Method and Device for Rodent Germplasm Vitrification

啮齿类种质玻璃化冷冻方法及装置

• 批准号: 7800253
• 负责人: Hongbin Ma
• 金额: $ 18.5万
• 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: 2009-04-15 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800253
• 关键词: Acrosome; Area; Binding; Biomedical Research; Caliber; Cell Survival; Cells; Characteristics; Chemicals; Complex; Cryopreservation; Cryoprotective Agents; Crystal Formation; Device Designs; Devices; Dimensions; Dimethyl Sulfoxide; Embryo; Engineering; Excision; Family suidae; Film; Glycerol; Goals; Heating; Ice; Investigation; Liquid substance; Measures; Medicine; Membrane; Metals; Methods; Microfabrication; Microfilaments; Modeling; Modification; Morphology; Mus; Nitrogen; Performance; Porosity; Procedures; Process; Pump; Reproductive Medicine; Research; Research Infrastructure; Research Personnel; Resources; Rodent; Silicon; Sperm Motility; Structure; Surface; Survival Rate; Suspension substance; Suspensions; System; Techniques; Temperature; Testing; Thick; Time; Toxic effect; Work; base; cell injury; cell type; cost; cryogenics; design; efficacy testing; evaporation; gene therapy; improved; melting; novel; oncology; operation; physical model; prevent; public health relevance; research study; sperm cell

DESCRIPTION (provided by applicant): The over-all goal of this application is to establish a universal and easy-to-use germplasm cryopreservation approach by developing a novel ultra-fast cooling device system. This device system achieves vitrification of germplasm suspensions without any cryoprotective agent (CPA) or with low concentration CPA (<1M). We will do this by combining cutting-edge engineering techniques including: (1) cryogenic thin film evaporation (TFE), (2) thermal fins, and (3) microfabrication techniques. Germplasm cryopreservation is essential to many areas of medicine such as reproductive medicine, oncology, and gene therapy. However, current cryopreservation methods and devices are very different for different types of germplasms or even within cell types and they are sometimes very difficult to use. Due to the complications and inefficiencies involved in these methods, many cells types cannot be successfully cryopreserved at all and many others respond very poorly to current methods (i.e., more than 50% of the cells are lost during the process). Therefore, it is important to establish a universal, efficient cell cryopreservation method and develop corresponding devices that require relatively simple operations. Direct vitrification (solidification without crystal formation) of cell suspensions without permeating CPAs (e.g. glycerol and DMSO), or with relatively low concentrations (<1M) of CPAs, is suitable for cryopreservation of essentially all cell types. This is the case because (1) vitrification prevents cell damages caused by ice formation; (2) a low concentration of CPA significantly lowers CPA toxicity and osmotic damages during CPA addition and removal procedures. However, vitrification with low concentration CPAs requires ultra-fast cooling rates (106-7K/min). Since none of the current cryopreservation methods and devices can achieve these ultra-fast cooling/warming rates, successful completion of this work will significantly enhance the underlying infrastructure for many areas of biomedical research and save a significant amount of resources by providing a universal, effective cell cryopreservation system. This work will be done by completing the following specific aims: (1) theoretical and experimental investigations on applications of the three cutting-edge techniques in cryopreservation, (2) determination of the optimal design of the proposed device system based on the above investigations, and (3) empirical testing the device system by cell survival rates. PUBLIC HEALTH RELEVANCE (provided by applicant): Germplasm cryopreservation is essential to many areas of medicine such as reproductive medicine, oncology, and gene therapy. However, current cryopreservation methods and devices are very different for different types of germplasms or even within cell types and they are sometimes very difficult to use. The over-all goal of this application is to establish a universal and easy-to-use germplasm cryopreservation approach by developing a novel ultra-fast cooling device system.

描述（申请人提供）：本申请的总体目标是通过开发一种新型的超快速冷却装置系统，建立一种通用且易于使用的种质冷冻保存方法。该装置系统实现了种质悬浮液的玻璃化冷冻，无需任何 冷冻保护剂 (CPA) 或低浓度 CPA (<1M)。我们将通过结合尖端工程技术来实现这一目标，包括：(1) 低温薄膜蒸发 (TFE)、(2) 散热片和 (3) 微加工技术。种质冷冻保存对于生殖医学、肿瘤学和基因治疗等许多医学领域至关重要。然而，目前的冷冻保存方法和装置对于不同类型的种质甚至细胞类型内有很大不同，并且有时非常难以使用。由于这些方法的复杂性和低效率，许多细胞类型根本无法成功冷冻保存，并且许多其他细胞类型对当前方法的反应非常差（即，超过 50% 的细胞在此过程中丢失）。因此，建立通用、高效的细胞冻存方法并开发操作相对简单的相应装置具有重要意义。不渗透 CPA（例如甘油和 DMSO）或使用相对较低浓度 (<1M) CPA 的细胞悬浮液的直接玻璃化（不形成晶体的凝固）适用于基本上所有细胞类型的冷冻保存。之所以会出现这种情况，是因为（1）玻璃化可以防止结冰造成的细胞损伤； (2)低浓度的CPA可显着降低CPA的毒性和CPA添加和去除过程中的渗透损伤。然而，低浓度 CPA 的玻璃化需要超快的冷却速率 (106-7K/min)。由于目前的冷冻保存方法和设备都无法实现这些超快的冷却/升温速率，因此这项工作的成功完成将显着增强生物医学研究许多领域的基础设施，并通过提供通用、有效的方法来节省大量资源。细胞冷冻保存系统。这项工作将通过完成以下具体目标来完成：（1）对三种尖端技术在低温保存中的应用进行理论和实验研究，（2）根据上述研究确定所提出的装置系统的优化设计， (3)通过细胞存活率对装置系统进行实证测试。 公共卫生相关性（由申请人提供）：种质冷冻保存对于生殖医学、肿瘤学和基因治疗等许多医学领域至关重要。然而，目前的冷冻保存方法和装置对于不同类型的种质甚至细胞类型内有很大不同，并且有时非常难以使用。该应用的总体目标是通过开发一种新型的超快速冷却装置系统，建立一种通用且易于使用的种质冷冻保存方法。