After expanding Crohn's disease patient-derived colonoids in a 48-well plate, gently remove the medium from the edge of the well without damaging the colonoid dome. Add 300 microliters of enzymatic dissociation reagent supplemented with 10 micromolar ROC1 and two inhibitor Y-27632 to each well. Using a P1000 pipette tip, scrape the surface of the well to break the colonoid dome and pipette the cell suspension up and down.
Transfer the cell suspension into a 15 milliliter tube. Incubate the collected colonoids in a 37 degrees Celsius water bath for five minutes. Centrifuge the colonoids at 400 x g for three minutes and remove the supernatant, leaving approximately 1.2 milliliters in the tube.
Next place the tip of the P1000 pipette into the suspension, holding it just above the bottom of the tube and rapidly pipette the suspension in and out of the tip to dissociate the colonoids. Observe the tube under the microscope to ensure no whole colonoids remain. And colonoids fragments are approximately 30 to 40 micrometers in size.
Next, add 10 milliliters of ice cold organoid wash media to the 15 milliliter tube. Centrifuge the tube at 400 x g for three minutes. Remove the supernatant and resuspend the pellet in one milliliter of ice cold organoid wash media.
Transfer the suspension to a 1.5 milliliter microcentrifuge tube and label it as a colonoid fragment tube. After mixing, transfer 50 microliters of the sample to a new tube and label it as a cell count tube. Centrifuge the tube at 400 x g for three minutes.
Remove the supernatant and resuspend the pellet in 500 microliters of enzymatic dissociation reagent, supplemented with 10 micromolar Y-27632. Incubate the single cell count tube in a 37 degrees Celsius water bath for five minutes. Using a P1000 pipette set to 400 microliters, rapidly pipette the sample.
Then observe the sample under a microscope to ensure a single cell suspension. Add one milliliter of organoid wash media to the single cell count tube. Then centrifuge the suspension and remove the supernatant before resuspending the pellet in 50 microliters of organoid wash media.
Add 50 microliters of trypan blue and count the cells using a hemocytometer. Based on this, calculate the concentration of cells in the colonoid fragment tube. Centrifuge the required volume in a new 1.5 milliliter micro centrifuge tube.
Then remove the supernatant. Resuspend the pellet in basement membrane extract, or BME. Now in a preincubated 96-well microtiter plate, reverse pipette 10 microliters of the colonoid BME solution per well and carefully mix the solution regularly to prevent uneven seeding.
Invert the plate and incubate at 37 degrees Celsius and 5%carbon dioxide. After 20 minutes, overlay the domes with 200 microliters of prewarmed organoid proliferation media and continue incubation for three days.
