Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification

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 mu L volume. Thawed oocysts exhibit similar to 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. We report here on the first successful method of cryopreservation of Cryptosporidium hominis parasites, one of the most common infectious causes of pediatric diarrhea in the developing world. This is significant as lack of cryopreservation methods for this species of parasite hinders progress of scientific and clinical research. In the absence of these methods, a reference isolate of C. hominis must be maintained periodically by passage in gnotobiotic piglets. Cryopreservation of this unique isolate is therefore a priority to increase sharing and availability for research, and to prevent catastrophic loss. Cryopreservation is also necessary to facilitate human challenge studies and clinical trials for the evaluation of therapeutics. Here, we successfully cryopreserved the parasite by vitrification using rapid cooling rates achieved in specially designed specimen containers. We showed that the thawed parasite retained viability and infectivity in gnotobiotic piglets. Notably these methods allowed us to establish a biobank of C. hominis oocysts of reference isolate TU502. The availability of biobanked optimized/standardized oocyst preparations may facilitate drug and vaccines evaluation.

Automated summary

Cryptosporidium hominis is a major cause of childhood diarrhea in developing countries, and the lack of cryopreservation and simple culturing methods hinders the development of therapeutics. This study successfully preserved Cryptosporidium hominis TU502 oocysts by vitrification, allowing for wider access to biological specimens for research purposes. The successful cryopreservation method is significant for the scientific and clinical research of this parasite.