For the treatment of acute and chronic liver failure, clinicians and investigators have been steadily working on transplantation of hepatocytes and development of bioartificial liver devices. Both of these approaches rely on a consistent, abundant, and readily available supply of suspended and cultured hepatocytes. Although there has been reasonable progress in cryopreservation of cultured hepatocytes, the freezing of isolated hepatocytes is still unreliable. We, and recently others, have shown that isolated hepatocytes have an unexpectedly high activation energy for water transport. As a result, hepatocytes become practically impermeable to water at relatively high subzero temperatures (approximately minus 30 degrees C), effectively trapping the water inside the cell, and catalyzing the nucleation of 'lethal' ice inside hepatocytes. All of these approaches to the cryopreservation of isolated hepatocytes include the addition of approximately 2.0 M of a penetrating cryoprotectant (e.g., DMSO). However, the disaccharide trehalose is known to stabilize many anhydrobiotic organisms against dehydration and desiccation stresses. The pharmaceutical industry has already made significant strides in storing proteinaceous drugs, liposomes, membranes, and viral particles as dried trehalose glasses. Unfortunately, mammalian cell membranes are impermeable to trehalose, and consequently, vital supramolecular structures, proteins, and lipids inside cells cannot benefit from the stabilizing properties of trehalose during freezing and/or drying. In our prior studies, we showed the use of a genetically engineered switchable membrane pore to introduce trehalose (and sucrose) into cells, and we obtained high levels of recovery and growth after cryopreservation. It is our hypothesis that trehalose, when introduced into hepatocytes, stabilizes cells against water loss (dehydration) due to its superior physico- chemical properties through (i) its high glass forming propensity and (ii) its ability to replace water in dehydrated cells ( the so-called """"""""water replacement hypothesis""""""""). Towards testing our hypothesis, we will first investigate the mechanism(s) by which trehalose mediates its protective effect on the recovery and function of cryopreserved isolated hepatocytes. As a result, we will be able to develop a reliable cryopreservation protocol for isolated rat and human hepatocytes. Second, we will determine the thermophysical properties of trehalose inside hepatocytes and fibroblasts. These parameters will enable us to specify the thermodynamic paths and conditions for achieving glassy state inside cells during drying. Third, we will investigate the mechanism(s) by which trehalose affords stability to dried hepatocytes and fibroblasts. These studies will help us elucidate the mechanism(s) of protection provided by trehalose during dry storage of cells loaded with trehalose, and result in a whole new approach to hepatocyte storage at ambient temperatures in dry state. Fourth, we will determine the optimal conditions for rehydration of dried cells. We anticipate that these optimization efforts will result in high recovery and stable, long-term function of isolated and cultured hepatocytes (rat and human) stored in dried glassy matrix.
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