Hepatocyte-based therapy including cell transplantation, bioartificial and engineered livers are limited by theinability to produce large quantities of high functioning primary human hepatocytes on demand. In addition, thepharmaceutical industry could benefit from a technology that can provide pooled donor population and the abilityto control supply with demand for drug metabolism and toxicity testing. The ultimate goal of this project todevelop the technology of vitrification and nanowarming of partial or whole human livers to produce abroad range of quantities of metabolically active, high quality primary hepatocytes on demand fortherapeutic and pharmaceutical applications.Vitrification, an ice-free cryopreservation method, shows promise but is currently not applicable to large tissueand organs due to damaging ice crystal formation during the slow warming. Recently, our group at the Universityof 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 tissueand 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 ratlivers that have been vitrified and rewarmed via nanowarming. This project will use the liver's own nativevascular system to load and unload the vitrification solution (VS) prior to hepatocyte isolation. This allowshomogeneous delivery of the VS to a large number of cells. Our preliminary results show that vitrification of awhole 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 hepatocytefunction. Lastly, nanowarmed vitrified livers showed largely normal architecture, displayed hepatocyte specificfunction (indocyanine green uptake) and homogeneous perfusion. This would suggest that large quantities ofviable and functioning cells can be isolated from the nanowarmed vitrified rat liver. The goals of the Phase Iproject can be achieved by accomplishing the following Specific Aims:Specific Aim 1: Determine the efficacy of loading and unloading three different concentrations (7, 8 and9M) of the vitrification solution (VS) in a rat liver on yield, viability and function of the isolatedhepatocytes.Specific Aim 2: Determine the IONP concentration that ensures uniform and rapid warming ratessufficient to avoid crystallization.Specific Aim 3: Determine the efficacy of vitrifying and nanowarming rat livers for obtaining largequantities of viable and high functioning hepatocytes.If successful, the Phase II project will scale-up the technology to porcine livers in collaboration with the MayoClinic. In addition, the project will collaborate with Lonza (world leading hepatocyte supplier) on not-suitable fortransplant donor human livers and/or segments by combining our sister company HepatoSys Inc's liverresuscitation method with vitrification/nanowarming. The innovation for this project is the vitrifying and rewarmingvia nanowarming of the whole liver prior to isolating the hepatocytes. This method was not possible beforenanowarming was developed due to the inability to generate sufficient warming rates in intact partial or wholeorgans to avoid the damaging effects of ice crystal formation.
Public Health Relevance Statement: 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.
Project Terms: Animals ; Architecture ; Engineering / Architecture ; Biomedical Engineering ; bio-engineered ; bio-engineers ; bioengineering ; Cell physiology ; Cell Function ; Cell Process ; Cellular Function ; Cellular Physiology ; Cellular Process ; Subcellular Process ; Cells ; Cell Body ; Consultations ; Cryopreservation ; Cryofixation ; cold preservation ; cold storage ; Crystallization ; Donor person ; transplant donor ; Drug Industry ; Pharmaceutic Industry ; Pharmaceutical Industry ; Drug toxicity ; Engineering ; Exhibits ; Freezing ; Goals ; Heating ; Human ; Modern Man ; Ice ; Indocyanine Green ; Ujoveridin ; Wofaverdin ; Ischemia ; Liver ; hepatic body system ; hepatic organ system ; Liver diseases ; Hepatic Disorder ; hepatic disease ; hepatopathy ; liver disorder ; liver transplantation ; Hepatic Transplantation ; Liver Grafting ; Liver Transplant ; Methods ; Minnesota ; United States National Institutes of Health ; NIH ; National Institutes of Health ; Legal patent ; Patents ; Perfusion ; Drug Kinetics ; Pharmacokinetics ; Production ; Rattus ; Common Rat Strains ; Rat ; Rats Mammals ; Reperfusion Therapy ; reperfusion ; Research Personnel ; Investigators ; Researchers ; Resuscitation ; Suspensions ; Suspension substance ; Family suidae ; Pigs ; Suidae ; Swine ; porcine ; suid ; Technology ; Temperature ; Testing ; Time ; Tissues ; Body Tissues ; Toxicology ; Transplantation ; transplant ; Universities ; Liver Failure ; Hepatic Failure ; Acute Liver Failure ; Acute Hepatic Failure ; Fulminant Liver Failure ; Fulminating Hepatic Failure ; Fulminating Liver Failure ; fulminant hepatic failure ; base ; Organ ; Vial device ; Vial ; improved ; Acute ; Chronic ; Phase ; Cell Transplantation ; Rewarming ; Ensure ; Hepatocyte ; Hepatic Cells ; Hepatic Parenchymal Cell ; Liver Cells ; liver function ; Toxicity Tests ; Toxicity Testing ; Funding ; uptake ; Collaborations ; cell mediated therapies ; cell-based therapeutic ; cell-based therapy ; cellular therapy ; Cell Therapy ; Therapeutic ; Metabolic ; Bioartificial Liver ; Artificial Liver ; Sister ; Clinic ; Protocol ; Protocols documentation ; Crystal Deposition ; Crystal Formation ; Vascular System ; Transplant Surgeon ; Cell Volumes ; Toxicities ; Toxic effect ; drug metabolism ; drug discovery ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; Hepatocyte transplantation ; Hepatocyte transplant ; scale up ; Population ; Coupled ; innovation ; innovate ; innovative ; drug testing ; drug detection ; commercialization ; radio frequency ; radiofrequency ; Industry Standard ; experimental study ; experiment ; experimental research ; iron oxide nanoparticle ; iron oxide nano particle ; nanowarming ;
