# Optimizing and expanding cryopreservation of Cryptosporidium oocysts

> **NIH NIH R21** · MASSACHUSETTS GENERAL HOSPITAL · 2020 · $266,600

## Abstract

PROJECT SUMMARY
Cryptosporidiosis, a diarrheal illness caused by infection with Cryptosporidium parasites, is a leading cause of
diarrheal morbidity and mortality in infants. Research progress is severely impacted by lack of basic biological
tools to study the parasite. As no robust in vitro cell culture platform exists, the parasite must be routinely
propagated in susceptible animals such as calves, pigs or mice. Further, the lack of cryopreservation methods
for Cryptosporidium oocysts, the external infectious form, restricts sharing of well-defined or genetically modified
strains and limits human challenge studies as each batch must be standardized. To address this limitation, we
propose to develop a robust method to cryopreserve Cryptosporidium oocysts by vitrification. Vitrification is an
`ice-free' approach to cryopreservation where cells are loaded with relatively high concentrations of
cryoprotective agents (CPAs) and rapidly cooled through the glass transition temperature. Recently, our lab
developed a method to vitrify oocysts by ultra-rapid vitrification. While high viability and infectivity was observed
in mice inoculated with thawed C. parvum oocysts, the technique is complicated, prone to user error, and limited
to very small sample volumes (2 µL), thereby restricting utilization of the technology in other labs. Thus, the
overall goals of this proposal are 1) to simplify and scale the protocol to larger volumes (>100 µL), and 2) modify,
as needed, to apply the methodology to cryopreserve C. hominis, the human restricted species. This will be
accomplished through two specific aims. In Specific Aim 1, we will tune the biochemical and phase-transition
properties of the system to identify the appropriate combinations of CPAs and freezing vessels to vitrify C.
parvum oocysts. We will utilize high aspect ratio freezing devices to enable increased sample volumes with
minimum impact on heat transfer properties. CPA solutions that lead to recovery of viable and infectious oocysts
after freezing will be prioritized for evaluation in Aim 2. In Specific Aim 2, we will optimize the vitrification
approach for compatibility with C. hominis oocysts and validate these methods in gnotobiotic piglets which
require the use of larger volumes, as would be for human challenge studies. As a result of this work, we anticipate
that a user-friendly vitrification protocol will be developed that can be easily utilized in other laboratories, thus
addressing a critical bottleneck of cryopreserving species of Cryptosporidium over a long periods of time.

## Key facts

- **NIH application ID:** 10041458
- **Project number:** 1R21AI154026-01
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Rebecca Sandlin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $266,600
- **Award type:** 1
- **Project period:** 2020-06-09 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10041458

## Citation

> US National Institutes of Health, RePORTER application 10041458, Optimizing and expanding cryopreservation of Cryptosporidium oocysts (1R21AI154026-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10041458. Licensed CC0.

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