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（通过应用提供）：人造组织，脐带血疗法，细胞移植程序以及对堤岸生殖细胞的愿望的冒险，促使人们需要建立一种方法来建立可靠的存储生物学材料的方法。冷冻保存是在长时间内储存生物材料而无需降解的有效手段。成功的冷冻保存的挑战是制定冷却方案，避免细胞脱水和细胞内冰的形成。尽管已开发模型来支持冷却协议的开发，但在实践中，模型预测与行为之间仍然存在显着差距。我们的假设是，这种差距的一个重要原因是，当前的最新模型不能解释细胞内溶液和细胞外培养基之间的化学成分差异。我们建议开发一种实验工具和技术，该工具和技术将允许直接测量细胞内相图。然后，我们将使用新的相图来开发新的冷冻保存协议。 细胞内图的测量将通过设计和构建微尺度差扫描量热仪（DSC）来完成，该量表能够检测介质中单个单元的相变。 „ DSC的操作基于Peltier效应，该效应允许在加热和冷却模式下进行精确的温度控制。我们已经通过实验确定了所提出的设计概念能够检测从单个猪卵母细胞释放的潜热，其细胞直径约为100€m。为了进行真实的DSC测量，我们有必要提高DSC组装过程的可重复性和可靠性。我们建议使用传统和微型制作技术的组合来设计和建立结构。改进的电子控制和快速测量设备将改善 测量质量也是如此。 在改进了DSC组装过程并以纯水为标准的设备进行校准之后，我们将通过测量小鼠卵母细胞的细胞内相图，小鼠和大鼠植入前胚胎和Zebrafish胚胎来证明新测量工具的功能。使用这样获得的相图，我们将为每个生物系统开发一个新的冷冻保存方案。为了制定新协议，我们将对当前的冷冻保存模型方法进行调整，这考虑了细胞内解决方案和细胞外培养基之间的相位差异。研究项目的总体成功将由冷冻保存增强水平来定义。 公共卫生相关性（由适用提供）：活细胞的代谢速率在低温下大大降低，使冷冻保存成为生物细胞和组织的有吸引力的长期存储选择。细胞移植在治疗获得性疾病和纠正遗传缺陷方面变得越来越普遍。人口老龄化的人要求这样的程序来改善其生活质量。因此，对有效的生物材料存储的需求变得越来越重要。存储需求的具体示例包括： - 在临床环境和恐怖分子或自然灾害的情况下使用大量活细胞/组织的库来键入。 - 从新生儿中保存脐带血，以产生完美匹配的基因疗法。 - 允许足够的时间在捐赠的生物材料中筛查可传播疾病。 - 促进细胞和组织从一个医疗中心到另一个医疗中心的运输 - 增加工程组织的保质期，例如兼容的心脏瓣膜，以降低制造成本。 - 保留濒危或转基因物种的精子和卵细胞。 尽管冷冻保存是一种诱人的生物保存方法，但冻结过程却是危险的。将生物学材料的温度从标称体内温度37 OC的温度带到-196oC的储存温度会导致细胞内水通过其液体/固相过渡点。取决于进行冷却的速度和细胞介质的化学性质，细胞内的水可能形成冰晶，脱水或玻璃（形成玻璃）。在这三种机制中，前两个通常会导致细胞/组织的死亡，而玻璃玻璃化态则提供了化学和机械稳定的结构，从而增加了细胞/组织存活的可能性。这是我们的目标是帮助了解生物样品是否倾向于玻璃化，以及工程冷却程序以帮助鼓励玻璃化。 我们的工作集中在开发一种称为微分差异量热仪的测量工具上，该工具将允许在不同的冷却条件和不同浓度的冷冻保护剂（例如甘油）的情况下测量内部细胞冰的形成。当前可用的机器本质上是宏观的，不允许这样的精细测量分辨率。我们的假设是，通过理解细胞尺度上的相变，我们将能够修改细胞冻结的数学模型以改善冷冻保存过程。我们将在小鼠卵母细胞，小鼠和大鼠胚胎以及斑马鱼胚胎上测试新机器。