In Vitro Sporozoite Isolation from Cryptosporidium parvum Oocysts

Abstract

Source: Dutta, D. et al., Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection. J. Vis. Exp. (2019)

In this video, we demonstrate an in vitro method to purify sporozoites from thick-walled oocysts of the pathogenic parasite Cryptosporidium parvum. The isolated sporozoites can be used for infection studies.

Protocol

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

Representative Results

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.

Materials

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)