131 A novel liquid nitrogen-free snap freezer INTRODUCTION Genomic, transcriptomic and (phospho)proteomic profiling of tumor biopsies plays an increasingly important role in translational cancer research and precision oncology, the selection of therapy for patients with cancer based on their molecular tumor profile1-3. Standardized high-quality (cryo)preservation to most accurately harness tumor biology of assessed tissue is a prerequisite for the generation of complex DNA, RNA and protein data4,5. Cryopreservation of cells and tissues demands swift cooling to sub-freezing temperatures at which biological and enzymatic processes are slowed down or completely stopped6,7. Liquid nitrogen (LN2, -196 °C or 77K), or pre-cooled isopentane (often -80 °C) are preferred coolants to control cooling rate and prevent cryo-artifacts in tissues, allowing their structural and biochemical preservation8-11. Tumor biopsies collected for research and precision oncology purposes are generally placed in a cryovial by trained staff and immediately immersed in LN2, This process is referred to as snap freezing and currently the golden standard12. Snap freezing is a laborious, potentially hazardous, and not user-friendly procedure. In addition, LN2 is not widely available and the use of sacrificial LN2 is non-sustainable due to its energy-intensive synthesis. There is an unmet need for a snap freezing device without these limitations that allows standardized optimal conservation of core needle biopsies or resected tissue for molecular profiling purposes. We have previously described an electrically powered, novel snap freezer that is not reliant on LN2 and has adjustable cold sink temperature that will influence the cooling rate8,13. This apparatus consists of a cryocooler, Thermal Energy Storage Unit (TESU) and a gas handling system, which is transportable and easy to handle. Cooling occurs through a narrow gas-gap between the cryovial and the thermal reservoir holding the vial. Recently, we showed that the cooling rate of a vial depends on the thermal properties of the vial material (e.g. aluminum, polypropylene) and on the coolant used. The cooling rate for a LN2-frozen tissue biopsy in an aluminum vial was about -25 °C/s13. We hypothesize that this novel snap freezer will preserve quality and molecular profiles of tissue biopsies similar to and is more user-friendly than the golden standard of LN2 quenching. To address this, we benchmarked the performance of the snap freezer prototype to the golden standard with regard to preservation of biology. Molecular profiles of snap frozen cell lines and human tissue biopsies were determined taking phosphoproteomic and transcriptomic profiles as a read out. The secondary aim of this study was to determine whether differences in freezing rate could influence the molecular profile of cancer cells. METHODS CELL CULTURE, LYSIS AND PROTEIN DIGESTION Cells from chronic myeloid leukemia (CML) K562 and the colorectal cancer cell line HCT116 were cultured according to standard methods as described in Supplementary Methods. 5
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