Human hepatocytes for drug toxicity screening from Cardiac Death Donor livers

描述（由申请人提供）：肝毒性是FDA批准许多新药的主要障碍。此外，由于肝脏是大多数异种药物的主要代谢部位，因此了解肝脏如何代谢新药是至关重要的。由于人类和常用的动物模型之间存在许多物种差异，因此在人类肝细胞上进行测试仍然是药物筛选的金标准。然而，由于这些肝细胞的来源通常来自已不再用于移植的肝脏，通常是因为缺血时间延长，因此高质量细胞的供应受到严重限制。该项目的长期目标是通过改进低温机器灌注（HMP）复苏系统，提高从不可移植的DCD肝脏中分离的原代人肝细胞的可用性和活力，我们已经开发出恢复缺血损伤DCD肝脏功能的系统，用于移植。人类肝细胞通常是从不可移植的DCD器官库中分离出来的，据估计，这些器官在目前的供体库中高达40%。然而，从这些供体中分离的肝细胞的活力不一致，导致可用性有限，导致这些细胞的成本更高。造成这种不一致的主要因素是这些器官经历了长时间的热缺血（WI）和保存过程，简单的冷藏，不能使这些器官复苏。HepatoSys溶液与HMP相结合，用于恢复耗尽的能量储存，维持膜稳定性，并最大限度地减少氧化损伤导致的肝脏复苏。初步研究表明，在60分钟WI大鼠DCD肝脏模型中，与单独使用24小时SCS和5小时HMP相比，在24小时SCS和5小时HMP后，HMP的活力提高了2倍以上（70比29%），产量提高了4倍（3.34比0.69 × 106个活细胞/肝脏）。此外，HMP组肝细胞的可移植性明显高于SCS细胞。更重要的是，这些细胞中也恢复了64%的ECOD活性。该项目的目的是为使用我们的方法从暴露于长时间热缺血的人类肝脏中分离出高产、活力、可移植性和保留肝细胞特异性功能的肝细胞提供概念证明。为此，我们提出两个目标。具体目标#1：比较从对照大鼠肝脏和大鼠DCD肝脏分离的肝细胞在WI 60或90分钟后的活力、产量和功能，用UW溶液冷藏24小时，用HepatoSys （HS）溶液冷藏5小时。在此目的中，我们将比较从对照大鼠肝脏和大鼠DCD肝脏中分离的肝细胞在WI 60或90分钟后的活力、产量和功能，用UW溶液冷藏24小时，用HepatoSys溶液冷藏5小时。此外，我们将测试在分离物中静脉周围和门周围肝细胞的相对贡献。这将通过测量总活性和确定谷氨酰胺合成酶阳性分离细胞的百分比（FACS分析）来评估静脉周围标记物谷氨酰胺合成酶的活性来完成。具体目标#2：比较从同一人DCD肝脏中分离的肝细胞的活力、产量、可移植性和功能：1)接受器官后立即，2)接受器官后，用HepatoSys （HS）溶液5小时HMP。该Aim将与Zen-Bio公司合作，Zen-Bio公司目前分离并销售用于研究目的的人类肝细胞。我们将把分离的细胞的功能特征与Zen-Bio公司目前销售的细胞的基准值进行比较。我们还将评估是否使用与Aim 1相同的方法保存静脉周围肝细胞。将从这些DCD动物中分离肝细胞，并评估其活力和产量。然后将细胞培养并评估尿素和白蛋白的产生。此外，还将评估细胞色素P450的活性和诱导作用。如果疗效确定，该项目将成为II期申请的基础，以开发灌注/细胞分离设备，使HMP功能恢复和肝细胞分离能够在单个设备中进行。此外，还将提出对细胞进行低温保存的研究，通过确定恢复的最大热缺血时间和最小灌注时间来优化恢复程序。该项目的成功将直接关系到HepatoSys肝脏生理学家和生物医学工程师之间建立的合作伙伴关系，以及与Zen-Bio公司建立的战略合作伙伴关系。

DESCRIPTION (provided by applicant): 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.