High-viability cryopreservation media for primary hepatocytes

原代肝细胞高活力冻存培养基

基本信息

批准号：
8711826
负责人：
Bruce Lamb
金额：
$ 22.44万
依托单位：
AFFINERGY, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8711826
关键词：
Adherence Adhesions Albumins Animal Model Animals Apoptosis Apoptotic Binding Cell Count Cell Death Cell Line Cell Survival Cells Collagen Complement Cryopreservation Development Drug Interactions Environment Enzyme Induction Enzymes Ethylene Glycols Extracellular Matrix Freezing Goals Harvest Hepatocyte Hepatotoxicity Hour Human Ice In Vitro Integrins Letters Life Liver Measures Mediating Metabolic Outcome Pathway interactions Peptides Pharmaceutical Preparations Pharmacologic Substance Phase Procedures Process Production Proto-Oncogene Proteins c-akt Protocols documentation RGD (sequence)Rattus Research Personnel Risk Schedule Signal Pathway Signal Transduction Solutions Staging Sterilization Stress Surface Technology Testing Time Tissue Donors Touch sensation Urea cell bank cost cost effective drug development drug discovery drug metabolism ethylene glycol immortalized cell improved in vivo in vivo Model interest prevent public health relevance research and development research study scale up success wasting

项目摘要

DESCRIPTION (provided by applicant): Cryopreserved hepatocytes are widely used in drug development as pharmaceutical companies strive to identify risk pre-clinically and shift product attrition to the earliest stages of drug discovery. Unlike immortalized cell lines, primary hepatocytes possess the full complement of metabolizing enzymes needed to represent the in vivo environment, making them cost-effective substitutes for expensive animal models in studying hepatotoxicity, drug-drug interactions, and drug metabolism. Unfortunately, supplies of fresh hepatocytes are greatly limited by donor availability. In addition, hepatocytes should be used within 5 hours of isolation, as signaling pathways that normally suppress apoptosis are disrupted when integrin-mediated binding to the extracellular matrix is eliminated. Cryopreservation alleviates the problems associated with time constraints and donor tissue availability. However, hepatocytes respond poorly when cryopreserved using current technology. Hepatocyte viability can be reduced by 20% or more, significant apoptosis is observed 24 hours after thawing, up to half of cryopreserved hepatocytes lose their ability to adhere to surfaces in vitro, and a 50% decrease in albumin and urea production is commonly observed. Together, these problems result in significant quantities of wasted hepatocytes, which ultimately increases costs for both hepatocyte suppliers and end users. In this application, we propose to improve cryopreservation outcomes by reducing apoptosis. We will establish proof-of-concept that supplementing commercially available media with hepatocyte-binding peptides will improve viability, adherence, and function of cryopreserved hepatocytes. At the conclusion of Phase 1, we expect to have a technology that can be scaled up in Phase 2 for the development of our own cryopreservation media. Ultimately, we plan to sell the cryopreservation media and to establish a primary hepatocyte cell bank that will significantly reduce the number of animal studies conducted as part of the drug development process, while simultaneously increasing the number of studies using human cells.

描述（由申请人提供）：冷冻保存的肝细胞广泛用于药物开发，因为制药公司努力在临床前识别风险并将产品损耗转移到药物发现的最早阶段。与永生化细胞系不同，原代肝细胞拥有代表体内环境所需的全套代谢酶，使其成为研究肝毒性、药物相互作用和药物代谢方面昂贵动物模型的经济有效的替代品。不幸的是，新鲜肝细胞的供应受到供体可用性的极大限制。此外，肝细胞应在分离后 5 小时内使用，因为当整合素介导的与细胞外基质的结合被消除时，通常抑制细胞凋亡的信号通路会被破坏。冷冻保存缓解了与时间限制和供体组织可用性相关的问题。然而，使用现有技术冷冻保存时，肝细胞反应较差。肝细胞活力可降低 20% 或更多，解冻 24 小时后观察到显着的细胞凋亡，多达一半的冷冻保存的肝细胞在体外失去粘附表面的能力，并且通常观察到白蛋白和尿素产量减少 50%。这些问题共同导致大量肝细胞浪费，最终增加了肝细胞供应商和最终用户的成本。在此应用中，我们建议通过减少细胞凋亡来改善冷冻保存结果。我们将建立概念验证，即用肝细胞结合肽补充市售培养基将提高冷冻保存肝细胞的活力、粘附性和功能。在第一阶段结束时，我们期望拥有一项可以在第二阶段扩大规模的技术，用于开发我们自己的冷冻保存介质。最终，我们计划出售冷冻保存介质并建立一个原代肝细胞库，这将显着减少作为药物开发过程一部分进行的动物研究数量，同时增加使用人体细胞的研究数量。