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.
1. Passage of Organoid Cultures
2. Microinjection of Organoid Culture.
Note: This protocol can be used to microinject bacteria into organoids. It may be helpful to start the injection with organoids that are more permissive to microinjection. For example, mouse gastric organoids can grow into very large cystic organoids that are easy to target.
3. Harvest Organoids for Any Analysis.
The authors have nothing to disclose.
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 |