Optimizing and expanding cryopreservation of Cryptosporidium oocysts

优化和扩大隐孢子虫卵囊的冷冻保存

基本信息

批准号：
10186701
负责人：
Rebecca Sandlin
金额：
$ 20.81万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-09 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10186701
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 porcine model prevent safety testing skills therapeutic evaluation time use tool user-friendly vaccine evaluation virtual wasting

项目摘要

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因缺乏基础生物学而受到严重影响。由于不存在强大的体外细胞培养平台，因此寄生虫必须是常规的。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压力和限制人类挑战研究，因为每批都必须标准化，为了解决这个限制。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。 “无冰”冷冻保存方法，其中细胞装载有相对高浓度的最近，我们的实验室使用了冷冻保护剂（CPA）并快速冷却到玻璃化转变温度。开发了一种通过超快速玻璃化来玻璃化卵囊的方法，同时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。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，应用该方法来冷冻人类限制物种 C. hominis。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. 我们将利用高纵横比冷冻装置来增加样本量。对传热特性的影响最小，从而恢复活的和传染性的卵囊。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人念珠菌卵囊相容的方法，并在知生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，我们预计这项工作的结果。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。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cryopreservation of infectious Cryptosporidium parvum oocysts achieved through vitrification using high aspect ratio specimen containers.

使用高纵横比标本容器通过玻璃化冷冻保存传染性隐孢子虫卵囊。

DOI：
发表时间：
2020-07-16
期刊：
影响因子：
4.6
作者：
Jaskiewicz, Justyna J;Sevenler, Derin;Swei, Anisa A;Widmer, Giovanni;Toner, Mehmet;Tzipori, Saul;Sandlin, Rebecca D
通讯作者：
Sandlin, Rebecca D

Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification.

通过玻璃化冷冻保存传染性隐孢子虫卵囊。

DOI：
发表时间：
2023-06
期刊：
影响因子：
6.7
作者：
Jaskiewicz, Justyna J;Dayao, Denise Ann E;Girouard, Donald;Sevenler, Derin;Widmer, Giovanni;Toner, Mehmet;Tzipori, Saul;Sandlin, Rebecca D
通讯作者：
Sandlin, Rebecca D

A highly antigenic fragment within the zoonotic Cryptosporidium parvum Gp900 glycoprotein (Domain 3) is absent in human restricted Cryptosporidium species.

人畜共患隐孢子虫 Gp900 糖蛋白（结构域 3）内的高抗原性片段在人类限制性隐孢子虫物种中不存在。