PRESERVATION OF LIVER SLICES BY VITRIFICATION

通过玻璃化保存肝切片

• 批准号: 6882959
• 负责人: MICHAEL John TAYLOR
• 金额: $ 14.81万
• 依托单位: CELL AND TISSUE SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-19 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882959
• 关键词: biotransformation; chemical synthesis; cryopreservation; cryoprotective agents; cytotoxicity; drug metabolism; histology; laboratory rat; liver preservation; method development; stainings; tissue /cell preparation

DESCRIPTION (provided by applicant): In-vitro drug testing on precision-cut liver slices (PCLS) provides the most realistic biochemical metabolic profile of in-situ conditions for predicting drug toxicity and metabolism during early stage drug development. An ample supply of cryopreserved human liver slices obtained from viable donor livers and hepatic resects of split liver transplant surgeries could be used for in-vitro drug testing and replace the need of experimental animals. However, imperfect preservation techniques that degrade viability and reduce drug testing incubation periods have limited the potential of PCLS. Prior research efforts to cryopreserve PCLS have encountered fundamental problems of ice formation at extra- and intracellular sites and toxic levels of cryoprotectants which have resulted in impaired xenobiotic metabolism and biotransformation capacities. The working hypothesis that this feasibility study seeks to address is that ice-free cryopreservation of precision-cut rat liver slices employing molecular ice control compounds will enhance xenobiotic metabolism and hepatic viability for extended incubation periods compared with existing vitrification and conventional cryopreservation technologies. It is hypothesized that vitreous cryopreservation of PCLS using synthetic molecular iceblockers will retain xenobiotic metabolic functions for longer incubation periods. The xenobiotic metabolism functions and cellular integrity of cryopreserved PCLS will be assessed using various viability and histological assays. Following successful completion of the aims of this Phase I SBIR feasibility proposal, scale up issues will be addressed in Phase II SBIR submission to fully develop an effective storage protocol for banking of human and large animal liver slices. It is anticipated that the PCLS banking will invariably reduce and in many case replace the need of experimental animals required for drug testing. This technology development will provide multiple benefits to the US health care system by expediting drug screening process and reducing drug development costs. The specific aims of Phase I of this proposal are the in- vitro evaluation of natural and synthetic molecular ice control compounds with selected cryoprotectants proven to produce ice-free cryopreservation and maintain tissue function and viability. First, the selected formulations will be tested in vitro for cytotoxicity. Second, formulations with the greatest biocompatibility will be evaluated for ice formation in PCLS during preservation. Rewarmed PCLS will be evaluated for metabolic function over extended incubation periods. The molecular ice control formulations which provide the highest tissue viability and least interstitial ice formation will be tested in larger animal and human PCLS tissues in a sub-sequent Phase II SBIR proposal.

描述（由申请人提供）： 精密切割肝脏切片 (PCLS) 的体外药物测试提供了最真实的原位条件生化代谢概况，用于预测药物开发早期的药物毒性和代谢。从活供体肝脏和劈裂肝移植手术的肝切除物中获得的大量冷冻保存的人类肝脏切片可用于体外药物测试并取代实验动物的需要。然而，不完善的保存技术会降低活力并缩短药物测试潜伏期，限制了 PCLS 的潜力。先前冷冻保存 PCLS 的研究工作遇到了细胞外和细胞内位点冰形成以及冷冻保护剂有毒水平的基本问题，这些问题导致外源代谢和生物转化能力受损。本可行性研究试图解决的工作假设是，与现有的玻璃化和传统冷冻保存技术相比，采用分子冰控制化合物对精确切割的大鼠肝脏切片进行无冰冷冻保存将增强外源代谢和肝脏活力，延长孵化期。据推测，使用合成分子冰阻剂对 PCLS 进行玻璃体冷冻保存将在更长的潜伏期内保留外源代谢功能。将使用各种活力和组织学测定来评估冷冻保存的 PCLS 的外源代谢功能和细胞完整性。在成功完成第一阶段 SBIR 可行性提案的目标后，将在第二阶段 SBIR 提交中解决扩大规模问题，以全面开发用于人类和大型动物肝脏切片库的有效存储方案。预计 PCLS 库将不可避免地减少并在许多情况下取代药物测试所需的实验动物的需求。这项技术的开发将加快药物筛选过程并降低药物开发成本，从而为美国医疗保健系统带来多重好处。 该提案第一阶段的具体目标是使用选定的冷冻保护剂对天然和合成分子冰控制化合物进行体外评估，这些冷冻保护剂被证明可以产生无冰冷冻保存并维持组织功能和活力。首先，将在体外测试所选制剂的细胞毒性。其次，将评估具有最大生物相容性的制剂在保存过程中在 PCLS 中的结冰情况。复温的 PCLS 将在延长的孵育期内评估代谢功能。 在随后的 II 期 SBIR 提案中，将在较大的动物和人类 PCLS 组织中测试提供最高组织活力和最少间质冰形成的分子冰控制配方。