Ice Nucelation in Biological Tissues - Implications to Cryopreservation

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代表了使用生物人工肝装置临时替代肝功能的一种有前途的方法。然而，生物人工肝装置需要一个现成的肝细胞和全肝组织切片来源，就像临床药物的异种研究一样。一种能够实现肝细胞和组织切片长期储存的技术是使用低温，即低温保存。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，但由于缺乏对全肝组织细胞对冷冻损伤反应的理化性质的基本理解，有效的冷冻保存方案的发展仍然受到阻碍。假设：了解肝组织切片细胞内冰的形成将提高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，我们将使用两种最新开发的技术，一种基于低温显微镜，另一种基于量热法，来测量肝组织切片冰核的动力学。如果这项技术成功，将使生物学家或生物工程师对组织切片的冷冻过程有一个完整的了解，并最终为天然组织提供更好的长期储存方案。具体目标：为了检验我们的假设，将完成以下目标。SA 1：所建议技术的验证。使用新技术获得的单个分离细胞的冰核数据与建立良好的低温显微镜程序的比较。SA2：扩展测量整个组织冰核动力学的技术。SA3：整个组织切片冰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.