperature of the medium for initial dilution of cells after thawing and resting of cells prior to ELISPOT. FVB/N mice were vaccinated with adjuvant only (CFA/IFA) or IGF-IR peptides, previously shown to be immunogenic by ELISPOT analysis. Mouse splenocytes were cryopreserved using five different media: Medium 1 (50% X-Vivo media, 40% FBS, 10% DMSO), Medium 2 (25% RMPI, 65% FBS, 10% DMSO), Medium 3 (90% FBS, 10% DMSO), Medium 4 (Amresco Media) and Medium 5 (EZ-CP2 Media). Prior to freezing, the cells were either rested on ice for 5 hours or immediately frozen and moved to liquid nitrogen. After four weeks, we used media at 37°C or 4°C for initial dilution of cells after thawing. The thawed cells were either rested overnight at 37°C or not rested. The recovery efficiency and T cell function were evaluated by determining cell viability, background levels, peptide responses, and mitogen responses in ELISPOT assays, respectively. We observed significantly lower cell viability when using freezing Medium 4 and 5, compared to the other media tested (Medium 5 vs Media 1-4: P<0.001; Medium 4 vs 1: P<0.001, Medium 4 vs 2 P<0.05). Resting cells for 5 hours prior to freezing resulted in higher viability, however this difference was not significant (P=0.072). Using media warmed to 37°C to dilute thawed samples resulted in significantly higher cell viability as compared to using media at 4°C (P<0.0001). Media 2 and 3 gave significantly lower background than Fresh cells (P<0.05). Medium 3 was the only freezing medium to result in similar levels of IGF1R peptide responses compared to Fresh. Overnight resting of cryopreserved cells before ELISPOT gave significantly lower T cells responses in both PHA and PMA/ionomycin controls compared to unrested cryopreserved cells (P<0.001).However, overnight resting of cells gave mitogen responses most similar to that of fresh cells when PHA or PMA/ionomycin controls were used. The method of cryopreservation can have a tremendous impact upon splenocytes viability and function. By using an optimized cryopreservation protocol, it is possible to obtain antigen-specific T cell function at levels similar to freshly isolated cells.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Gad, E., Rastetter, L., Herendeen, D. et al. Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT. j. immunotherapy cancer 1 (Suppl 1), P211 (2013).

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