Optimizing and expanding cryopreservation of Cryptosporidium oocysts

项目摘要 隐孢子虫病是一种由隐孢子虫寄生虫感染引起的慢性疾病，是导致人类生殖系统疾病的主要原因。 婴儿腹泻发病率和死亡率。研究进展受到缺乏基本生物学的严重影响 研究寄生虫的工具。由于不存在稳健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，限制了明确定义的或遗传修饰的 菌株和限值人体挑战研究，因为每个批次必须标准化。为了解决这个问题，我们 建议开发一种稳定的方法，通过玻璃化冷冻保存隐孢子虫卵囊。玻璃化是一种 冷冻保存的“无冰”方法，其中细胞装载有相对高浓度的 冷冻保护剂（CPA），并迅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来玻璃化卵囊的方法。虽然观察到高活力和感染性 在接种解冻的C.卵囊，该技术是复杂的，容易出现用户错误， 非常小的样品体积（2微升），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）简化方案并将其扩展到更大的体积（>100微升），以及2）修改， 根据需要，将该方法应用于冷冻保存C。hominis，人类的限制物种。这将是 通过两个具体目标实现。在具体目标1中，我们将调整生化和相变 该系统的属性，以确定适当的组合的CPA和冷冻容器玻璃化C。 小卵囊我们将利用高纵横比冷冻设备，以增加样品体积， 对传热性能的影响最小。CPA解决方案，可回收活的和感染性卵囊 将优先考虑在目标2中进行评价。在具体目标2中，我们将优化玻璃化 与C.人卵囊，并在无菌仔猪中验证这些方法， 需要使用更大的体积，就像人体挑战研究一样。由于这项工作，我们预计 将开发一个用户友好的玻璃化方案，可以很容易地在其他实验室使用， 解决了长时间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.