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.