A Technique to Develop a Parasite Infection in Intestinal Organoids via Microinjection

Take a human intestinal organoid culture. The organoids consist of epithelial cells facing the central lumen, mimicking in vivo architecture.

Take a microinjector containing crescent-shaped sporozoites  — the infective form — of Cryptosporidium parvum, a parasite.

Inject sporozoites into the organoid lumen.

Parasite lectins bind to specific carbohydrates on epithelial cells, facilitating their entry into a parasitophorous vacuole.

Internalized parasite transforms into a trophozoite  — the feeding stage — which acquires nutrients and undergoes asexual reproduction to form type I meront, containing merozoites  — the invasive form.

Released merozoites infect neighboring epithelial cells and develop type II meronts containing merozoites.

The merozoites enter neighboring cells and differentiate into either microgamont or macrogamont  — male and female reproductive stages.

The microgamont undergoes division to form microgametes, which are released outside the vacuole.

A microgamete fertilizes the macrogamont, producing a zygote.

The zygote undergoes division, forming a sporozoite-containing oocyst. Upon release, the sporozoites repeat the infection cycle.

Harvest the organoids to assess the infection progress.

To microinject the parasites into the apical side of a 3D organoid, first, use a micropipette puller to prepare glass injection capillaries. Use forceps to cut the tip of the capillary to a 9- to 12-micrometer diameter, to enable an easy flow of sporozoites, or oocysts if you choose to inject oocysts directly, and use micro-loader tips to fill each capillary with a fast green dye-labeled oocyst or sporozoite suspension.

Then, load the first sporozoite-filled capillary into a microinjector, and use an inverted microscope at a 5x magnification and a constant pressure to microinject 100 to 200 nanoliters of the suspension into each organoid.