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Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. While cryopreservation of Cryptosporidium oocysts by vitrification was recently achieved, the method is restricted to small sample volumes, thereby limiting widespread implementation of this procedure. Here, a second-generation method is described for cryopreservation of C. parvum oocysts by vitrification using custom high aspect ratio specimen containers, which enable a 100-fold increase in sample volume compared to previous methods. Oocysts cryopreserved using the described protocol exhibit high viability, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice. Importantly, the course of the infection is comparable to that observed in mice infected with unfrozen oocysts. Vitrification of C. parvum oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials.
Bibliographical noteFunding Information:
We thank the Bill and Melinda Gates Foundation for financial support (grant OPP1197110). We sincerely thank Octavio Hurtado (Massachusetts General Hospital) for laboratory guidance and Dr. Bruno Miranda Oliveira (Tufts University) for help with the animal work.
© 2020, The Author(s).
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ATP-Bio: NSF Engineering Research Center for Advanced Technologies for the Preservation of Biological Systems (ATP-Bio)
9/1/20 → 8/31/25
Project: Research project