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

Published: November 30, 2023

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…

Açıklamalar

The authors have nothing to disclose.

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

Play Video

Bu Makaleden Alıntı Yapın
Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria. J. Vis. Exp. (Pending Publication), e21793, doi: (2023).

View Video