PRESERVATION OF LIVER SPHEROIDS

肝球体的保存

• 批准号: 7107042
• 负责人: LIA H CAMPBELL
• 金额: $ 14.79万
• 依托单位: CELL AND TISSUE SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7107042
• 关键词: bioassay; cell aggregation; cell cell interaction; cell component structure /function; cell population study; cryopreservation; cytotoxicity; evaluation /testing; freezing; laboratory rat; light microscopy; liver cells; method development; tissue /cell culture; water

DESCRIPTION (provided by applicant): Assays are being developed in efforts to reduce the number of animals required for research. In particular, cell based systems that can evaluate a variety of compounds and/or screen single compounds with many cell types would provide the most advantageous platforms. These types of assay systems would be applicable to drug discovery, toxicology testing and environmental screening. While many cell types can be used in these systems, there are some cell types that are more relevant. Hepatocytes are one such cell type. However, their functions in culture become compromised very quickly and they have not been particularly amenable to cryopreservation when frozen as individual cells either in suspension or as a monolayer. Hepatocytes will aggregate in culture forming spheroids and studies have shown that spheroids maintain their specific functions longer in culture than freshly isolated hepatocytes. Therefore, spheroids have wide applicability for many different assay systems. Even better would be preserved spheroids that could be used whenever they were needed. To date, only one published study has been performed evaluating liver spheroids after cryopreservation. In the current study, both freezing and vitrification cryopreservation methods for liver spheroids will be developed using primary hepatocytes from rats. Freezing involves preservation with formation of ice. A protocol derived from studies cryopreserving hepatocytes will be employed. Vitrification is an ice-free preservation method that has been used successfully in some tissues. Two vitrification solutions and two rewarming methods will be evaluated using two non-specific viability assays and two liver cell-specific function assays. Either approach could prove successful and the goal of this study is to demonstrate viability and specific function of liver spheroids by either method. Phase II studies will involve optimization for the best method of cryopreservation by either freezing or vitrification in anticipation of commercialization of mammalian liver spheroids.

描述（由申请人提供）：正在开发试验，以减少研究所需的动物数量。特别地，可以评估多种化合物和/或用许多细胞类型筛选单一化合物的基于细胞的系统将提供最有利的平台。这些类型的分析系统将适用于药物发现，毒理学测试和环境筛选。虽然许多细胞类型可用于这些系统中，但有一些细胞类型更相关。肝细胞就是这样一种细胞类型。然而，它们在培养中的功能很快就受到损害，并且当它们作为悬浮液中的单个细胞或作为单层细胞冷冻时，它们并不特别适合于冷冻保存。肝细胞将在培养物中聚集形成球状体，并且研究表明球状体在培养物中比新鲜分离的肝细胞保持其特异性功能更长时间。因此，球状体对于许多不同的测定系统具有广泛的适用性。更好的是保存球体，可以在需要的时候使用。迄今为止，只有一项已发表的研究评价了冷冻保存后的肝脏球状体。在本研究中，将使用大鼠原代肝细胞开发肝球体的冷冻和玻璃化冷冻保存方法。冷冻包括通过形成冰来保存。将采用源自冻存肝细胞研究的方案。玻璃化冷冻是一种无冰保存方法，已成功用于某些组织。将使用两种非特异性活力试验和两种肝细胞特异性功能试验评价两种玻璃化冷冻溶液和两种复温方法。这两种方法都可以证明是成功的，本研究的目的是通过这两种方法证明肝球体的活力和特异性功能。II期研究将包括优化冷冻或玻璃化冷冻保存的最佳方法，以预期哺乳动物肝脏球状体的商业化。