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.