In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

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1. In Vitro Purification of Sporozoites from C. parvum Oocysts

1. Prepare Cryptosporidium oocysts for infecting cells (Figure 1A).
   NOTE: Cryptosporidium oocysts were purchased from a commercial source (see the Table of Materials). These oocysts are produced in calves and are stored in phosphate-buffered saline (PBS) with antibiotics. They can be stored for about 3 months at 4 °C and should never be frozen. We normally use oocysts within one month. Organoids can be infected with either intact oocysts, or sporozoites may be isolated from excysted oocysts and used to infect organoids if it is important not to have oocysts carryover from the original inoculum.
   1. Keep oocysts on ice throughout all manipulations until they are added to the organoids.
   2. Calculate the number of oocysts needed for a full six-well plate of organoids (usually about 5 x 10⁵–2.5 x 10⁵ for the plate). Count the number of oocysts in a hemocytometer to verify the quantity and transfer to a centrifuge tube.
      NOTE: To aid in visualization, oocysts may be mixed 1:1 with an oocyst-specific fluorescent antibody (see the Table of Materials) before being loaded onto the hemocytometer. The fluorophore-labeled oocysts can then be easily visualized and enumerated using a fluorescence microscope. We suggest injecting about 100–1,000 oocysts/organoids. In general, 1,000–2,000 organoids can be grown in a six-well plate.
   3. Bring the volume of the oocyst suspension up to 900 µL with PBS. Add 100 µL of sodium hypochlorite (e.g., Clorox) bleach (at 4 °C). Incubate for 10 min on ice.
   4. Centrifuge for 3 min in a microcentrifuge at 8,000 x g at 4 °C. Orient the tubes in the centrifuge with the cap opening facing inward. The pellet can be hard to see so knowing where the parasites have pelleted in the tube is essential.
   5. Remove the supernatant with a pipette being careful to avoid the pellet. Add 1 mL of Dulbecco's modified Eagle's medium (DMEM) and vortex to mix.
   6. Centrifuge for 3 min in a microcentrifuge at 8,000 x g at 4 °C.
   7. Repeat washes with DMEM two more times.
   8. Prepare expansion medium (OME) or differentiation organoid medium (OMD) to which taurocholate has been added to a final concentration of 0.5% (w/v) (See Table of Materials). Taurocholate should always be prepared and added fresh.
      NOTE: We have successfully used 0.5% taurocholate in our infection assays where the inoculum is intact oocysts and saw improved rates of infection without deleterious effects on the host cells. However, taurocholate may have unanticipated effects on cells and lower concentrations have been used successfully in infection assays.
   9. Resuspend oocysts in 100 μL of organoid culture medium supplemented with 0.5% (w/v) sodium taurocholate. Count oocysts again as described in step 1.1.2.
   10. Add Fast Green dye to the suspension in order to visualize injection.
2. Purify sporozoites from C. parvum oocysts after bleaching and washing out the bleach as described above.
   1. Transfer the oocysts to a 15 mL tube. Resuspend oocysts in room temperature excystation medium (0.75% w/v sodium taurocholate in DMEM) to obtain 1 x 10⁷ oocysts/mL. The addition of taurocholate improves the excystation rate of the oocysts, improving sporozoite yield.
   2. Incubate oocyst suspension at 37 °C for 1–1.5 h.
   3. Check the sample microscopically for the extent of excystation; 60–80% excystation is reasonable for good recovery of sporozoites. If the level of excystation is low, incubate longer (another 30 min to 1 h).
   4. Determine the percent excystation relative to the number of starting oocysts. Excystation is calculated as:
      % excystation = [1 – (number of intact oocysts/number of oocysts at start)] x 100
   5. Wash cells to remove excystation reagents by adding 14 mL of PBS or medium, mixing, and recovering cells (intact oocysts, oocyst shells, and sporozoites) by centrifugation at 3,400 x g for 20 min to recover sporozoites. Aspirate carefully to avoid losing cells.
   6. Resuspend the sporozoite pellet in 1–2 mL of DMEM to obtain 3 x 10⁷ oocysts/mL (based on the number of starting oocysts).
   7. To remove the remaining oocysts and shells, filter the suspension through a 3 µm filter (Figure 1B). Use a 47 mm filter holder apparatus fitted with a polycarbonate filter (3 µm pore size) attached to a 10 mL syringe barrel. Place the filter holder apparatus on top of a 15 mL tube. Place the assembly in an ice bucket or in a cold room.
   8. Add 7.5 mL of the sporozoite suspension to the filter assembly and allow it to filter through by gravity. Wash through with another 7.5 mL of DMEM.
      NOTE: To ensure success in sporozoite isolation fresh oocysts and good excystation are critical. If there are too many unexcysted oocysts, the suspension will not flow through by gravity. Applying pressure on the syringe can force unexcysted oocysts through. Microinjection of sporozoites is more challenging than that of oocysts because sporozoites may clump and block the capillary. To avoid this, we recommend making a wider capillary tip when injecting organoids with sporozoites. To achieve sufficient levels of infection, 2–4 times the number of sporozoites need to be injected into each organoid as compared to organoids infected with oocysts.
   9. Centrifuge the filtered sporozoite suspension at 3,400 x g using a swinging bucket rotor for 20 min to pellet sporozoites.
   10. Resuspend in 50–100 μL of OME or OMD organoid culture medium (see the Table of Materials) supplemented with 0.05% (w/v) Fast Green dye and L-glutathione, betaine, L-cysteine, linoleic acid and taurine-containing reducing buffer (see the Table of Materials).
       NOTE: Incubating oocysts for too long may result in the lysis of sporozoites and poor recovery and therefore should be avoided.

