Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

The overall goal of this procedure is to generate organoids and use them as a three dimensional model for infection biology. A number of years ago, my lab discovered LDR five as a marker for stem cells in a number of different adult organs in mice. And man, since we have learned to take single stem cells from these organs and culture them in the form of organoids, small versions of those organs, and with organoid technology, we've now shown that we can study cancer in grade detail, but also infectious disease problems.

The main advantage of this technique over modeling infection biology using cancer cell lines is that here the cells used are primary non transformed epithelial cells, and that a three dimensional structure is preserved, Beginning with approximately one square centimeter of fresh gastric tissue. In a dry 10 centimeter Petri dish, use forceps to carefully remove the mucus and muscle layer. Wash the tissue when cold chelating buffer with gentle back and forth movements.

Move the washed tissue to a new dry 10 centimeter dish. Cut the tissue into 25 to 50 small pieces, approximately two to five square millimeters in size and use forceps to transfer them to a 50 milliliter tube. After wetting a 10 milliliter pipette in chelating buffer, add 10 milliliters of the buffer to the 50 milliliter tube and wash the pieces by vigorously pipetting up and down 10 times.

Allow the pieces to settle and remove the supra natant. Repeat washing until the supra natant is clear. Next, pipette 20 milliliters of one X chelating buffer.

Supplemented with 10 millimolar EDTA onto the tissue pieces and incubate them for 10 minutes. At room temperature, invert the tube gently every two minutes following this incubation, pipette gently up and down. One time using a 10 milliliter pipette, allow the pieces to settle and remove the supernatant.

Then transfer the pieces to a clean 10 centimeter dish and remove all of the liquid. Place a glass slide on top of the tissue pieces and observe the tissue at 10 x magnification on an inverted microscope. Pick up the dish and apply gentle pressure to the glass.

Slide the rim of the tissue will appear cloudy indicating gland release. Collect the released glands and tissue pieces in 30 milliliters of cold basal medium. Allow the tissue fragments to settle and collect the snat now containing the glands in two 15 milliliter tubes.

Centrifuge the tubes at 200 times G for five minutes at four degrees Celsius. Discard the supinate and store the resulting pellets on ice to see the glands. Suspend the pellet in 60 microliters of basement matrix on ice.

Separately prepare five sterile 1.5 milliliter tubes. Each containing 50 microliters of basement matrix. Transfer 10 microliters of the suspended glands into one of the tubes containing 50 microliters of basement matrix.

Make a serial dilution by transferring 10 microliters from this first dilution to the next 1.5 milliliter tube and repeating for the remaining tubes. Now pipette 50 microliters of the suspended gland dilution from each 1.5 milliliter tube into individual wells of a Prewarm 24 well plate. The drop should form a dome carefully.

Place the plate into a cell culture incubator and let the basement matrix solidify for 10 minutes. After preparing the appropriate culture medium, carefully remove the plate containing the now solidified basement matrix and add 500 microliters of the medium to each. Well return the plate to the cell culture incubator.

Refresh the medium every two to three days to prepare to passage. The organoid cultures. Hold the tip of a pasture pipette in the flame of abundance and burner and leave it to cool.

The tip should be narrowed from 1.5 millimeters to approximately 0.5 millimeters. After incubation, remove the plate now containing organoids from the cell culture incubator. Aspirate the medium from one.

Well add one milliliter of cold basal medium to the well and vigorously pipette up and down to break up the basement matrix. Transfer the contents of the well to a 15 milliliter tube and place it on ice. Wet the pipette in basal, medium and optimally narrowed pipette will take up the medium visibly slower than an un narrowed pipette.

Then break up the organoids by pipetting vigorously up and down 10 times. Add nine milliliters of cold basal medium and centrifuge the tube for five minutes. Add 200 times G and four degrees celsius.

After discarding the snat, carefully re suspend the pellet in 250 microliters of basement matrix and place the tube on ice. Place a 50 microliter drop of the basement matrix containing cells into each well of a prewarm four well cell culture plate. Place the plate carefully back into the incubator and allow the basement matrix to solidify for 10 minutes.

Now remove the plate from the incubator and add 500 microliters of basal medium supplemented with growth factors. Return the plate to the incubator and replace the medium every two to three days. To begin microinjection of organoids, harvest h pylori I bacteria and wash them in basal medium according to standard protocols.

Following optical density measurements to determine bacterial number dilute the culture to one times 10 to the ninth bacteria per milliliter. In basal medium, pull a glass capillary into two injection needles using a micro pipette 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 stereo microscope.

Set up inside a sterile culture hood. Insert the needle into the injection holder and fix it to the micro manipulator. Take up approximately 10 microliters of the bacterial solution into the needle.

Then place the four well cell culture plate containing gastric organoids under the stereo microscope. Navigating with the micro manipulator, 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 five 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. Gastric organoids are formed in culture following isolation of gastric glands and growth in basement membrane containing appropriate growth factors.

The human gastric organoids seen here typically start as small cysts and expand into spheres with a diameter of 50 to 300 micrometers. After injecting organoids with h pylori I cells immunohistochemical staining for the bacterial protein cytotoxicity associated gene A or CAG A shows spiraled shape h pylori in close proximity to the epithelial cells that form the organoid. Now, it is important to remember that functional growth factors and inhibitors are crucial for the growth of organoids.

Especially the wind should be tested for every single batch in a separate assay. Now, after this procedure, different assays can be performed. For example, whole mound immunofluorescence, RNA analysis or even protein analysis.

Organoids are also amenable to genetic modifications.