Intracellular Phase Diagram Creation Using a Micro-Differential Scanning Calorime

使用微差扫描热量创建细胞内相图

• 批准号: 7895048
• 负责人: Gary L. Solbrekken
• 金额: $ 18.14万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895048
• 关键词: Accounting; Arts; Behavior; Biocompatible Materials; Biological; Biological Preservation; Caliber; Cell Death; Cell Transplantation; Cells; Chemicals; Chemistry; Clinical; Cryopreservation; Dehydration; Development; Devices; Disease; Electronics; Embryo; Engineering; Equipment; Failure; Family suidae; Freezing; Germ Cells; Glass; Glycerol; Goals; Heart Valves; Heating; Histocompatibility Testing; Ice; Individual; Life; Liquid substance; Measurement; Measures; Medical center; Metabolic; Modeling; Mus; Mutation; Natural Disasters; Nature; Newborn Infant; Oocytes; Phase; Phase Transition; Potassium Chloride; Probability; Procedures; Process; Property; Proteins; Protocols documentation; Quality of life; Rattus; Research Project Grants; Resolution; Sampling; Scanning; Solid; Solutions; Structure; Techniques; Temperature; Testing; Thermodynamics; Time; Tissue Engineering; Tissue Survival; Tissues; Transgenic Organisms; Umbilical Cord Blood; Water; Work; Zebrafish; aging population; base; behavior prediction; biological systems; blastocyst; cold temperature; cost; cryobiology; design; egg; extracellular; gene therapy; improved; in vivo; mathematical model; operation; phase change; public health relevance; solute; sperm cell; success; tool; tv watching

DESCRIPTION (provided by applicant): The advent of artificial tissues, cord blood therapies, cell transplantation procedures, and the desire to bank germ cells have driven the need to establish a means to reliably store biological materials. Cryopreservation is an effective means to store biological materials without functional degradation over a prolonged period of time. The challenge with successful cryopreservation is developing cooling protocols that avoid cellular dehydration and intracellular ice formation. While models have been developed to facilitate the development of cooling protocols, there is still a significant gap between model predictions and behavior in practice. It is our hypothesis that one significant reason for this gap is that current state-of-the-art models do not account for the difference in chemical composition between intracellular solution and extracellular media. We are proposing to develop an experimental tool and technique that will allow direct measurement of the intracellular phase diagram. We will then use that new phase diagram to develop new cryopreservation protocols. The measurement of the intracellular phase diagram will be accomplished by designing and building a micro-scale differential scanning calorimeter (¿DSC) that is capable of detecting the phase transition of a single cell in media. The operation of the ¿DSC is based on the Peltier effect, which allows precise temperature control during both heating and cooling modes. We have experimentally established that the proposed design concept is capable of detecting the latent heat released from a single porcine oocyte with a cell diameter of about 100 ¿m. In order for true DSC measurements to be taken it is necessary for us to improve the repeatability and reliability of the ¿DSC assembly process. We are proposing to design and build the structures using a combination of traditional and micro-fabrication techniques. Improved electronic control and fast measurement equipment will improve the measurement quality as well. After the ¿DSC assembly process has been improved and the device calibrated using pure water as a standard, we will demonstrate the capabilities of the new measurement tool by measuring the intracellular phase diagram of mouse oocytes, mouse and rat preimplantation embryos, and zebrafish embryos. Using the phase diagrams thus obtained, we will develop a new cryopreservation protocols for each biological system. In order to develop the new protocols we will make adjustments to the current cryopreservation modeling approach, accounting for the phase diagram difference between the intracellular solution and the extracellular media. The overall success of the research project will be defined by the level of cryopreservation enhancement. PUBLIC HEALTH RELEVANCE (provided by applicant): The metabolic rate of living cells diminishes dramatically at low temperatures, making cryopreservation an attractive long term storage option for biological cells and tissues. Cell transplantation is becoming more prevalent for treatment of acquired diseases and for correction of genetic defects. An aging population is demanding such procedures for improving their quality of life. Therefore the need for effective biological material storage has become increasingly important. Specific examples of storage needs include: - Banking of large quantity of living cells/tissues for typing to use in clinical settings and in case of terrorist or natural disasters. - Preservation of umbilical cord blood from newborns for the potential of generating perfectly matched genetic therapies. - Allowing sufficient time for the screening of transmissible diseases in donated biological materials. - Facilitating the transport of cells and tissues from one medical center to another - Increasing the shelf-life of engineered tissues, such as compliant heart valves, to reduce manufacturing costs. - Preserving the sperm and egg cells of endangered or transgenic species. Although cryopreservation is an attractive means to biopreservation, the freezing process is rather hazardous. Bringing the temperature of biological material from a nominal in vivo temperature of 37 oC to a storage temperature of -196oC causes the intracellular water to pass through its liquid/solid phase transition point. Depending on the rate at which cooling takes place and the chemistry of the cell media, the water inside a cell may form ice crystals, dehydrate, or vitrify (form a glass). Of those three mechanisms, the first two generally result in the death of the cell/tissue, while the glassy vitrification state provides a chemically and mechanically stable structure that increases the probability of cell/tissue survival. Thus, it is our goal to help understand if a biological sample will tend to vitrify, and to engineering cooling procedures to help encourage vitrification. Our work is focusing on developing a measurement tool, called a micro-differential scanning calorimeter, which will allow the measurement of internal cell ice formation under different cooling conditions and for different concentrations of cryoprotectant agents, such as glycerol. Currently available machines are macroscopic in nature and do not allow such fine measurement resolution. Our hypothesis is that by understanding the phase transitions at the scale of a cell, we will be able to modify mathematical models of cellular freezing to improve the cryopreservation process. We will test the new machine on mouse oocytes, mouse and rat embryos, and zebrafish embryos.

