Fast Freezing Processes in Tissues

项目总结(原件) 这项研究的目标是推进生物系统冷冻保存方案的制定 使用基本的生物物理参数。目前，我们对生物物理学的认识存在着严重的空白。 在快速冻结过程中控制细胞内和细胞外水状态的参数。这项建议 旨在通过采用两项新的有效技术来弥补这一重大差距：(A)计量 基于微型热电传感器的技术，采用微制造技术 适用于人造组织和(B)基于量热法组合的测量技术 以及适用于天然组织的低温显微镜。具体来说，该项目将：(1)设计 制造和测试微型热电传感器以测量隔离细胞中的快速冷冻过程 (新生儿真皮成纤维细胞，NHDF)和人造组织(NHDF包埋在胶原胶中) 薄片)使用和不使用冷冻保护剂(CPA)以及(2)开发量热和低 温度显微镜(定向凝固)方法评估NHDF和NHDF中的快速冻结过程 含和不含CPA的天然(哺乳动物肝脏)组织切片。CPA在两种组织中的添加和移除 (人工和自然)系统将使用不可逆热力学模型进行建模。这些集成在一起 来自不同学科的方法将导致合理设计和开发冷冻过程 冷冻保存捐赠的组织，这是一种极其稀缺的资源，以及人工工程组织。 美国国立卫生研究院第15区提案的教育和基础设施发展部分包括 以及路易斯安那州立大学本科生的研究内容。 项目总结(NIGMS补充奖) NIGMS补充(设备)奖申请资金购买最先进的差速器 使用扫描量热计(DSC 8500)，以提高项目目标2中获得的数据的质量。这个 新的仪器将允许我们限制测量误差并评估细胞的真实贡献 异质性。此外，新的仪器将可控地将细胞冷冻到更高的速度(~750 ˚C/分钟)，从而使我们能够完全实现原始提案的目标，即评估快速冻结 组织中的过程。

PROJECT SUMMARY (ORIGINAL) The goal of the research is to advance the formulation of cryopreservation protocols for biological systems using fundamental biophysical parameters. At present, crucial gaps exist in our knowledge of the biophysical parameters governing the states of intra- and extra-cellular water during a fast freezing process. This proposal aims to redress this crucial gap by introducing two new and effective techniques: (a) a measurement technique based on microscale thermoelectric sensors, engineered using microfabrication techniques suitable for use with artificial tissues and (b) a measurement technique based on a combination of calorimetry and low temperature microscopy suitable for use with native tissues. Specifically, the project will: (1) design fabricate and test microscale thermoelectric sensors to measure fast freezing processes in isolated cells (Neonatal Human Dermal fibroblasts, NHDFs) and artificial tissues (NHDFs embedded in a collagen gel sheet) with and without Cryoprotective Agents (CPAs) and (2) develop a combination of calorimetric and low temperature microscopy (directional solidification) methods to assess fast freezing processes in NHDFs and native (mammalian liver) tissue sections with and without CPAs. CPA addition and removal in both tissue (artificial and native) systems will be modeled using irreversible thermodynamic models. These integrated methods from disparate disciplines will lead to rational design and development of freezing processes for cryopreservation of donated tissues, an extremely scarce resource, as well as artificially engineered tissues. The education and infrastructure development part of this NIH AREA 15 proposal includes both instructional and research components for LSU undergraduate students. PROJECT SUMMARY (NIGMS Supplemental Award) The NIGMS supplemental (equipment) award requests funds to purchase a state of the art differential scanning calorimeter (DSC 8500) to enhance the quality of the data obtained in Aim 2 of the project. The new instrument will allow us to limit measurement errors and to assess the true contribution of cell heterogeneity. In addition, the new instrument will controllably freeze cells to a much higher rates (~750 ˚C/min) thus allowing us to fully accomplish the goals of the original proposal, i.e., to assess fast freezing processes in tissues.