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

Published: October 31, 2023

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.

Açıklamalar

The authors have nothing to disclose.

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

Etiketler

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Bu Makaleden Alıntı Yapın
A Technique to Develop a Parasite Infection in Intestinal Organoids via Microinjection. J. Vis. Exp. (Pending Publication), e21720, doi: (2023).

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