Hepatocyte production from ice-free cryopreserved and nanowarmed livers

基于肝细胞的治疗，包括细胞移植、生物人工和工程化肝脏， 不能按需生产大量高功能原代人肝细胞。此外该 制药行业可以受益于一种技术，可以提供汇集的供体群体和能力， 控制药物代谢和毒性试验的供需。该项目的最终目标是 发展部分或整个人类肝脏的玻璃化冷冻和再冷冻技术， 广泛数量的代谢活性、高质量原代肝细胞， 治疗和制药应用。 玻璃化冷冻是一种无冰冷冻保存方法，显示出前景，但目前不适用于大型组织 和器官，因为在缓慢升温过程中冰晶的形成会造成损害。最近，我们在大学的小组 明尼苏达州的一家研究机构开发了一种“放射性武器”， (RF)实现足以避免玻璃化组织结晶和破裂的均匀升温速率的技术 并且具有按比例放大到部分和/或整个人体器官的能力。 该I期项目的目标是确定从整个大鼠中分离肝细胞的有效性 玻璃化并通过冷冻复温的肝脏。这个项目将使用肝脏自身的 在肝细胞分离之前，将玻璃化冷冻液（VS）装载到血管系统中。这允许 将VS均匀递送至大量细胞。我们的初步结果表明， 获得整个大鼠肝脏和足以避免结晶和破裂的均匀复温速率。 在低温下加载和卸载VS可获得高产率、活率和肝细胞 功能最后，无臂玻璃化冷冻肝脏显示基本正常的结构，显示肝细胞特异性， 功能（吲哚菁绿色摄取）和均匀灌注。这意味着大量的 可以从去臂玻璃化冷冻的大鼠肝脏中分离出有活力和功能的细胞。第一阶段的目标 项目可以通过实现以下具体目标来实现： 具体目标1：确定加载和卸载三种不同浓度（7、8和10）的功效。 9 M）的玻璃化溶液（VS）对大鼠肝脏中分离的 肝细胞 具体目标2：确定确保均匀和快速升温速率的IONP浓度 足以避免结晶。 具体目标3：确定玻璃化冷冻和解冻大鼠肝脏以获得大的 大量有活力的高功能肝细胞。 如果成功，第二阶段项目将与马约合作，将该技术扩大到猪肝 诊所此外，该项目将与Lonza（世界领先的肝细胞供应商）合作， 通过结合我们的姐妹公司HepatoSys Inc.的肝脏， 玻璃化/冷冻复苏法。本项目的创新之处在于玻璃化和复温 通过在分离肝细胞之前对整个肝脏进行预处理。这种方法以前是不可能的 由于无法在完整的部分或全部区域产生足够的变暖率， 器官，以避免冰晶形成的破坏性影响。

Hepatocyte-based therapy including cell transplantation, bioartificial and engineered livers are limited by the inability to produce large quantities of high functioning primary human hepatocytes on demand. In addition, the pharmaceutical industry could benefit from a technology that can provide pooled donor population and the ability to control supply with demand for drug metabolism and toxicity testing. The ultimate goal of this project to develop the technology of vitrification and nanowarming of partial or whole human livers to produce a broad range of quantities of metabolically active, high quality primary hepatocytes on demand for therapeutic and pharmaceutical applications. Vitrification, an ice-free cryopreservation method, shows promise but is currently not applicable to large tissue and organs due to damaging ice crystal formation during the slow warming. Recently, our group at the University of Minnesota developed “nanowarming” using iron oxide nanoparticles (IONPs) coupled with radio frequency (RF) technology to achieve uniform warming rates sufficient to avoid crystallization and cracking in vitrified tissue and has the ability to scale up to partial and/or whole human organs. The goal for this Phase I project will be to determine the efficacy of isolating hepatocytes from whole rat livers that have been vitrified and rewarmed via nanowarming. This project will use the liver’s own native vascular system to load and unload the vitrification solution (VS) prior to hepatocyte isolation. This allows homogeneous delivery of the VS to a large number of cells. Our preliminary results show that vitrification of a whole rat liver and uniform rewarming rates sufficient to avoid crystallization and cracking were achieved. Loading and unloading of the VS at hypothermic temperatures resulted in high yield, viabiligy and hepatocyte function. Lastly, nanowarmed vitrified livers showed largely normal architecture, displayed hepatocyte specific function (indocyanine green uptake) and homogeneous perfusion. This would suggest that large quantities of viable and functioning cells can be isolated from the nanowarmed vitrified rat liver. The goals of the Phase I project can be achieved by accomplishing the following Specific Aims: Specific Aim 1: Determine the efficacy of loading and unloading three different concentrations (7, 8 and 9M) of the vitrification solution (VS) in a rat liver on yield, viability and function of the isolated hepatocytes. Specific Aim 2: Determine the IONP concentration that ensures uniform and rapid warming rates sufficient to avoid crystallization. Specific Aim 3: Determine the efficacy of vitrifying and nanowarming rat livers for obtaining large quantities of viable and high functioning hepatocytes. If successful, the Phase II project will scale-up the technology to porcine livers in collaboration with the Mayo Clinic. In addition, the project will collaborate with Lonza (world leading hepatocyte supplier) on not-suitable for transplant donor human livers and/or segments by combining our sister company HepatoSys Inc’s liver resuscitation method with vitrification/nanowarming. The innovation for this project is the vitrifying and rewarming via nanowarming of the whole liver prior to isolating the hepatocytes. This method was not possible before nanowarming was developed due to the inability to generate sufficient warming rates in intact partial or whole organs to avoid the damaging effects of ice crystal formation.