# High Quality Long Term Preservation of Sporozoites via Deep Supercooling

> **NIH NIH R21** · MASSACHUSETTS GENERAL HOSPITAL · 2020 · $210,000

## Abstract

Abstract
Malaria is caused by several species of intracellular protozoan parasites of the Plasmodium genus and is
responsible for nearly 500,000 deaths each year, primarily among young children. The first obligatory stage of
malaria occurs in the liver, when sporozoites transmitted by infected mosquitoes invade hepatocytes. While liver
stage malaria research has been relatively underexplored, recent technological advances have enabled the
development of sophisticated assays to model liver infection, an attractive target for drug and vaccine
development. Despite these advances, a major hurdle in liver stage research is the short shelf-life of ex vivo
sporozoites, where decreased viability is observed within ~2h after dissection from mosquito salivary glands.
Therefore, live mosquitoes must be maintained by researchers, creating many safety concerns when using
human-infective species. Logistical challenges are also encountered for high-throughput screening efforts,
where strict timelines prevent large pooling of parasites, limiting the standardization of large screening efforts,
despite the development of robust assays. There is a clear need for the development of preservation methods
for sporozoites. Here, we propose to develop a sporozoite preservation method based on our recent
breakthrough in deep supercooling of aqueous solutions where we achieve very low temperatures (down to -20
oC) in the absence of ice crystallization. We hypothesize that at these supercooled temperatures, the diminished
metabolic activity of sporozoites will prolong their functional viability, allowing their storage for up to 7-14 days.
As ice crystallization is avoided, this may be a ‘gentler’ method to preserve parasites compared to standard
cryopreservation approaches. The proposed stabilization period is sufficient time for global dissemination of
parasites and would allow pooling of parasites over several days.
We will accomplish this goal through two Specific Aims. In Aim 1, we will prioritize cold storage solutions that
lead to improved sporozoite viability and infectivity following 72 h storage under supercooled conditions (-4°C),
for both free sporozoites and whole glands. We will then optimize the solutions through supplementation with
biomolecules known to mitigate cell damage under hypothermic storage conditions. Using these optimized
storage solutions, in Aim 2 we will explore deeper supercooling temperatures (-20°C) with a goal to preserve
>50% infectivity following 7-14 d storage for free sporozoites and whole glands. We will further explore the
preservation of whole mosquitoes to enable dissections at a later time. We expect that by the completion of this
innovative exploratory project we will provide at least one solution towards the successful preservation of
sporozoites for multiple species. As the methods to achieve supercooling are straightforward, this can be readily
disseminated to other labs for immediate implementation.

## Key facts

- **NIH application ID:** 9822952
- **Project number:** 5R21AI142415-02
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Rebecca Sandlin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $210,000
- **Award type:** 5
- **Project period:** 2018-11-14 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9822952

## Citation

> US National Institutes of Health, RePORTER application 9822952, High Quality Long Term Preservation of Sporozoites via Deep Supercooling (5R21AI142415-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9822952. Licensed CC0.

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