A Technique to Develop a Parasite Infection in Intestinal Organoids via Microinjection

Abstract

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

This video demonstrates a microinjection technique to develop a human intestinal organoid infection model. The infectious sporozoite stage of the parasite Cryptosporidium parvum is microinjected into the organoid lumen. Upon invading the epithelial cells of the organoid, it undergoes asexual and sexual reproduction to complete its life cycle and spreads the infection.

Protocol

1. In Vitro Purification of Sporozoites from C. parvum Oocysts Purify sporozoites from C. parvum oocysts after bleaching and washing out the bleach as described above. 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.</li…

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

Basement membrane extract (extracellular matrix)	amsbio	3533-010-02
Dynamag 15 rack	Thermofisher Scientific	12301D
Dynamag 2 rack	Thermofisher Scientific	12321D
EMD Millipore Isopore Polycarbonate Membrane Filters- 3µm	EMD-Millipore	TSTP02500
Fast green dye	SIGMA	F7252-5G
Femtojet 4i Microinjector	Eppendorf	5252000013
Glass capillaries of 1 mm diameter	WPI	TW100F-4
Matrigel (extracellular matrix)	Corning	356237
Microfuge tube 1.5ml	Eppendorf	T9661-1000EA
Micro-loader tips	Eppendorf	612-7933
Micropipette puller P-97	Shutter instrument	P-97
Penstrep	Gibco	15140-122
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
Vortex Genie 2	Scientific industries, Inc	SI0236
Adv+++ (DMEM+Penstrep+Glutamax+Hepes)			Final amount
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
INTESTINAL ORGANOID MEDIA-OME (Expansion media)			Final concentration
A83-01	Tocris	2939-50mg	0.5µM
Adv+++			make upto 100 ml
B27	Invitrogen	17504044	1X
EGF	Peprotech	AF-100-15	50ng/mL
Gastrin	Tocris	3006-1mg	10 nM
NAC	Sigma	A9125-25G	1.25mM
NIC	Sigma	N0636-100G	10mM
Noggin CM	In house*		10%
P38 inhibitor (SB202190)	Sigma	S7076-25 mg	10µM
PGE2	Tocris	2296-10	10 nM
Primocin	InvivoGen	ant-pm-1	1ml/500ml media
RSpoI CM	In house*		20%
Wnt3a CM	In house*		50%
In house* – cell lines will be provided upon request
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)
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