To coat the plate with EME, dilute EME 1 to 20 in cold Advanced DMEM+For coating with collagen, dilute five milligrams per milliliter of collagen I in Advanced DMEM+to 100 micrograms per milliliter. Coat with the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids.
Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME. Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids and transfer them to the same conical tube.
Centrifuge the sample at 350 G for two minutes and remove the supernatant, including any EME residue. Then, add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM+to neutralize trypsin.
Centrifuge at 350 G for five minutes and aspirate the supernant before resuspending the pellet in 4.8 milliliters of rIOM2D. Remove excess EME or collagen in Advanced DMEM+from the wells. Then, add 200 microliters of organoids in rIOM2D and 10 micromolar Y27632 to each pre-coated well.
After 4 to 16 hours, collect the media and centrifuge at 1, 000 G for one minute. Transfer the supernatant into a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged rIOM2D to each well.
Change the rIOM2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells. In contrast, a collagen I substrate was insufficient to reform 3D structures.
