JoVE Encyclopedia of Experiments
Immunology
0 views • 2:33 min • July 8th, 2025
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.