Ultra-fast cooling- a novel approach to cryopreservation of Cryptosporidium.

The efforts to cryopreserve a gastrointestinal parasite of Cryptosporidium species using traditional cryogenics have eluded scientists for decades. In this paper, a novel method of ultra-fast cooling was applied to freeze Cryptosporidium oocysts. Application of the ultra-fast freezing rate of 4000 K/s allowed for recovery of live and infectious oocysts after thawing.

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The original paper in Nature Communications is here.

Cryptosporidium, an apicomplexan parasite of growing importance worldwide, is a cause of debilitating gastrointestinal infection in children and immunosupressed individuals. It is second only to Rotavirus among enteric pathogens that cause acute infant diarrhea in the developing world. The malnutrition status resulting from cryptosporidiosis contributes further to cognitive impairment and stunted growth in children. Nitazoxanide is the only FDA-approved treatment for cryptosporidiosis but has limited efficacy in immunosupressed and malnourished hosts. The growing prevalence of cryptosporidiosis in recent years has brought attention to the need for drug and vaccine development. However, the lack of cryopreservation methods and the short shelf life of oocysts make it difficult to generate and distribute genetically characterized oocysts. Consequently, laboratory strains of Cryptosporidium must be maintained by propagation in susceptible animals (mice, calves and piglets) every 6-8 weeks, an expensive and time-consuming process. This limitation hinders progress of research and most significantly, evaluation of therapeutics and vaccines in animal and human challenge studies.

Numerous attempts to cryopreserve Cryptosporidium oocysts have been made during the last four decades using traditional methods of slow cooling, though none resulted in robust recovery of infectious oocysts. As an alternative, we explored ultra-fast cooling as a novel approach to cryopreserve C. parvum oocysts. Compared to slow cooling, ultra-fast cooling results in formation of a vitreous or ‘ice-free` state, and often leads to increased cell viability and function. 

A major hurdle to the development of the ultra-fast cooling protocol was the impermeable nature of the oocyst wall, which prevented cryoprotective agent (CPA) uptake. To overcome this limitation, a bleaching protocol was developed where sodium hypochlorite exposure results in oocyst permeability to CPAs. Bleached oocysts were then incubated in a CPA solution consisting of trehalose and DMSO, commonly used extra- and intra-cellular CPAs, respectively. Trehalose allows to eliminate source of ice crystals formation by severe dehydration of oocysts, while DMSO diffusion into oocysts provides cryoprotective function. Ultra-fast cooling was then achieved by loading the oocysts into microcapillaries (0.2 mm diameter) and plunging into liquid nitrogen, resulting in a cooling rate of nearly 4000 K/s, such that the extracellular solution is vitrified nearly instantaneously.  Using this method, thawed oocysts exhibit 50-80% viability and yield sporozoites that appear morphologically normal. Inoculation of thawed oocysts into interferon-γ knockout mice demonstrates that infectivity is maintained. This cryopreservation protocol overcomes a major bottleneck in the study of Cryptosporidium biology and the development of therapeutics. 

Silica micro capillary containing Cryptosporidium parvum oocysts before plunging into liquid nitrogen.

Justyna Jaskiewicz

PhD student, Cummings School of Veterinary Medicine at the Tufts University