Hepatocyte production from ice-free cryopreserved and nanowarmed livers

Hepatocyte-based therapy including cell transplantation, bioartificial and engineered livers are limited by the inability to produce large quantities of high functioning primary human hepatocytes on demand. In addition, the pharmaceutical industry could benefit from a technology that can provide pooled donor population and the ability to control supply with demand for drug metabolism and toxicity testing. The ultimate goal of this project to develop the technology of vitrification and nanowarming of partial or whole human livers to produce a broad range of quantities of metabolically active, high quality primary hepatocytes on demand for therapeutic and pharmaceutical applications. Vitrification, an ice-free cryopreservation method, shows promise but is currently not applicable to large tissue and organs due to damaging ice crystal formation during the slow warming. Recently, our group at the University of Minnesota developed “nanowarming” using iron oxide nanoparticles (IONPs) coupled with radio frequency (RF) technology to achieve uniform warming rates sufficient to avoid crystallization and cracking in vitrified tissue and has the ability to scale up to partial and/or whole human organs. The goal for this Phase I project will be to determine the efficacy of isolating hepatocytes from whole rat livers that have been vitrified and rewarmed via nanowarming. This project will use the liver’s own native vascular system to load and unload the vitrification solution (VS) prior to hepatocyte isolation. This allows homogeneous delivery of the VS to a large number of cells. Our preliminary results show that vitrification of a whole rat liver and uniform rewarming rates sufficient to avoid crystallization and cracking were achieved. Loading and unloading of the VS at hypothermic temperatures resulted in high yield, viabiligy and hepatocyte function. Lastly, nanowarmed vitrified livers showed largely normal architecture, displayed hepatocyte specific function (indocyanine green uptake) and homogeneous perfusion. This would suggest that large quantities of viable and functioning cells can be isolated from the nanowarmed vitrified rat liver. The goals of the Phase I project can be achieved by accomplishing the following Specific Aims: Specific Aim 1: Determine the efficacy of loading and unloading three different concentrations (7, 8 and 9M) of the vitrification solution (VS) in a rat liver on yield, viability and function of the isolated hepatocytes. Specific Aim 2: Determine the IONP concentration that ensures uniform and rapid warming rates sufficient to avoid crystallization. Specific Aim 3: Determine the efficacy of vitrifying and nanowarming rat livers for obtaining large quantities of viable and high functioning hepatocytes. If successful, the Phase II project will scale-up the technology to porcine livers in collaboration with the Mayo Clinic. In addition, the project will collaborate with Lonza (world leading hepatocyte supplier) on not-suitable for transplant donor human livers and/or segments by combining our sister company HepatoSys Inc’s liver resuscitation method with vitrification/nanowarming. The innovation for this project is the vitrifying and rewarming via nanowarming of the whole liver prior to isolating the hepatocytes. This method was not possible before nanowarming was developed due to the inability to generate sufficient warming rates in intact partial or whole organs to avoid the damaging effects of ice crystal formation.The long-term goal of this project is to develop the technology of vitrification and nanowarming of human livers to provide a broad range of quantities of high functioning primary hepatocytes (liver cells) on demand. Cell transplantation, bioartificial livers, and engineered whole livers are promising solutions to end-stage liver disease and acute liver failure. However, the inability to produce large quantities of high functioning hepatocytes on demand has been a major hurdle. Pooled donor population of hepatocytes, potential product of this technology could impact the the pharmaceutical industry’s testing of drug metabolism and toxicity. Our group at the University of Minnesota has developed a technology that has the potential to vitrify (an ice-free cryopreservation method) partial and/or whole human and animal livers and then rewarm the liver rapidly and produce large quantities of high functioning hepatocytes. This technology combines vitrification and “nanowarming.” Nanowarming utilizes iron oxide nanoparticles (IONP) with radio frequency technology to rapidly and uniformly warm large volumes of tissues and organs. Our preliminary study demonstrate that we can load and unload the vitrification solution and obtain high yield, viability and functioning hepatocytes. Results also suggest that we can vitrify a whole rat liver with IONP, rapidly nanowarm, and during reperfusion observe homogeneous perfusion and hepatocyte specific function from the liver which are important for isolating hepatocytes. Based on these exciting preliminary results, the goal for this Phase I project will be to determine the efficacy of vitrifying and nanowaming rat livers for obtaining large quantities of viable and high functioning hepatocytes. If successful, the Phase II project will scale-up the technology to porcine livers and then combine our sister company, HepatoSys Inc’s liver resuscitation method with the IONP technology on not-suitable for transplant donor human livers and/or segments.