Vitrification Cassette For The Cryopreservation of Cryptosporidium Parvum Oocysts

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Scientists at Tufts University and Harvard Medical School have developed a method to cryopreserve infectious Cryptosporidium oocysts. The results were published in Scientific Reports.

Cryptosporidium is a microscopic parasite that causes the diarrheal disease cryptosporidiosis. While this parasite can be spread in many different ways water is the main source of transmission. Infants and immunocompromised adults are most at risk for severe disease.

Constant propagation of CO in laboratory animals is the only way for research labs to have access to viable strains of the parasite. All attempts to cryopreserve CO have so far led to loss of viability. These failures have been attributed to inefficient loading of cryopreserving agents.

A hypochlorite treatment followed by rapid cooling in the presence of cryopreserving additives resulted in sporozoites that displayed normal motility and morphology and were able to infect interferon-gamma (IFN-γ) knockout mice.

The researchers used a customized polycarbonate vitrification cassette (RD500011) manufactured by Grace Bio-Labs. The cassette consists of two sheets of polycarbonate bonded by a pressure-sensitive adhesive, which is cut out to form a 130µm deep chamber with an internal volume of ~ 200 µL. Solutions are pipetted into the cassette through a PDMS punch, which seals the cassette port and creates a vacuum.  This cassette increased the sample volume to approximately 200 µL with minimal reduction of the cooling rate.

The researchers reported that “this volume is sufficient to produce multiple inocula from a single cryopreserved sample (e.g. 4 doses for pigs or > 20 human doses for clinical trials), thus overcoming a major bottleneck in the development and testing of therapeutics for the treatment of cryptosporidiosis.”

References:

Jaskiewicz, J.J., Sevenler, D., Swei, A.A. et al. Cryopreservation of infectious Cryptosporidium parvum oocysts achieved through vitrification using high aspect ratio specimen containers. Sci Rep 10, 11711 (2020). https://doi.org/10.1038/s41598-020-68643-6