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