Hepatocyte production from ice-free cryopreserved and nanowarmed livers

从无冰冷冻和纳米加热的肝脏中生产肝细胞

• 批准号: 10156435
• 负责人: Charles Y Lee
• 金额: $ 37.43万
• 依托单位: VITRISTOR LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-05 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156435
• 关键词: Acute; Acute Liver Failure; Animals; Architecture; Artificial Liver; Biomedical Engineering; Cell Therapy; Cell Transplantation; Cell Volumes; Cell physiology; Cells; Chronic; Clinic; Collaborations; Consultations; Coupled; Cryopreservation; Crystal Formation; Crystallization; Donor person; Drug Industry; Drug Kinetics; Drug toxicity; Engineering; Ensure; Exhibits; Family suidae; Freezing; Funding; Goals; Heating; Hepatocyte; Hepatocyte transplantation; Human; Ice; Indocyanine Green; Industry Standard; Ischemia; Legal patent; Liver; Liver Failure; Liver diseases; Metabolic; Methods; Minnesota; Organ; Perfusion; Pharmacologic Substance; Phase; Population; Production; Protocols documentation; Rattus; Reperfusion Therapy; Research Personnel; Resuscitation; Rewarming; Sister; Suspensions; Technology; Temperature; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicity Tests; Toxicology; Transplant Surgeon; Transplantation; United States National Institutes of Health; Universities; Vascular System; Vial device; base; commercialization; drug discovery; drug metabolism; drug testing; experimental study; improved; innovation; iron oxide nanoparticle; liver function; liver transplantation; nanowarming; radio frequency; scale up; uptake

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.

基于肝细胞的疗法，包括细胞移植，生物人工和工程生活，受到限制 无法按需产生大量高功能的原代人肝细胞。另外， 制药行业可以从一项可以提供汇总捐助者和能力的技术中受益 控制对药物代谢和毒性测试的需求的供应。该项目的最终目标 开发对部分或整个人类的玻璃化和纳米武术的技术，以产生 大量代谢活性，高质量的原发性肝细胞的需求 治疗和药物应用。 Vitriation是一种无冰的冷冻保存方法，显示出希望，但目前不适用于大型组织 和器官在缓慢变暖过程中破坏了冰晶形成。最近，我们的小组在大学 明尼苏达州使用氧化铁纳米颗粒（IONP）与射频相结合的“纳米臂” （RF）实现足以避免结晶和玻璃体组织破裂的均匀变暖速率的技术 并具有扩展到部分和/或整个人体器官的能力。 该阶段I项目的目标是确定与全鼠隔离肝细胞的效率 通过纳米武器玻璃化和重新加热的肝脏。该项目将使用肝脏自己的本地 血管系统在肝细胞分离之前加载和卸载玻璃化溶液（VS）。这允许 VS均匀递送到大量细胞。我们的初步结果表明 全鼠肝脏和均匀的重新加热速率足以避免结晶和开裂。 在低温温度下VS的负载和卸载导致高产，可生活和肝细胞 功能。最后，纳米武器玻璃化的寿命在很大程度上表现出正常的建筑，显示了特定于肝细胞 功能（吲哚氨酸绿色摄取）和均匀的灌注。这表明大量 可以从纳米臂的玻璃体肝脏中分离出可行的和功能的细胞。第一阶段的目标 可以通过完成以下特定目标来实现项目： 特定目标1：确定加载和卸载三种不同浓度的效率（7、8和 大鼠肝脏中的玻璃化溶液（VS）的9m），其产量，生存力和功能 肝细胞。 特定目标2：确定确保均匀和快速变暖速率的IONP浓度 足以避免结晶。 特定目标3：确定玻璃化和纳米武器的效率，以获得大量 数量可行且功能高的肝细胞。 如果成功，第二阶段项目将扩大技术，以与蛋黄酱合作生活 诊所。此外，该项目将与Lonza（世界领先的肝细胞供应商）合作，不适合 通过结合我们的姊妹公司Hepatosys Inc的肝脏，移植人类的生命和/或细分市场 玻璃化/纳米臂的复苏方法。该项目的创新是玻璃化和重新加热 通过在隔离肝细胞之前对整个肝脏进行纳米武术。这种方法是不可能的 由于无法在完整的部分或整体上产生足够的变暖率，因此开发了纳米武器 器官避免冰晶形成的破坏作用。