描述（由申请人提供）：人造组织、脐带血疗法、细胞移植程序的出现以及储存生殖细胞的愿望推动了建立可靠储存生物材料的方法的需求。冷冻保存是储存生物材料的有效方法。成功冷冻保存的挑战是开发避免细胞脱水和细胞内冰形成的冷却方案，虽然已经开发了模型来促进冷却方案的开发，但仍然存在很大的差距。我们的假设是，造成这种差距的一个重要原因是当前最先进的模型没有考虑细胞内溶液和细胞外介质之间化学成分的差异。开发一种实验工具和技术，可以直接测量细胞内相图，然后我们将使用该新相图来开发新的冷冻保存方案。 细胞内相图的测量将通过设计和构建能够检测介质中单个细胞的相变的微型差示扫描量热计（DSC）来完成。 DSC 基于珀耳帖效应，可在加热和冷却模式下实现精确的温度控制。我们通过实验证明，所提出的设计概念能够检测细胞直径约为 100 ¿ 的单个猪卵母细胞释放的潜热。 m. 为了进行真正的 DSC 测量，我们有必要提高 ¿我们建议结合使用传统和微制造技术来设计和构建结构，改进的电子控制和快速测量设备将改善结构。 测量质量也是如此。 之后 ¿ DSC组装工艺得到了改进，并且使用纯水作为标准校准了设备，我们将通过测量小鼠卵母细胞、小鼠胚胎和大鼠植入前以及斑马鱼胚胎的细胞内相图来展示新测量工具的功能。根据由此获得的图表，我们将为每个生物系统开发新的冷冻保存方案为了开发新的方案，我们将对当前的冷冻保存建模方法进行调整，考虑细胞内溶液之间的相图差异。研究项目的整体成功将取决于冷冻保存增强的水平。 公共健康相关性（由申请人提供）：活细胞的代谢率在低温下急剧降低，使得冷冻保存成为生物细胞和组织有吸引力的长期储存选择细胞移植对于获得性疾病的治疗和纠正变得越来越普遍。人口老龄化需要此类程序来提高其生活质量，因此有效的生物材料储存需求变得越来越重要，具体例子包括： - 储存大量活细胞/组织，用于临床环境以及恐怖或自然灾害情况下的打字。 - 保存新生儿的脐带血，以实现产生完美匹配的基因疗法的潜力。 - 留出足够的时间对捐赠的生物材料中的传染性疾病进行筛查。 - 促进细胞和组织从一个医疗中心运输到另一个医疗中心 - 延长工程组织（例如顺应性心脏瓣膜）的保质期，以降低制造成本。 - 保存濒危或转基因物种的精子和卵细胞。 尽管冷冻保存是生物保存的一种有吸引力的方法，但冷冻过程相当危险，将生物材料的温度从 37 oC 的标称体内温度降低到 -196 oC 的储存温度会导致细胞内的水通过其液/固相。根据冷却速度和细胞介质的化学性质，细胞内的水可能会形成冰晶、脱水或玻璃化（形成玻璃）。两种状态通常会导致细胞/组织死亡，而玻璃态玻璃化状态提供了化学和机械稳定的结构，增加了细胞/组织存活的可能性。因此，我们的目标是帮助了解生物样品是否倾向于死亡。玻璃化，并设计冷却程序以帮助促进玻璃化。 我们的工作重点是一种称为微差示扫描量热计的测量工具，它可以测量不同冷却条件下和不同浓度的冷冻保护剂（例如甘油）的内部细胞冰的形成，目前可用的机器本质上是宏观的。并且不允许如此精细的测量分辨率。我们的假设是，通过了解细胞尺度的相变，我们将能够修改细胞冷冻的数学模型，以改进冷冻保存过程。我们将在小鼠上测试新机器。卵母细胞、小鼠和大鼠胚胎以及斑马鱼胚胎。