Hepatocyte production from ice-free cryopreserved and nanowarmed livers

基于肝细胞的治疗，包括细胞移植、生物人工肝和工程肝，都受到 无法按需大量生产高功能原代人类肝细胞。此外， 制药业可以受益于一种技术，这种技术可以提供汇集的捐赠者群体和能力 控制药物代谢和毒性检测的供需。该项目的最终目标是 开发部分或整个人体肝脏的玻璃化冷冻和纳米温控技术，以生产 广泛数量的代谢活跃的高质量原代肝细胞按需 治疗和制药应用。 玻璃化冷冻是一种无冰冷冻保存方法，前景看好，但目前还不适用于大型组织 和器官，因为在缓慢升温期间破坏了冰晶的形成。最近，我们大学的小组 明尼苏达州开发了使用纳米氧化铁(IONPs)和射频相结合的“纳米武器” (RF)技术，可实现足以避免玻璃化组织结晶和破裂的均匀升温速度 并具有放大到部分和/或整个人体器官的能力。 这个第一阶段项目的目标将是确定从整个大鼠中分离肝细胞的效果 通过纳米温控技术玻璃化和复温的肝脏。这个项目将使用肝脏本身的原生体 在分离肝细胞之前，血管系统加载和卸载玻璃化液(VS)。这使得 将VS均匀地输送到大量细胞。我们的初步结果表明，玻璃化的一种 整个大鼠肝脏和均匀的复温速度足以避免结晶和破裂。 在低温下装载和卸载VS可获得高产量、存活率和肝细胞 功能。最后，纳米级玻璃化冷冻的肝脏显示出基本正常的结构，显示出肝细胞特异性。 功能(吲哚青绿摄取)和均匀灌流。这表明，大量的 可以从纳米武装玻璃化冷冻的大鼠肝脏中分离出具有活性和功能的细胞。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：确定三种不同浓度(7、8和 9m)大鼠肝脏玻璃化冷冻液(VS)对离体肝细胞产量、存活率和功能的影响 肝细胞。 具体目标2：确定确保均匀和快速升温速度的IONP浓度 足以避免结晶。 具体目标3：确定玻璃化冷冻和纳米温控大鼠肝脏获得大鼠肝脏的有效性 大量有活力和高功能的肝细胞。 如果成功，二期项目将与Mayo合作将这项技术扩大到猪肝 诊所。此外，该项目将与龙沙(世界领先的肝细胞供应商)合作，研究不适合于 通过结合我们的姊妹公司HepatoSys Inc.的肝脏来移植供体人体肝脏和/或节段 玻璃化冷冻/纳米武器复苏法。这个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纳米温控。这种方法以前是不可能的。 纳米武器的发展是由于无法在完整、部分或全部情况下产生足够的升温速度 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.