PRESERVATION OF LIVER SPHEROIDS

描述（由申请人提供）：为了减少研究所需动物的数量，正在开发检测方法。特别是，基于细胞的系统可以评估多种化合物和/或筛选具有多种细胞类型的单一化合物，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。虽然在这些系统中可以使用许多细胞类型，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会受到损害，当它们作为单个细胞在悬浮液中或作为单层冷冻时，它们不特别适合冷冻保存。肝细胞在培养中聚集形成球状体，研究表明球状体在培养中比新鲜分离的肝细胞保持其特定功能的时间更长。因此，球体广泛适用于许多不同的分析系统。更好的方法是保存球体，以便在需要时随时使用。迄今为止，只有一项已发表的研究对冷冻保存后的肝球体进行了评估。在目前的研究中，冷冻和玻璃化冷冻保存肝球体的方法将利用大鼠的原代肝细胞。冷冻包括通过冰的形成来保存。将采用从低温保存肝细胞的研究中获得的方案。玻璃化是一种无冰保存方法，已成功地应用于一些组织。两种玻璃化溶液和两种复温方法将使用两种非特异性活力测定和两种肝细胞特异性功能测定进行评估。任何一种方法都可能被证明是成功的，本研究的目标是通过任何一种方法证明肝球体的生存能力和特定功能。II期研究将包括通过冷冻或玻璃化来优化最佳冷冻保存方法，以预期哺乳动物肝球体的商业化。

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.