These studies aim to provide robust and reproducible model systems to perform orientation specific studies of the intestinal epithelium. Apical organoids can be generated in high numbers and hold promise for high throughput screening. Monolayers are easier to be manipulated and offer access to both apical and basal signs.
Make sure that the size of the organoids is 150 to 250 micrometers in diameter prior to beginning the inversion protocol. Carefully remove and discard the medium from each well containing organoids without disrupting the ECM dome. Add one milliliter of ice cold dissociation solution to each well and incubate the plate at room temperature for one minute.
Carefully dislodge the domes by pipetting slowly with a coated tip, taking care to not disrupt and fragment the organoids. Transfer the organoid suspension to a plate treated with anti adherence solution and place the plate on a shaker at four degrees Celsius for 30 minutes. After 30 minutes remove the plate and gently pipette the solution up and down using a one milliliter pipette tip coated with anti adherence solution.
Place the plate on the shaker at four degrees Celsius for another 30 minutes. Remove the plate and let the organoids settle by gravity for one to two minutes at room temperature. After the organoids settle, remove as much of the dissociation solution as possible and wash them by adding 1.5 milliliters of DMEM F-12.
Allow the organoids to sediment and remove the supernatant. Then repeat the wash. Remove as much of the DMEM F-12 as possible and add 0.5 milliliters of intestinal organoid expansion medium.
Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. The following day perform a partial medium change by tilting the plate at a 25 to 30 degree angle and removing medium along the wall of the well taking care to not remove suspended organoids. Add 0.4 milliliters of intestinal organoid expansion medium and incubate at 37 degrees Celsius and 5%carbon dioxide for three days.
If aggregates have formed use a one milliliter pipette tip coated with anti adherent solution to shear the aggregates by pipetting up and down 20 times while pressing the end of the tip to the bottom of the plate. Add one milliliter of pre-warmed 0.05%Trypsin-EDTA to resuspend organoids. And mix thoroughly to ensure an even suspension.
Add up to an additional one milliliter of Trypsin-EDTA for a large number of cells, or if a significant amount of ECM remains. Incubate at 37 degrees Celsius for five to 10 minutes, then mix thoroughly with a one milliliter pipette to disrupt the organoids so that they're completely dissociated into single cells or small fragments. To completely dissociate fragments or whole organoids continue the incubation with Trypsin-EDTA at 37 degrees Celsius for another three to five minutes.
Once the organoids are sufficiently dissociated add an equal volume of DMEM F-12 and pipette up and down to mix thoroughly. Inactivate the Trypsin-EDTA by adding 10%FBS to the cells. Centrifuge fragments at 200 times G for five minutes at two to eight degrees Celsius.
If the dissociated organoids failed to pellet mix the cells thoroughly by pipetting up and down and centrifuge them again. Carefully remove as much supernatant as possible. leaving only the cell pellet.
Resuspend the cells in 100 microliters of intestinal organoid differentiation medium for each well to be seeded. Adjusting the volume appropriately for larger or smaller well sizes. Remove the coated plates from the incubator and remove the excess basement membrane matrix solution from each well.
Add 100 microliters of the cell suspension to the upper well of each cell culture insert and 500 microliters of intestinal organoid differentiation medium to the lower well. Then incubate at 37 degrees Celsius and 5%carbon dioxide. Replace the medium in both the upper and lower wells every two to three days.
To establish an ALI culture remove medium from the upper and lower wells, and add fresh intestinal organoid differentiation medium to the lower well leaving the upper well empty. The intestinal organoids cultured with intestinal organoid expansion medium exhibited a cystic morphology. When ECM is removed some organoids tend to aggregate during the first three days and need to be sheered at to increase the organoid number.
Intestinal organoids cultured in ECM continued to expand and exhibit spontaneous formation of secondary budding structures. Organoids maintained for five days in the absence of extracellular matrix exhibited elongated, cystic, and irregular forms. The expression of atypical markers, such as villin and ZO-1 was detected the outer side of the epithelium that was exposed to the medium.
ECM embedded organoids stained for nuclei, villin, and ZO-1 demonstrating an apical basal polarity where the apical side was facing the lumen of the organoid. Once the monolayer was established cells formed tight junctions and a confluent layer. The confluent layer also orients its villin containing brush border toward the apical side of the epithelium, forming ZO-1 multi protein complexes in between cells.
Transition to an ALI culture induced further differentiation with more prominent goblet cells which was visualized by staining for the secreted mucin protein MUC2 To induce the polarity inversion it is crucial to remove all ECM without disrupting or fragmenting the organoids. Media changes should also be done with care so as to avoid removing any of the suspended organoids. Ensuring that there are sufficient single cells for the establishment of the monolayer culture is a major determinant of its quality.
Apical specific functions such as nutrient absorption or host pathogen interactions can now he studied in relevant systems that suit the researchers'needs.
