Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

With this protocol, we present an easy method to induce polarity reversal of adult stem cell-derived organoids and explain how to assess the proliferation. One major challenge using intestinal organoids is that the apical cell surface remains hidden on the inside and is usually hard to probe. Apical-out organoids present the apical cell surface to the outside and make it accessible. Compared to frequently used organoid-derived monolayers and Transwell support systems, apical-out organoids retain the 3D structure.

[Instructor] To begin, obtain a culture of adult stem cell-derived intestinal organoids from dogs. Carefully pipette out the medium from the basement membrane extract embedded organoids. Add 500 microliters of organoid harvesting solution to each well. Pipette the suspension up and down repeatedly to detach all matrix domes from the plastic surface. Now, transfer the organoids into a 15-milliliter tube. Resuspend well to fully dissociate any remaining hydrogel matrix. Incubate the tube on ice for 1.5 hours. Shake the tube well every 10 minutes to prevent clumping of the organoids and ensure even dissociation of remaining hydrogel components. While the tube is incubating, coat a 96-well plate with an anti-adherence solution. For each well of a trypsinized 24-well plate, coat eight wells of a 96-well plate, then incubate. Next, add at least twice the volume of PBS as the organoid harvesting solution used to the 15-milliliter tube. Centrifuge at eight degrees Celsius at 150g for five minutes. Pipette out the supernatant, and resuspend the organoids in one milliliter of PBS. Now, transfer 500 microliters of the suspension into a separate 15-milliliter tube and centrifuge again. Remove all anti-adherence solution from the 96-well plate, then remove as much supernatant as possible after centrifugation. Use one tube for the generation of floating basal-out organoids and the other for apical-out organoids. For basal-out organoids, add 100 microliters of refined medium containing 7.5% basement membrane extract per well of the 96-well plate to one 15-milliliter tube. Mix well and disperse the organoids evenly across the desired number of wells. For apical-out organoids, add 100 microliters of plain refined medium per well of the 96-well plate to the other 15-milliliter tube. Mix well and disperse the organoids evenly across the desired number of wells. Incubate the organoids at 37 degrees Celsius and 5% carbon dioxide for three days. At defined time points of 24-hour intervals, add 50 microliters of three micromolar EdU diluted in refined medium to all wells containing organoids. Incubate at 37 degrees Celsius and 5% carbon dioxide for 1.5 hours. Collect EdU-labeled organoids using wide-bore tips and transfer them into a 15-milliliter tube. Add an equal volume of 4% PFA to each tube. Pipette up and down to mix well. After incubating at room temperature for 15 minutes, add two volumes of PBS to the tube and centrifuge. Aspirate the supernatant, then resuspend the organoids in one milliliter of PBS before another round of centrifugation. Resuspend the organoids in one milliliter of PBS after removing the supernatant, and transfer the suspension into a 1.5-milliliter tube. Store the organoids at four degrees Celsius until performing the EdU staining reaction. A very specific feature of apical-out suspension cultures is the number of dead cells floating around the organoids. This is due to apical-out organoids extruding terminally differentiated and dead cells into the surrounding medium, while basal-out organoids accumulate dead cells in the organoid lumen. Basal-out and apical-out polarity orientations influence the organoid structural organization with basal-out structures displaying higher cell proliferation over time. Confocal images revealed well-defined cellular structures with nuclear staining confirming organized polarity in basal-out organoids. Quantification of EdU-positive cells showed that basal-out organoids had significantly higher proliferation rates, peaking at 48 hours.