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

1. Passage of Organoid Cultures

1. 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.
2. 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.

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.

1. Preparation of Material
   1. Pull a glass capillary into two injection needles using a micropipette puller, according to the manufacturers' recommendations. The micropipette puller will heat the glass capillary in the middle and will pull the capillary into two parts, thereby generating two glass needles. It is practical to pull several needles and store them in a clean petri dish.
   2. To allow bacterial infection, remove the antibiotics from all media for at least 3 medium changes (=one week). This will dilute antibiotics from the basement matrix. If using a bacterial strain that is resistant to a specific antibiotic, add this specific antibiotic to minimize the chances of contamination.
   3. Prepare the workbench for microinjections. For work at different biosafety levels, it may be necessary to place a stereomicroscope inside a sterile bench. In this protocol, inject H. pylori under biosafety level 2 conditions under a stereomicroscope inside a sterile bench.
   4. Culture the bacteria according to standard protocols.
   5. To estimate the multiplicity of infection (MOI), estimate two parameters: The number of bacteria and the number of cells per organoid as outlined below. The MOI can correlate with results, for example with the host's IL-8 mRNA expression.
      1. To calculate the bacterial density in the infection solution, first establish the standard curve of optical density and colony forming units (CFU) according to standard protocols. An optical density of 0.1 at 550 nm should yield approximately 10⁷ CFU/ml.
      2. To estimate the number of cells per organoid, count the number of organoids in 1 well. Disrupt organoids as outlined in steps 1.1-1.4. After centrifugation for 5 min at 200 x g and 4 °C, add 1 ml of enzymatic dissociation solution and resuspend.
         1. Incubate at 37 °C with repeated shaking for 5-10 min. Determine whether the cells have dissociated into single cells under the microscope (inverted microscope such as Leica DMIL at 10X magnification). If necessary prolong the incubation.
         2. Count cells per organoid using a hemocytometer and calculate the number of cells per organoid. Human gastric organoids with a diameter of about 200 µm have approximately 4,000 cells. Injection of 0.2 µl of a bacterial solution with 1 x 10⁹ bacteria per ml results in an approximate MOI of 50.
2. Injection of Organoids.
   Note: For microinjection, the glass needle is stabilized in a holder, which can be maneuvered in 3 dimensions by a micromanipulator. Organoids remain within the basement matrix within the well. Due to size and positioning in the well, not all organoids are equally amenable to injection. Usually, the 30 largest organoids in one well can be targeted in 5 min.
   1. Harvest bacteria and wash them in the basal medium according to standard protocols.
   2. Calculate the number of bacteria per ml according to the optical density (see step 2.1.5.1). Dilute the bacteria to 1 x 10⁹ bacteria per ml in a basal medium.
   3. Take a glass needle. Using forceps break the tip so that the opening will be approximately 10 µm wide.
   4. Insert the needle into the injection holder and fix it to the micromanipulator.
   5. Take up approximately 10 µl bacterial solution into the needle.
   6. Place a 4-well plate with low edges containing organoid cultures under the stereomicroscope.
   7. Navigating with the micromanipulator, target a single organoid with the needle. Position the needle close to the organoid and then insert the needle into the organoid with one swift movement. Inject approximately 0.2 µl bacterial solution into each organoid using the microinjector.

3. Harvest Organoids for Any Analysis.

1. For example, fix organoids after 4 hr. Remove supernatant and add 1 ml 2% formaldehyde and incubate 20 min at RT or O/N at 4 °C. Organoids can be processed for immunohistochemistry according to standard procedures.