This article details a method for isolating sporozoites from oocysts of Cryptosporidium parvum in vitro. The process involves excystation using specific media and subsequent purification steps to prepare sporozoites for injection procedures.
To isolate sporozoites from oocysts of Cryptosporidium parvum in vitro, take bleach-treated oocyst suspension.
Add excystation media containing sodium taurocholate and trypsin.
Sodium taurocholate weakens the glycocalyx layer and oocyst walls, while trypsin degrades oocyst wall proteins, resulting in oocyst excystation and sporozoite release.
Microscopically assess the excystation efficiency.
Centrifuge to pellet the sporozoites, intact oocysts, and oocyst shells.
Resuspend the pellet and pass it through a filtering assembly placed on ice to keep the sporozoites dormant.
Larger oocyst shells and unruptured oocysts are retained in the membrane filter, while small-sized sporozoites pass through the filter via gravity and are collected in the filtrate.
Centrifuge the filtrate.
Resuspend the sporozoite pellet in media containing a dye and reducing buffer constituents.
The dye aids in visualizing the sporozoite injection procedure, while the reducing buffer creates a gastrointestinal tract-like environment, enhancing sporozoite viability and infectivity.
The prepared sporozoites are ready for injection procedures.
To purify C. parvum sporozoites, transfer the oocysts to a 15-milliliter tube, and resuspend the cells at a 1 x 107 oocysts 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 sporozoite pellet at a 3 times 10 to the seventh oocysts per 1 to 2 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 3-micrometer pore-size polycarbonate filter into a 15-milliliter tube at 4 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.
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