Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

This protocol demonstrates the utility of the stem cell-derived intestinal organoid system for modeling epithelial invasion by enteric pathogens, and modification of this process using cytokines. Micro-injection allows the pathogen of interest to be delivered directly into the lumenal cavity of the organoid, replicating more closely the process of infection in vivo. This method could be applied to the study of different enteric pathogens or alternative cytokines of interest.

I would suggest completing a trial run of the procedure with phenol red only in order to master the micro-injection technique prior to using pathogens. On day two, begin the differentiation by changing the media to 10 milliliters of stem cell culture medium, supplemented with growth factors. On day six, change the media to 10 milliliters of RPMI-B27 media, supplemented with six micromolar CHIR99021, plus three micromolar retinoic acid to begin patterning the posterior endoderm to the hindgut.

On differentiation day 10, embed the hindgut in the basement membrane matrix. First, remove the media from the hindgut plate, and wash the plate with calcium magnesium-free DPPS once. Add five milliliters of collagenase solution to the plate and incubate at 37 degrees Celsius for five minutes.

Next, inactivate the collagenase by adding five milliliters of the organoid base growth media to the plate. The hindgut cells should be floating at this point. Collect the hindgut suspension in a 15-milliliter conical tube.

Then centrifuge at 240-times-G for one minute. After centrifugation, pipette off the supernatant, add 10 milliliters of the organoid base growth media, break up the hindgut into smaller pieces by gently pipetting, and centrifuge again at 95-times-G for one minute. Wash the cells in the organoid base growth media twice.

Resuspend the cells in 300 to 500 microliters of the base growth medium, and add approximately 100 microliters of this solution to 1.5 milliliters of the basement membrane matrix. Set up a 24-well plate on a plate heater at 37 degrees Celsius. and spot out 60 microliters of the hindgut cell matrix mixture into one well of the 24-well plate.

Allow it to set briefly and check the density under a microscope. After spotting out the rest of the matrix into the 24-well plate, incubate the plate at 37 degrees Celsius for 10 minutes. Then add 800 microliters of the base growth media containing growth factors to each well of the 24-well plate.

To passage organoids, first remove the media from them, and replace it with 500 microliters per well of the cell lifting solution. Incubate at four degrees Celsius for 40 to 50 minutes, at which point the organoids should be floating in the solution. Gently pipette the organoids and the cell lifting solution into 15-milliliter conical tubes, trying not to break up the organoids.

After allowing the organoids to settle for three to five minutes, remove the supernatant and the single cells. Resuspend the organoids in five milliliters of the base growth medium, and pipette them gently to wash. Centrifuge at 95-times-G for two minutes.

Next, set up the 24-well plate on a plate heater at 37 degrees Celsius within the hood, and remove the supernatant from the organoid pellet. Then, using a P1000 pipette, resuspend the organoids in 300 to 500 microliters of the base growth media to break up the organoids into smaller chunks. Place approximately 100 microliters of the organoids into 1.5 milliliters of the basement membrane matrix and pipette briefly to mix.

Spot out 60 microliters of the basement membrane matrix into one well of the 24-well plate. After leaving it to solidify for 30 seconds, check the density under the microscope. After spotting out the rest of the matrix into a 24-well plate, place the plate in an incubator at 37 degrees Celsius for 10 minutes, and then overlay it with 800 microliters of the base growth medium with growth factors according to the manuscript.

For micro-injection assays with recombinant human IL-22 pre-stimulation, add recombinant human IL-22 to the culture media to a final concentration of 100 nanograms per milliliter 18 hours prior to injections. Load the micro-injection dish containing organoids onto the microscope stage, remove the lid, and bring the organoids into focus, ready for the injection to begin. Turn the injector and the arm-control stations on.

Ensure the injector is set to a pressure of 600 kilopascals and an injection time of 0.5 seconds. If it is not already backed away from the microscope stage, rotate the injection arm to make sure it is, and remove its grip-head. Load the drill tip with 10 microliters of the inoculum, gripping the drill tip gently at its blunt end.

Place the drill tip into the grip-head and reattach it to the micro-injection arm. Gently move the arm into a position where the needle is situated one to two centimeters above the micro-injection dish. Use the arm control to position the needle tip in the center of the dish, and lower it until it is just over the surface of the media.

Program the arm-control station to return the needle to this point after all injections. Focus the microscope on the organoids and select the target to inject. Position the needle just above and the the right of the organoid to be injected, and move the needle downward and laterally into the organoid lumen.

Press the inject button on the micro-injector to let the phenol-stained bacteria mixture emerge from the needle. And inject each organoid three times. When all required organoids are injected, remove the micro-injection plate from the stage, replace the lid, and incubate the plate at 37 degrees Celsius for 90 minutes.

After the incubation, aspirate the growth media and replace it with three milliliters of cell lifting solution. Then, incubate at four degrees Celsius for 45 minutes. Next, gently move the organoids and the cell lifting solution to a 15-milliliter conical tube containing five milliliters of DPBS.

Centrifuge at 370-times-G for three minutes. Remove the supernatant, and add one milliliter of the base growth media containing 0.1 milligrams per milliliter gentamicin. Next, with a P1000, pipette up and down approximately 50 times to break up the organoids.

Add four milliliters more media, and incubate at 37 degrees Celsius for one hour to kill extracellular bacteria. Next, centrifuge the organoids at 370-times-G for three minutes. Aspirate the supernatant, leaving as little as possible.

Wash the organoids with DPBS once and centrifuge again. Add 500 microliters of the lysis buffer, and pipette up and down approximately 50 times to manually dissociate the human intestinal organoids. After incubating at room temperature for five minutes, serially dilute the resulting solution tenfold in DPBS.

Pipette three 20-micron droplets of the neat and diluted solutions onto pre-warmed LB agar plates. Finally, incubate at 37 degrees Celsius overnight and proceed with colony-counting. The human intestinal organoids were micro-injected with the phenol red bacterial solution.

Retention of this red color by the organoids prevents duplicate injections. Pre-treatment of the human intestinal organoids derived from the COF-2 cell line with recombinant human IL-22 restricts the invasion of S.typhimurium SL1344 into intestinal epithelial cells. Infected organoids were processed for immuno-staining, or transmission electron microscopy, in order to facilitate the visualization of host IEC bacterial interactions.

RNA can be extracted from the injected organoids to look at transcriptomic response to infection. Fluorescence and electron microscopy can also be used to observe host pathogen interactions in more detail. The most important thing to remember is to ensure that you've had enough practice on the micro-injector to be able to complete your injections rapidly and efficiently for consistent results.

Micro-injection drill tips have a fine, sharp point, and care should be taken when loading and unloading them from the micro-injector. This technique allows for epithelial cell pathogen interactions to be studied in previously unobtainable detail. This infection model will be of particular use to those studying human-specific pathogens.