establishing an infection model through microinjection of gastric organoids with pathogenic bacteria

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

To begin microinjection of organoids, harvest H. pylori bacteria, and wash them in basal medium according to standard protocols. Following optical density measurements to determine bacterial number, dilute the culture to 1 x 109 bacteria per milliliter in basal medium.
Pull the glass capillary into two injection needles using a micropipette puller, and store in a clean petri dish. Then using forceps, break the tip of one injection needle to produce an opening that is approximately 10 micrometers wide.
Working at a stereomicroscope, set up inside a sterile culture hood, insert the needle into the injection holder, and fix it to the micromanipulator. Take up approximately 10 microliters of the bacterial solution into the needle. Then, place the 4-well cell culture plate containing gastric organoids under the stereomicroscope.
Navigating with the micromanipulator, position the needle close to an organoid. Insert the needle into the organoid with one swift movement, and then, inject approximately 0.2 microliters of bacterial solution into the center of the organoid. With experience, approximately 30 of the largest organoids in a well can be injected in 5 minutes. Incubate the injected organoids for the desired time in a cell culture incubator.
Often, individuals new to this technique will struggle because they find it difficult to efficiently target the organoids. Mouse gastric organoids grow cystic and are the easiest to target. So, injection of mouse gastric organoids with, for example, a dye, may be a good practice for this technique.

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1. Passage of Organoid Cultures
<ol>
	<li>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.</li>
	<li>Take up organoids in the pipette and vigorously pipette 10x up and down. The breaking of the organoids can be observed by the eye.</li>
</ol>
2. Microinjection of Organoid Culture.
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Source: Bartfeld, S. et al. <a href="https://app.jove.com/v/53359">Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori.</a> 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

	Add 1 ml of cold basal medium to the basement matrix in the well. Using a 1 ml micropipette, vigorously pipette up and ...

Under a stereomicroscope, observe gastric organoids, three-dimensional cellular structures composed of differentiated gastric cells, and stem cells.
Introduce a microinjector loaded with Helicobacter pylori, a gastric pathogen.
Insert the needle into the target organoid and inject the bacterial suspension.&#160;
Observe a cloudy solution develop within the lumen.
H.pylori employs a specific adhesin protein on its surface to bind to the gastric cell receptor, establishing a stable interaction between the bacteria and host cells.
This interaction triggers the elongation of the bacterial secretion pilus, a large tunnel-shaped structure that extends toward the host cell membrane.
H.pylori uses the secretion pilus to inject virulence factors, including cytotoxic associated gene A or CagA into the host cell.
Once inside the cell, CagA undergoes phosphorylation by the host cell kinase.
Phosphorylated CagA deregulates multiple intracellular signaling cascades that alter host cell functions, promoting cancerous transformation.&#160;
Collect the infected organoids for further analysis.

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

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

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