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

总结 除了保护“完整的人”之外，在这一领域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， 现时有超过12万名病人正在轮候器官移植？2)我们能设计出一种有效的保护方法吗 计划，以减轻迅速增长的市场，工程组织产品的传播？3)我们能 保存具有与新鲜细胞相似的代谢和酶活性的原代细胞稳健且便宜？最后， 人们不禁要问：这三个问题，能否用“统一的方法”来回答？目前，临床 器官保存的黄金标准是静态冷藏（+4 ℃），肾脏最长可保存72小时，理想情况下为12小时。 肝脏手术需要几个小时，这在后勤和灵活性方面是有限的。另一方面， 冷冻保存和玻璃化--尽管某些细胞类型在细胞水平上取得了成功--并不预示成功 在器官和组织层面上的应用当前的技术差距需要一个上级和统一的 生物保存方法是从头开始设计的，其长期目标是储存细胞， 组织/组织产品和器官的采购时间超过一周，以缓解目前的后勤限制。基于我们 最近发表的作品，我们提出，非冻结（过冷）保存（SCP）的生活物质提供 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 开发一种新的“深度过冷”（DSC）保存方法，从单细胞和多细胞开始 组织构建体。在这一探索期之后，中期目标是将这些应用于a） 商业组织构建体的储存，和B）从肾脏到心脏和肝脏的成功器官储存。 对于小样品（~<1 ml），在温度（-4至-6 ℃）下最多可实现~7天的过冷 不像我们以前的工作那样冻结。然而，不可能保持大体积（10毫升） 保存溶液在非常低的温度下（~-20 ℃）长时间（~100天）以实用的方式。 最近，我们在一项突破中解决了这一问题，称为“通过表面密封的深度过冷（DSC）”， 其中我们将水与不混溶的液体（油、烷烃、醇）密封在固体容器中，以实现实际上 稳定的过冷温度（低至-20 ° C），适用于大容量（高达100 ml）和长时间（高达100 天）。因此，本研究的目的是开发一种用于3 肝细胞/组织模型（悬浮细胞、2D平板细胞、3D球状体），然后比较长期 （5天）这三个模型的成功。在超低温（-10至-20 ℃）下的深度过冷可以显著地 与冷藏（4 ℃）相比，减缓新陈代谢和损伤过程，我们预计这将 大大提高了存储时间和质量。我们假设原代肝细胞要么附着在 而那些在文化上极化良好的人将更成功地生存更长时间 在较低的温度下。因此，我们预计2D平板肝细胞和3D球体可以保存 在DSC（-10至-20 ℃）条件下，其性能逐渐优于细胞悬浮液。我们将测试这个假设， 这两个目标是我们首先比较并找到所有模型的最佳温度和保存解决方案 然后研究不同的保存成功率差异的潜在分子特征， 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: