Using this sporozoite purification and microinjection procedure, essentially any host microbe can be studied in any tissue-derived organ system. Using this technique, we can control the amount of pure sporozoites injected into the organoid lumen or the apical side of the organoid. To purify C.parvum sporozoites, transfer the oocyst to a 15-milliliter tube and resuspend the cells at a one times 10 to the seventh oocyst per milliliter excystation medium concentration.
After 60 to 90 minutes at 37 degrees Celsius, mix 14 milliliters of an appropriate wash solution with the oocysts, and sediment the cells by centrifugation. Aspirate the supernatant carefully to avoid losing oocysts and resuspend the sporozoic pellet at a three times 10 to the seventh oocyst per one to two milliliters of DMEM concentration. To remove any remaining oocysts and shells, place a 10-milliliter syringe barrel equipped with a 47-milliliter filter holder apparatus fitted with a three-micrometer pore size polycarbonate filter into a 15-milliliter tube at four degrees Celsius.
Add 7.5 milliliters of the sporozoite suspension to the filter assembly. When the entire volume has passed through the strainer by gravity, wash any remaining cells through the strainer with another 7.5 milliliters of DMEM. When all of the wash has passed through the strainer, collect the filtered sporozoite suspension by centrifugation for 10 minutes and label the pellet in 50 to 100 microliters of an appropriate organoid culture medium, supplemented with 0.05%fast green dye, and L-glutathione, betaine, L-cysteine, linoleic acid, and taurine containing reducing buffer.
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 nine 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.
The excystation protocol results in the release of sporozoites from approximately 70 to 80%of the oocysts. Therefore, it is essential to filter out the remaining oocyst shells through a three-micrometer filtration. Remove almost 100%of the unexcysted oocysts.
Moreover, the addition of a green dye helps ensure the injection of all of the organoids, and allows visualization of the injected organoids for at least 24 hours after the injection. Although this is a well-practiced method, scanning electron microscopy can be used to ensure that the excystation process did not damage the sporozoites or the oocysts. The injection of equal amounts of oocysts into the organoid lumen can be visually confirmed by simple microscopic imaging.
Immunofluorescence assays can also be used to explore the types of cells that are infected by Cryptosporidium. After the differentiated organoids have been infected for five days, the presence of sporozoites within the oocysts can be confirmed by drying down a portion of the oocysts onto an adhesive slide, fixing the oocysts with methanol, and combining DAPI staining with a suitable oocyst-specific antibody. For maximum results, use fresh oocysts, use the same capillary for all of the injections, and change the media every day after injection.
Using this technique, various labs are now starting to use organoids to study host-microbe interactions. We are studying commensals as well as other pathogens in similar organoid microculture settings. As Cryptosporidium is a human parasite, it is important to perform these experiments according to Level 2 safety regulations.
