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…
Divulgazioni
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 |
Citazione di questo articolo
Establishing an Infection Model Through Microinjection of Gastric Organoids with Pathogenic Bacteria. J. Vis. Exp. (Pending Publication), e21793, doi: (2023).