Optimizing and expanding cryopreservation of Cryptosporidium oocysts

优化和扩大隐孢子虫卵囊的冷冻保存

• 批准号: 10041458
• 负责人: Rebecca Sandlin
• 金额: $ 26.66万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-09 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041458
• 关键词: 2 year old; AIDS/HIV problem; Address; Africa South of the Sahara; Animals; Area; Asia; Biochemical; Biological; Biological Assay; Biology; Cell Culture Techniques; Child; Clinical; Cryopreservation; Cryopreserved Cell; Cryoprotective Agents; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; Data; Devices; Diarrhea; Dimethyl Sulfoxide; Engineering; Enteral; Etiology; Evaluation; Family suidae; Formulation; Foundations; Freezing; Genomics; Geometry; Glass; Glycerol; Gnotobiotic; Goals; Growth; Human; Ice; Impaired cognition; In Vitro; Individual; Infant; Infection; Laboratories; Laboratory Animals; Lead; Life; Logistics; Methodology; Methods; Modeling; Morbidity - disease rate; Multicenter Studies; Mus; Oocysts; Parasites; Pathogenesis; Pharmaceutical Preparations; Phase Transition; Phylogenetic Analysis; Predisposition; Preventive measure; Procedures; Process; Property; Protocols documentation; Public Health; Recovery; Reporting; Reproducibility; Research; Research Personnel; Rotavirus; Safety; Sampling; Shigella; Site; Solid; Source; Standardization; Staphylococcus hominis; System; Techniques; Technology; Testing; Therapeutic; Time; Toddler; Toxic effect; Transition Temperature; Translating; Vaccines; Work; base; clinically relevant; comparative; cryobiology; crystallinity; diarrheal disease; drug discovery; enterotoxigenic Escherichia coli; experimental study; improved; mortality; mouse model; novel; pathogen; prevent; safety testing; skills; therapeutic evaluation; time use; tool; user-friendly; vaccine evaluation; virtual; wasting

PROJECT SUMMARY Cryptosporidiosis, a diarrheal illness caused by infection with Cryptosporidium parasites, is a leading cause of diarrheal morbidity and mortality in infants. Research progress is severely impacted by lack of basic biological tools to study the parasite. As no robust in vitro cell culture platform exists, the parasite must be routinely propagated in susceptible animals such as calves, pigs or mice. Further, the lack of cryopreservation methods for Cryptosporidium oocysts, the external infectious form, restricts sharing of well-defined or genetically modified strains and limits human challenge studies as each batch must be standardized. To address this limitation, we propose to develop a robust method to cryopreserve Cryptosporidium oocysts by vitrification. Vitrification is an `ice-free' approach to cryopreservation where cells are loaded with relatively high concentrations of cryoprotective agents (CPAs) and rapidly cooled through the glass transition temperature. Recently, our lab developed a method to vitrify oocysts by ultra-rapid vitrification. While high viability and infectivity was observed in mice inoculated with thawed C. parvum oocysts, the technique is complicated, prone to user error, and limited to very small sample volumes (2 µL), thereby restricting utilization of the technology in other labs. Thus, the overall goals of this proposal are 1) to simplify and scale the protocol to larger volumes (>100 µL), and 2) modify, as needed, to apply the methodology to cryopreserve C. hominis, the human restricted species. This will be accomplished through two specific aims. In Specific Aim 1, we will tune the biochemical and phase-transition properties of the system to identify the appropriate combinations of CPAs and freezing vessels to vitrify C. parvum oocysts. We will utilize high aspect ratio freezing devices to enable increased sample volumes with minimum impact on heat transfer properties. CPA solutions that lead to recovery of viable and infectious oocysts after freezing will be prioritized for evaluation in Aim 2. In Specific Aim 2, we will optimize the vitrification approach for compatibility with C. hominis oocysts and validate these methods in gnotobiotic piglets which require the use of larger volumes, as would be for human challenge studies. As a result of this work, we anticipate that a user-friendly vitrification protocol will be developed that can be easily utilized in other laboratories, thus addressing a critical bottleneck of cryopreserving species of Cryptosporidium over a long periods of time.

项目概要 隐孢子虫病是一种由隐孢子虫寄生虫感染引起的腹泻病，是导致以下疾病的主要原因 婴儿腹泻发病率和死亡率。由于缺乏基础生物学知识，研究进展受到严重影响 研究寄生虫的工具。由于不存在强大的体外细胞培养平台，因此必须对寄生虫进行常规培养 在易感动物如小牛、猪或小鼠中繁殖。此外，缺乏冷冻保存方法 对于隐孢子虫卵囊，外部感染形式，限制了明确定义或转基因的共享 由于每批都必须标准化，因此会增加和限制人类挑战研究。为了解决这个限制，我们 建议开发一种通过玻璃化冷冻保存隐孢子虫卵囊的可靠方法。玻璃化冷冻是一种 “无冰”冷冻保存方法，其中细胞负载相对高浓度的 冷冻保护剂 (CPA) 并快速冷却至玻璃化转变温度。最近，我们实验室 开发了一种通过超快速玻璃化来玻璃化卵囊的方法。虽然观察到高活力和传染性 在接种解冻的小隐孢子虫卵囊的小鼠中，该技术很复杂，容易出现用户错误，并且受到限制 样品量非常小 (2 µL)，从而限制了该技术在其他实验室的使用。因此， 该提案的总体目标是 1) 简化协议并将其扩展到更大的体积 (>100 µL)，以及 2) 修改， 根据需要，应用该方法来冷冻保存人类限制物种 C. hominis。这将是 通过两个具体目标来实现。在具体目标 1 中，我们将调整生化和相变 系统的特性，以确定 CPA 和冷冻容器的适当组合以玻璃化 C. 微小卵囊。我们将利用高纵横比冷冻装置来增加样品体积 对传热性能的影响最小。 CPA 解决方案可回收有活力且具有传染性的卵囊 冷冻后将优先用于目标2中的评估。在具体目标2中，我们将优化玻璃化 与 C. hominis 卵囊相容的方法，并在无菌仔猪中验证这些方法， 需要使用更大的体积，就像人类挑战研究一样。作为这项工作的结果，我们预计 将开发一个用户友好的玻璃化协议，可以在其他实验室轻松使用，从而 解决了长期冷冻保存隐孢子虫物种的一个关键瓶颈。