Human hepatocytes for drug toxicity screening from Cardiac Death Donor livers

用于心脏死亡供体肝脏药物毒性筛查的人肝细胞

• 批准号: 8314669
• 负责人: MARK G CLEMENS
• 金额: $ 36.13万
• 依托单位: HEPATOSYS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-04 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8314669
• 关键词: Abbreviations; Address; Albumins; Animal Model; Animals; Benchmarking; Biomedical Engineering; Blood Circulation; Cardiac Death; Cell Line; Cell Separation; Cell Survival; Cells; Characteristics; Clinical; Clinical Trials; Collaborations; Coupled; Cryopreservation; Cryopreserved Cell; Cytochrome P450; Development; Devices; Drug Approval; Drug toxicity; Failure; Glutamate-Ammonia Ligase; Goals; Gold; Harvest; Health Care Costs; Hepatocyte; Hepatotoxicity; Hour; Human; Ice; Investments; Ischemia; Lead; Left; Legal patent; Letters; Life; Liver; Measures; Membrane; Metabolism; Modeling; Modification; Organ; Organ Preservation; Perfusion; Pharmaceutical Preparations; Phase; Population; Positioning Attribute; Preclinical Drug Evaluation; Procedures; Process; Production; Rattus; Recovery; Relative (related person); Research; Resuscitation; Rodent; Screening procedure; Site; Solutions; Source; System; Technology; Testing; Time; Toxicity Tests; Translating; Transplantation; Universities; University of Wisconsin-lactobionate solution; Urea; Warm Ischemia; Wisconsin; Xenobiotics; base; cost; design; design and construction; drug development; drug testing; experience; functional restoration; improved; in vivo; innovation; innovative technologies; oxidation; pre-clinical; preclinical study; restoration; species difference; success

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.

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生物的代谢的主要部位，因此必须了解肝脏如何代谢新药。由于人类与常用动物模型之间存在许多物种差异，因此对人肝细胞的测试仍然是药物筛查的金标准。但是，由于这些肝细胞的来源通常来自肝脏被降低用于移植的肝脏，通常是由于延长的缺血时间，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修改低温机器灌注（HMP）复苏系统，改善从不可移植DCD肝脏中分离出的原发性肝细胞的可用性和生存能力，我们已经开发出该系统以恢复缺血损坏的DCD肝脏的功能，以恢复用于移植的DCD肝脏。人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，该器官估计高达当前供体池的40％。但是，这些供体的分离肝细胞的生存力不一致导致可用性有限，从而导致这些细胞的成本更高。导致这种不一致的主要因素是这些器官经历的温暖缺血（WI）的延长时期和保存过程，简单的冷藏，这不会复苏这些器官。开发了与HMP结合的Hepatosys溶液，以恢复耗尽的能量储存，保持膜稳定性并最大程度地减少氧化损伤，从而导致肝脏复苏。初步研究表明，在60分钟的Wi大鼠DCD肝模型HMP中，HEPATOSYS溶液提高了可行性大于2倍（70 vs. 29％）和产量为4倍（3.34 vs. 0.69 x 106 Live细胞/GLIVER）在24小时SCS和5 hrs HRS HMP之后与单独进行24小时相比。此外，来自HMP组的肝细胞具有明显高于SCS细胞的高质量。更重要的是，在这些细胞中还回收了64％的ECOD活性。该项目的目的是为使用我们的过程提供概念验证，以分离出高产量，生存能力，平稳性和保留肝细胞特定功能的肝细胞，从暴露于延长的温暖缺血的人肝脏中。为此，我们提出了两个目标。具体目标＃1：比较从对照大鼠肝脏和大鼠DCD肝脏中分离出60或90分钟的WI，24小时冷储存的肝细胞的生存力，产量和功能，使用UW溶液和5小时HMP与Hepatosys（HS）溶液进行5小时HMP。在此目标中，我们将比较从对照大鼠肝脏和大鼠DCD肝脏中分离出60或90分钟的WI，24HR冷藏在UW溶液中，并与Hepatosys溶液中分离出的肝细胞的生存力，产量和功能。此外，我们将测试分离株中骨膜与紫pat肝细胞的相对贡献。这将通过评估活性通过测量总活性并确定谷氨酰胺合成酶阳性的分离细胞百分比（FACS分析）来评估活性来实现。具体目标＃2：比较从同一人类DCD肝脏中分离出的肝细胞的生存力，产量，平稳性和功能1）接收器官后立即与2）接收器官和5小时HMP和肝（HS）溶液后立即进行。这个目标将与目前隔离并出售人类肝细胞的公司Zen-Bio，Inc。合作。我们将比较Zen-Bio，Inc.当前在目前出售的细胞中分离的细胞中的功能特性。我们还将使用与AIM 1中相同的方法评估是否保留了有孔的肝细胞。将从这些DCD动物中隔离肝细胞，并将评估肝细胞。然后，将对细胞进行培养并评估尿素和白蛋白的产生。另外，还将评估细胞色素P450活性和诱导。如果确定功效，该项目将构成II期应用开发灌注/细胞隔离装置的基础，以允许在单个设备中进行HMP功能恢复和肝细胞隔离。此外，将提出研究以冷冻保存细胞，通过确定最大温暖缺血时间和最小灌注时间的恢复时间来优化恢复程序。该项目的成功将与Hepatosys的肝生理学家和生物医学工程师之间建立的合作伙伴关系直接相关，并与Zen-Bio，Inc。建立战略合作伙伴关系。 公共卫生相关性：该项目的长期目标是提高从心脏死亡（DCD）肝脏后不可转移捐赠中分离出的原发性肝细胞的质量和可用性。这旨在满足对新药物开发中大量用于代谢和毒性测试的高质量人肝细胞供应的非常重要的需求。 Hepatosys，Inc。开发了一种低温机器灌注溶液，该溶液可以通过与这些DCD肝脏相似的温暖缺血时期复苏。初步研究表明，这一过程允许从缺血受损的肝细胞分离肝细胞，如果使用当前的标准程序，则无法从中分离出肝细胞。该项目将测试从大鼠DCD肝脏中收获的细胞的功能和生存力，并在用肝素溶液灌注后，具有60和90分钟的温暖缺血，并确认能够隔离代表该体内该细胞群的能力。我补充，我们将与目前生产出用于研究目的的孤立人类细胞的公司Zen-Bio Inc.合作。在这项合作中，我们将把啮齿动物的研究扩展到技术对人肝脏的应用。如果成功，将提出一个II期项目来设计设备，以允许使用单个设备恢复功能并隔离肝细胞。将提出其他研究以优化复苏过程，并确定在被丢弃的人肝中允许的最大温暖缺血时间。 Hepatosys的肝脏生理学家与生物医学工程师之间的合作以及与Zen-Bio，Inc。的战略合作伙伴关系应该使我们能够快速将这种创新技术转化为商业上可行的产品。