Deep Supercooling of Liver Cells and 2D and 3D Tissue Constructs: Effect of Attachment

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Summary Apart from the preservation of a “whole human” there are three outstanding questions in the field of preservation 1) Can we preserve whole organs to reduce quality and logistics issues in transplantation where over 120000 patients are currently waiting for a replacement organ? 2) Can we devise an effective preservation scheme to ease the dissemination for the rapidly growing market of engineered tissue products? 3) Can we preserve primary cells with metabolic and enzymatic activity similar to fresh cells robustly and cheaply? Finally, one can ask the question: Can we answer these three questions with a “unified method”? Currently, the clinical gold standard for organ preservation is static cold storage (+4 oC) with up to 72 hours for kidney and ideally 12 hours for liver which is limiting in terms of logistics and flexibility. On the other end of the spectrum, cryopreservation and vitrification - despite successes at the cell level for some cell types - do not project success at the organ and tissue level soon. This current technological gap necessitates a superior and unified biopreservation method that is designed from the ground up with the long-term goal of storage of cells, tissues/tissue products and organs beyond one week to relieve the current logistic constraints. Based on our recent published works, we propose that non-freezing (supercooling) preservation (SCP) of living matter provides such a solution that bridges the gap between preservation of cells, tissue and organs. Our short-term goal is to develop a novel “Deep Supercooling” (DSC) preservation methods starting with single cells and multicellular tissue constructs. The intermediate-term goal, following up this exploratory period, is then to apply these to a) storage of commercial tissue-constructs, and b) successful organ storage from kidneys to hearts and livers. Supercooling can be achieved for small samples (~<1ml) at temperatures (-4 to -6 oC) for ~7 days at most without any freezing as in our earlier work. Yet, it has been impossible to keep a large volume (10s of milliliters) of preservation solution at very low temperatures (~-20 oC) for long times (~100 days) in a practical manner. Recently we addressed this in a breakthrough, dubbed “Deep Supercooling (DSC) via Surface Sealing”, where we seal water with an immiscible liquid (oils, alkanes, alcohols) in a solid container to achieve a practically stable supercooling temperature (down to -20 oC) for large volumes (up to 100 ml) for long period (up to 100 days). The objective of the proposed study is thus to develop “Deep Supercooling” preservation method for 3 models of hepatic cells/tissues (suspended cells, 2D plated cells, 3D spheroids) and then compare the long-term (5 day) success of these 3 models. Deep supercooling at ultralow (-10 to -20 oC) temperatures can drastically slow down metabolism and injury processes compared to cold storage (at 4 oC) and we expect this will significantly improve storage time and quality. We hypothesize that primary hepatocytes that are either attached to a surface or each other, and those that are well polarized in culture will survive with better success for longer at lower temperatures. Accordingly, we expect that the 2D plated hepatocytes and 3D Spheroids to preserve progressively better than cell suspensions under DSC (-10 to -20 oC) conditions. We will test this hypothesis in the two aims where we first compare and find optimum temperatures and preservation solutions for all models and then investigate the underlying molecular signatures for differences in preservation success of the different models: