Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria

Abstract

Source: Bartfeld, S. et al. Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori. J. Vis. Exp.(2015).

This video demonstrates a method to establish a gastric organoid infection model by microinjecting gastric pathogen Helicobacter pylori into the lumen of the organoids. This model helps in studying infection-induced changes in gastric cells.

Protocol

1. Passage of Organoid Cultures Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and down until the basement matrix is broken up. Transfer to a 15 ml tube. Place on ice. Take up organoids in the pipette and vigorously pipette 10x up and down. The breaking of the organoids can be observed by the eye. 2. Microinjection of Organoid Culture. <st…

Materials

Medium
HEPES	Invitrogen	15630-056
Advanced DMEM/F12	Invitrogen	12634-028
Matrigel, GFR, phenol free	BD	356231
GlutaMAX	Invitrogen	35050-079	Stock concentration 200 mM, final concentration 2 mM
B27	Invitrogen	17504-044	Stock concentration 50 x, final concentration 1x
N-Acetylcysteine	Sigma-Aldrich	A9165-5G	Stock concentration 500 mM, final concentration 1 mM
Murine recombinant EGF	Invitrogen	PMG8043	Stock concentration 500 µg/ml, final concentration 50 ng/ml
Human recombinant FGF10	Peprotech	100-26	Stock concentration 100 µg/ml, final concentration 200 ng/ml
Tweezers	Neolabs	2-1033	Tweezers with fine tips are helpful for the removal of muscle layer from the tissue.
4 Well Multidishes	Thermo Scientific	144444	You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator.
Microinjector	Narishige	IM-5B
Stereomicroscope	Leica	MZ75
Micropipette Puller	Sutter Instruments	Flaming Brown Micropipette Puller
anti Cag A antibody	Santa Cruz	sc-25766