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下的生化代谢谱，用于预测早期药物开发期间的药物毒性和代谢。从活体供肝和劈离式肝移植手术的肝切除中获得的大量冷冻保存的人肝切片可用于体外药物测试，并取代实验动物的需求。然而，不完善的保存技术，降低活力和减少药物测试孵育期限制了PCLS的潜力。先前冷冻保存PCLS的研究工作遇到了在细胞外和细胞内位点形成冰的基本问题，以及导致外源性代谢和生物转化能力受损的冷冻保护剂的毒性水平。这项可行性研究试图解决的工作假设是，采用分子冰控制化合物的精密切割大鼠肝切片的无冰冷冻保存将增强异生素代谢和肝脏活力，与现有的玻璃化和传统的冷冻保存技术相比，延长了孵育期。据推测，玻璃体冷冻保存的PCLS使用合成分子冰阻滞剂将保留外源性代谢功能较长的潜伏期。将使用各种活力和组织学测定来评估冷冻保存的PCLS的异生物质代谢功能和细胞完整性。在成功完成本第I阶段SBIR可行性提案的目标后，将在第II阶段SBIR提交文件中解决规模扩大问题，以全面制定有效的人类和大型动物肝脏切片库储存方案。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。这项技术的发展将通过加快药物筛选过程和降低药物开发成本，为美国医疗保健系统带来多重好处。 本提案第I阶段的具体目标是体外评价天然和合成分子冰控制化合物，其中选定的冷冻保护剂已被证明可实现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，将评价具有最大生物相容性的制剂在保存期间在PCLS中的冰形成。将在延长的孵育期内评价复温PCLS的代谢功能。 将在sub-ESTA II期SBIR提案中，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对照制剂。