Supporting Information Data files.

Cryptosporidium hominis is a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The human C. hominis TU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiring C. hominis. Here, we report cryopreservation of C. hominis TU502 oocysts by vitrification using specially designed specimen containers scaled to 100 μL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens.

人隐孢子虫（Cryptosporidium hominis）是发展中国家儿童腹泻的重要致病原。治疗药物的研发因多项重大技术瓶颈受阻，其中包括缺乏成熟的冷冻保存（cryopreservation）方案及简易培养方法。这制约了适用于研究及人体挑战试验的、经优化/标准化的单一感染性寄生虫卵囊（oocysts）来源的供应。目前，人隐孢子虫TU502分离株仅在1个实验室的无菌仔猪（gnotobiotic piglets）中传代培养，进一步限制了卵囊的可及性。简化高效的冷冻保存技术可助力建立生物样本库，为相关研究提供卵囊来源，并向其他需要人隐孢子虫的研究人员分发样本。本研究报道了采用体积适配100微升的特制样本容器，通过玻璃化（vitrification）法实现人隐孢子虫TU502卵囊的冷冻保存。解冻后的卵囊存活率约为70%，且具有良好的脱囊（excystation）活性，在无菌仔猪中的感染率可达100%。经优化/标准化的卵囊来源实现供应后，可通过拓宽生物样本的可及性，简化药物及疫苗的评估流程。