Optimizing and expanding cryopreservation of Cryptosporidium oocysts

项目总结 隐孢子虫病是一种由感染隐孢子虫引起的腹泻疾病，是导致 婴儿腹泻发病率和死亡率。研究进展因缺乏基本生物学知识而受到严重影响 研究寄生虫的工具。由于没有强大的体外细胞培养平台，寄生虫必须常规 在牛犊、猪或老鼠等易感动物中繁殖。此外，缺乏冷冻保存方法 对于隐孢子虫卵囊这种外部感染形式，限制共享明确定义的或转基因的 筛选和限制人类挑战研究，因为每一批都必须标准化。为了解决这一限制，我们 提出一种玻璃化冷冻保存隐孢子虫卵囊的有效方法。玻璃化是一种 “无冰”冷冻保存方法，即细胞内负载相对较高浓度的 低温保护剂(CPA)和快速冷却通过玻璃化转变温度。最近，我们的实验室 开发了一种通过超快速玻璃化冷冻卵囊的方法。同时观察到高活性和传染性 在接种解冻的微小弧菌卵囊的小鼠中，这项技术很复杂，容易出现用户错误，而且受到限制 样品体积非常小(L 2微米)，从而限制了该技术在其他实验室的应用。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容量(&gt；100µL)，以及2)修改， 根据需要，将该方法应用于人类限制物种人毛虫的冷冻保存。这将是 通过两个具体的目标来实现。在具体目标1中，我们将调整生化和相变 系统的性质，以确定适当的CPA和冷冻容器的组合，以玻璃化C. 微小卵囊。我们将利用高宽高比的冷冻设备来增加样品体积 对换热性能的影响最小。可恢复活体和感染性卵囊的CPA溶液 在目标2中，将优先考虑冷冻后的情况。在具体目标2中，我们将优化玻璃化 与人毛滴虫卵囊的配伍方法，并在诺生仔猪身上验证这些方法 需要使用更大的卷，人类挑战研究也是如此。作为这项工作的结果，我们预计 将开发一种用户友好的玻璃化冷冻方案，以便在其他实验室中轻松使用，从而 解决长期冷冻保存隐孢子虫物种的关键瓶颈。

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.