Ice Nucelation in Biological Tissues - Implications to Cryopreservation

描述(由申请人提供)：摘要：使用肝组织细胞作为分离的肝细胞悬浮液或整个肝组织切片是使用生物人工肝装置暂时替代肝功能的一种很有前途的方法。然而，生物人工肝设备需要随时可用的肝细胞和全肝组织切片，临床药物的异种研究也是如此。一种能够实现肝细胞和组织切片长期保存的技术是使用低温，即冷冻保存。尽管已经进行了广泛的研究来确定冷冻方案和冷冻保存剂对肝细胞和全肝切片活力的影响，但由于缺乏对控制全肝组织细胞对冷冻损伤反应的物理化学性质的基本了解，有效的低温保存方案的发展仍然受到阻碍。假设：了解肝组织切片中细胞内冰的形成将增强我们合理设计和优化冷冻/储存方案的能力。为了验证这一假设，我们将使用最近开发的两种技术的组合来测量肝组织切片中冰核的动力学，一种基于低温显微镜，另一种基于热量计。这项拟议的技术如果成功，将使生物学家或生物工程师对组织切片中的冷冻过程有一个完整的了解，并最终将导致更好的天然组织的长期存储方案。具体目标：为了验证我们的假设，将实现以下目标。SA 1：对提议的技术进行验证。对使用新技术获得的单个分离细胞中的冰核数据与成熟的冷冻显微镜程序进行了比较。SA2：测量整个组织中冰成核动力学的技术的扩展。SA3：整个组织切片中冰成核动力学的建模和预测。

DESCRIPTION (provided by applicant): Summary: The use of liver tissue cells either as isolated hepatocyte suspensions or whole liver tissue slices represents a promising approach to temporary replacement of liver function using bioartificial liver devices. However, the bioartificial liver devices need a readily available source of hepatocytes and whole liver tissue slices, as do the xenobiotic studies of clinical drugs. One technique capable of achieving long term storage of liver cells and tissue slices is the use of low temperatures, i.e. cryopreservation. Although extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of hepatocytes and whole liver slices, the development of efficient cryopreservation protocols is still hindered by a lack of fundamental understanding of the physicochemical properties governing the response of whole liver tissue cells to freezing injury. Hypotheses: Knowledge of intracellular ice formation in liver tissue sections will enhance our ability to rationally design and optimize freeze/storage protocols. To test the hypothesis, we will measure the kinetics of ice nucleation in liver tissue sections using a combination of 2 recently developed techniques, one based on low temperature microscopy and another on calorimetry. The proposed technique, if successful, will give the biologist or bioengineer a complete understanding of the freezing processes in tissue sections and will ultimately, lead to better long-term storage protocols for native tissues. Specific Aims: In order to test our hypotheses, the following aims will be accomplished. SA 1: Validation of the proposed technique. Comparison of the ice nucleation data in single isolated cells obtained using the new technique and with a well established cryomicroscopy procedure. SA2: Extension of the technique to measure ice nucleation kinetics in whole tissues. SA3: Modeling and prediction of ice nucleation kinetics in whole tissue sections.