title: Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.
abstract: The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions.
authors.affiliation: (a)Fraunhofer Institute for Biomedical Engineering, Ensheimerstr. 48, 66386 St. Ingbert, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Germann
authors.forename: Young-Joo
authors.initials: YJ
authors.lastname: Oh
authors.forename: Tomm
authors.initials: T
authors.lastname: Schmidt
authors.forename: Uwe
authors.initials: U
authors.lastname: Schön
authors.forename: Heiko
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Hagen
authors.initials: H
authors.lastname: von Briesen
mesh_terms: D001793:Blood Preservation / Q000295:instrumentation / Q000379:methods*; D002470:Cell Survival; D003080:Cold Temperature; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005615:Freezing; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
keywords: Cryopreservation; ELISpot; Optimal sample storage; PBMC functionality; PBMC recovery