Alterations in Human Liver Metabolome during Prolonged Cryostorage.

Tissue metabolomics requires high sample quality that crucially depends on the biobanking storage protocol. Hence, we systematically analyzed the influence of realistic storage scenarios on the liver metabolome with different storage temperatures and repeated transfer of samples between storage and retrieval environments, simulating the repeated temperature changes affecting unrelated samples stored in the same container as the sample that is to be retrieved. By cycling between storage (-80 °C freezer, liquid nitrogen, cold nitrogen gas) and retrieval (room temperature, -80 °C), assuming three cycles per day and sample, we simulated biobank storage between 3 months and 10 years. Liver tissue metabolome was analyzed by liquid chromatography/mass spectrometry. Most metabolite concentrations changed <5% for the first "year" of time-compressed biobanking simulation, predominantly due to hydrolysis of peptides and lipids. Interestingly, storage temperature affected metabolite concentrations only little, while there was a linear dependence on the number of temperature change cycles. Elevated sample temperature during (prolonged) retrieval time led to a distinctly different signature of metabolite changes that were induced by cycling. Our findings allow giving recommendations for optimized storage protocols and provide signatures that allow detection of deviations from protocol.

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

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.