결과

Figure 1: Preparation and purification of Cryptosporidium oocysts and sporozoites. (A) Schematic representation of the method used for oocyst and sporozoite preparation for infection. (B) Image showing in vitro excystation of oocysts. Filtration of unexcysted oocysts and shells gives a purified solution of sporozoites. Scale bar = 10 µm.

자료

Name	Company	Catalog Number	Comments
EMD Millipore Isopore Polycarbonate Membrane Filters- 3µm	EMD-Millipore	TSTP02500
Fast green dye	SIGMA	F7252-5G
Microfuge tube 1.5ml	Eppendorf	T9661-1000EA
Micro-loader tips	Eppendorf	612-7933
Sodium hypoclorite (use 5%)	Clorox	50371478
Super stick slides	Waterborne, Inc	S100-3
Swinnex-25 47mm Polycarbonate filter holder	EMD-Millipore	SX0002500
Taurocholic acid sodium salt hydrate	SIGMA	T4009-5G
Tween-20	Merck	8221840500
DMEM	Invitrogen	12634-010	500ml
Penstrep	Gibco	15140-122	5ml of stock in 500ml DMEM
Glutamax	Gibco	35050038	5ml of stock in 500ml DMEM
Hepes	Gibco	15630056	5ml of stock in 500ml DMEM
Reducing buffer (for resuspension of oocysts and sporozoites for injection)			Final concentration
L-Glutathione reduced	Sigma	G4251-10MG	0.5 μg/μl of OME/OMD/LOM
Betaine	Sigma	61962	0.5 μg/μl of OME/OMD/LOM
L-Cysteine	Sigma	168149-2.5G	0.5 μg/μl of OME/OMD/LOM
Linoleic acid	Sigma	L1376-10MG	6.8 μg/ml of OME/OMD /LOM
Taurine	Sigma	T0625-10MG	0.5 μg/μl of OME/OMD/LOM
INTESTINAL ORGANOID MEDIA-OMD (Differentiation media)			To differentiate organoids, expanding small intestinal organoids were grown in a Wnt-rich medium for six to seven days after splitting, and then grown in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, prostaglandin E2 from a Wnt-rich medium or OME)