Liver toxicity is a major barrier to the FDA approval of many new drugs. Moreover, since the liver is the major site of metabolism of most xenobiotics it is essential to know how the liver metabolizes new drug. Because of the many species differences between humans and commonly used animal models, testing on human hepatocytes remains the gold standard for drug screening. However, because the source of these hepatocytes is usually from livers that have been declined for use in transplantation, typically because of extended ischemic time, the supply of high quality cells is severely limited. The long-term goal of this project is to improve the availability and viability of primary human hepatocytes isolated from non-transplantable DCD livers through the modification of a hypothermic machine perfusion (HMP) resuscitation system that we have developed to restore function to ischemia damaged DCD livers for use in transplantation. Human hepatocytes are typically isolated from the pool of non-transplantable DCD organs which has been estimated to be as high as 40% of the current donor pool. However, inconsistent viability of the isolated hepatocytes from these donors has led to limited availability, resulting in higher costs for these cells. Major factors contributing to this inconsistency are the extended periods of warm ischemia (WI) experienced by these organs and the preservation process, simple cold storage, which does not resuscitate these organs. The HepatoSys solution coupled with HMP was developed to restore depleted energy stores, maintain membrane stability, and minimize oxidation damage resulting in a resuscitated liver. Preliminary studies show that in a 60 min WI rat DCD liver model HMP with the HepatoSys solution improved viability greater than 2-fold (70 vs. 29%) and yield 4-fold (3.34 vs. 0.69 x 106 live cells/gliver) after 24hr SCS and 5 hrs HMP when compared to 24hr SCS alone. In addition the hepatocytes from the HMP group had significantly higher plateability than SCS cells. More importantly, 64% of ECOD activity was also recovered in these cells. The objective of this project is to provide proof of concept for the use of our process to isolate hepatocytes with high yield, viability, plateability and retention of hepatocyte specific function from human livers exposed to extended warm ischemia. To accomplish this we propose two aims. Specific Aim #1: Compare viability, yields, and functionality of hepatocytes isolated from control rat livers and rat DCD livers after 60 or 90 mins of WI, 24hr cold storage with the UW solution and 5hr HMP with the HepatoSys (HS) solution. In this aim we will compare viability, yields, and functionality of hepatocytes isolated from control rat livers and rat DCD livers after 60 or 90 mins of WI, 24hr cold storage with the UW solution and 5hr HMP with the HepatoSys solution. In addition, we will test the relative contribution of perivenous vs. periporta hepatocytes in the isolate. This will be accomplished by assessing the activity the perivenous marker glutamine synthetase by measuring total activity and determining the percentage of isolated cells positive for glutamine synthetase (FACS analysis). Specific Aim #2: Compare the viability, yield, plateability and function of hepatocytes isolated from the same human DCD liver 1) immediately after receiving the organ and 2) after receiving the organ and 5hr HMP with the HepatoSys (HS) solution. This Aim will be in collaboration with Zen-Bio, Inc., a company that currently isolates and sells human hepatocytes for research purposes. We will compare functional characteristics in cells that we isolate to benchmark values in cells currently being sold by Zen-Bio, Inc. We will also assess whether perivenous hepatocytes are preserved using the same approach as in Aim 1. Hepatocytes will be isolated from these DCD animals and viability and yield will be assessed. The cells will then be cultured and assessed for urea and albumin production. In addition, cytochrome P450 activity and induction will also be evaluated. If efficacy is established, this project will form the basis for a Phase II application to develop a perfusion/cell isolation device to allow both HMP functional restoration and hepatocyte isolation to be conducted in a single device. In addition, studies will be proposed to cryopreserve the cells, optimize the recovery procedure by determining the maximum warm ischemic time and minimum perfusion time for recovery. The success of this project will be directly related to the established partnership among the liver physiologist and biomedical engineer at HepatoSys and the establishment of a strategic partnership with Zen-Bio, Inc. PUBLIC HEALTH RELEVANCE: The long term goal of this project is to improve the quality and availability of primary human hepatocytes isolated from non-transplantable Donation after Cardiac Death (DCD) livers. This is designed to address the very important need for an abundant supply of high quality human hepatocytes for metabolism and toxicity testing in new drug development. HepatoSys, Inc. has developed a hypothermic machine perfusion solution that resuscitates livers with extended periods of warm ischemia similar to those from these DCD livers. The preliminary studies show that this process allowed isolation of hepatocytes from ischemia damaged livers from which no hepatocytes could be plated if the current standard procedure was used. This project will test the functionality and viability of the cells harvested from rat DCD livers with 60 and 90 mins of warm ischemia after perfusion with the HepatoSys solution and confirm the ability to isolate a population of cells representative of that in vivo. I addition, we will collaborate with Zen-Bio Inc., a company that currently produces isolated human cells sold for research purposes. In this collaboration we will extend the rodent studies to the application of the technology to human livers. If successful, a Phase II project will be proposed to design a device to allow the restoration of function and isolation of hepatocytes using a single device. Additional studies will be proposed to optimize the resuscitation process and determine the maximum warm ischemic time allowable in discarded human livers. The collaborations between the liver physiologist and biomedical engineer in HepatoSys along with the strategic partnership with Zen-Bio, Inc. should allow us to quickly translate this innovative technology into a commercially viable product.