Deep Supercooling of Liver Cells and 2D and 3D Tissue Constructs: Effect of Attachment

摘要 除了保存一个“完整的人”之外，还有三个悬而未决的问题 保存1)我们能否保存整个器官以减少移植中的质量和后勤问题 目前有超过120000名患者在等待移植器官？2)我们能设计出有效的保存方法吗？ 为快速增长的组织工程产品市场的传播提供便利的方案？3)我们是否可以 保持原代细胞具有与新鲜细胞相似的代谢和酶活性，坚固而廉价？最后， 我们可以问，这三个问题能不能用一个“统一的方法”来回答？目前，临床上 器官保存的黄金标准是静态冷藏(+4oC)，肾脏最多保存72小时，理想情况下为12小时 在后勤和灵活性方面受到限制的肝脏工作时间。在光谱的另一端， 冷冻保存和玻璃化冷冻--尽管某些类型的细胞在细胞水平上取得了成功--并不意味着成功 在器官和组织层面上。当前的技术差距需要一种优越而统一的 生物保存法是一种从头开始设计的生物保存方法，其长期目标是保存细胞， 组织/组织产品和器官超过一周，以缓解目前的后勤限制。基于我们的 在最近发表的著作中，我们提出生物的非冷冻(过冷)保存(SCP)提供了 这种解决方案弥合了细胞、组织和器官保存之间的差距。我们的短期目标是 发展一种从单细胞和多细胞开始的新型深过冷(DSC)保存方法 组织结构。在这一探索期之后，中期目标是将这些应用于) 商业组织结构的存储，以及b)从肾脏到心脏和肝脏的器官成功存储。 对于小样品(~&lt；1ml)，在-4至-6 oC的温度下，最多可实现~7天的过冷 不会像我们之前的工作那样结冰。然而，保持大容量(10毫升)是不可能的 在很低的温度(~-20oC)下长时间(~100天)保存保存液是一种实用的方法。 最近，我们在一项突破性的研究中解决了这一问题，这项突破性研究被称为“表面密封深度过冷(DSC)”， 我们在固体容器中用不相容的液体(油、烷烃、醇)密封水，以实现实际上的 大容量(高达100毫升)长期(高达100毫升)的稳定过冷温度(低至-20摄氏度) 天数)。因此，拟议研究的目标是开发3种“深过冷”保存方法 肝细胞/组织模型(悬浮细胞、2D平板细胞、3D球体)，然后比较长期 (5天)以上3种模型均成功。超低(-10到-20摄氏度)温度下的深度过冷可能会极大地 与冷藏(4摄氏度)相比，新陈代谢和损伤过程会变慢，我们预计这将 显著提高存储时间和质量。我们假设连接在一起的原代肝细胞 在表面上或彼此之间，那些在文化上两极分化良好的人将以更好的成功存活更长时间 在较低的温度下。因此，我们期望2D平板肝细胞和3D球体能够保存 在DSC(-10~-20oC)条件下，逐渐优于细胞悬液。我们将在以下方面测试这一假设 这两个目标是我们首先比较和找到所有模型的最佳温度和保存解决方案 然后调查潜在的分子特征，找出不同保存成功率的差异 型号：

Summary Apart from the preservation of a “whole human” there are three outstanding questions in the field of preservation 1) Can we preserve whole organs to reduce quality and logistics issues in transplantation where over 120000 patients are currently waiting for a replacement organ? 2) Can we devise an effective preservation scheme to ease the dissemination for the rapidly growing market of engineered tissue products? 3) Can we preserve primary cells with metabolic and enzymatic activity similar to fresh cells robustly and cheaply? Finally, one can ask the question: Can we answer these three questions with a “unified method”? Currently, the clinical gold standard for organ preservation is static cold storage (+4 oC) with up to 72 hours for kidney and ideally 12 hours for liver which is limiting in terms of logistics and flexibility. On the other end of the spectrum, cryopreservation and vitrification - despite successes at the cell level for some cell types - do not project success at the organ and tissue level soon. This current technological gap necessitates a superior and unified biopreservation method that is designed from the ground up with the long-term goal of storage of cells, tissues/tissue products and organs beyond one week to relieve the current logistic constraints. Based on our recent published works, we propose that non-freezing (supercooling) preservation (SCP) of living matter provides such a solution that bridges the gap between preservation of cells, tissue and organs. Our short-term goal is to develop a novel “Deep Supercooling” (DSC) preservation methods starting with single cells and multicellular tissue constructs. The intermediate-term goal, following up this exploratory period, is then to apply these to a) storage of commercial tissue-constructs, and b) successful organ storage from kidneys to hearts and livers. Supercooling can be achieved for small samples (~<1ml) at temperatures (-4 to -6 oC) for ~7 days at most without any freezing as in our earlier work. Yet, it has been impossible to keep a large volume (10s of milliliters) of preservation solution at very low temperatures (~-20 oC) for long times (~100 days) in a practical manner. Recently we addressed this in a breakthrough, dubbed “Deep Supercooling (DSC) via Surface Sealing”, where we seal water with an immiscible liquid (oils, alkanes, alcohols) in a solid container to achieve a practically stable supercooling temperature (down to -20 oC) for large volumes (up to 100 ml) for long period (up to 100 days). The objective of the proposed study is thus to develop “Deep Supercooling” preservation method for 3 models of hepatic cells/tissues (suspended cells, 2D plated cells, 3D spheroids) and then compare the long-term (5 day) success of these 3 models. Deep supercooling at ultralow (-10 to -20 oC) temperatures can drastically slow down metabolism and injury processes compared to cold storage (at 4 oC) and we expect this will significantly improve storage time and quality. We hypothesize that primary hepatocytes that are either attached to a surface or each other, and those that are well polarized in culture will survive with better success for longer at lower temperatures. Accordingly, we expect that the 2D plated hepatocytes and 3D Spheroids to preserve progressively better than cell suspensions under DSC (-10 to -20 oC) conditions. We will test this hypothesis in the two aims where we first compare and find optimum temperatures and preservation solutions for all models and then investigate the underlying molecular signatures for differences in preservation success of the different models: