High-throughput in vitro culture system for Cryptosporidium oocysts: replacing animals in research

In vitro culture of Cryptosporidium has previously failed to generate oocysts suitable for research purposes. Supply of the pathogen relies upon maintenance in calves, a usage which has now reached crisis point because of increasing demand.

A novel in vitro methodology, developed by Prof Yarlett (US), can potentially produce large numbers of oocysts, replacing maintenance of Cryptosporidium cultures in animals. The method is based on a hollow fibre bioreactor, seeded with cultured human colon tumour cells. The cells form a monolayer on the outside of the hollow microfibers, fed by culture medium flowing through the inside of the microfibers and diffusing through to the cells. Between the microfibers the cells create a stagnant micro-oxygenic environment within which Cryptosporidium sporozoites, following hatching from the oocyst, survive and invade host cells. This method has advantages over conventional cell culture: the biphasic architecture gives Cryptosporidium a micro-oxygenic environment to inhabit while providing host cells with adequate oxygen and nutrients, and the parasite is able to undergo multiple cycles of host cell invasion, growth, reproduction and release, before oocysts are produced. These steps are limited in other culture systems, leading to reduced or no oocyst production. Yarlett's method can generate 108 oocysts/ml/day for 6+ months; this could replace commercial production in calves, where the normal purchased quantity is 106 oocysts per batch.

This project will fund transfer of the Yarlett system to Cardiff University generating a European hub for Cryptosporidium oocyst supply. We will test the genetic stability of Cryptosporidium maintained in this system for 100 days by resequencing the genome at 20 fold coverage for SNP comparison with the published C. parvum genome from parasites maintained in calves. The culture method will also be tested with a C. hominis isolate from the NHS Cryptosporidium Reference Strain Laboratory.

• 2019 Research Review: Bioreactor for Cryptosporidium oocysts