High-viability cryopreservation media for primary hepatocytes

描述（由申请人提供）：冻存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，原代肝细胞具有代表体内环境所需的全部代谢酶，使其成为研究肝毒性，药物相互作用和药物代谢的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被破坏。冷冻保存解决了与时间限制和供体组织可用性相关的问题。然而，当使用当前技术冷冻保存时，肝细胞的反应很差。肝细胞活力可降低20%或更多，解冻后24小时观察到显著的细胞凋亡，多达一半的冻存肝细胞失去体外粘附表面的能力，通常观察到白蛋白和尿素产生减少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。在本申请中，我们提出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改善冻存肝细胞的活力、粘附性和功能。在第一阶段结束时，我们希望有一种技术可以在第二阶段扩大规模，用于开发我们自己的冷冻保存培养基。最终，我们计划出售冷冻保存培养基，并建立原代肝细胞库，这将大大减少作为药物开发过程一部分进行的动物研究数量，同时增加使用人类细胞的研究数量。

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.