In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes the first apical-out in-a-dish model and is a critical improvement over basal lateral out enteroid in-a-dish models when establishing in vitro modeling of potential therapeutics intended for oral administration. This method allows access to the apical surface of enteroids. Compounds can be added to cell media and the compounds are taken up apically as they would be in vivo.

Investigators interested in establishing an in vitro system of intestinal inflammation to test potential oral therapeutics may find this technique especially useful. Tissue sourced from different ages and species may require further standardization. So patients while establishing polarity reversal times may be required.

For reversing the polarity of 3D enteroids aspirate media from each well of a 24 well plate of established 3D basolateral out enteroids. Add 500 microliters of five millimolar ice cold, EDTA or PBS to each well of a 24 well plate and gently mechanically disrupt the basement membrane extract or extracellular matrix dome by pipetting up and down five to six times with a P 200 pipette. Transfer the contents of four wells to a 15 milliliter chronicled tube.

Then add eight milliliters of ice cold EDTA slash PBS to the tube. Transfer the remaining 20 wells in the same manner. Incubate the tubes at four degrees Celsius for one hour on a rotating platform or shaker at 330 RPM.

After incubation centrifuge the tubes and discard the supernatant from each tube. Combine and wash the pellets with five milliliters of DMEM F12. Then centrifuge the cells suspension and remove the supernatant before adding 12 milliliters of antibiotic free medium to the tube ensuring that the cell pellets are resuspended.

Pippet 500 microliters of the enteroid suspension into each well of a 24 well ultra-low attachment plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for two to five days or until the enteroids have reversed polarity. On day one abstaining, transfer the contents of four wells of a 24 well plate to a 1.5 milliliter micro centrifuge tube.

Repeat for all desired wells with each treatment in a separate tube. Centrifuge the tubes and resuspend the pellet in 300 microliters of 4%from aldehyde fixative at room temperature. After 30 minutes wash the pellets with 500 microliters of PBS.

Add 500 microliters of 0.1%Triton X 100 to the tubes. Incubate the tubes for one hour at room temperature. Next, place the tubes on a rotator or shaker at 200 RPM for 15 minutes at two to eight degrees Celsius.

After the last incubation, add 500 microliters of 10%NDS in PBS T and incubate at room temperature for 45 minutes. During incubation, prepare primary antibody solution. The next day add 250 to 500 microliters PBS T to the tubes depending on the size of the pellet, and place them on the rotator or shaker at 200 RPM for one hour at two to eight degrees Celsius.

Add 200 microliters of the secondary antibody solution and incubate the tubes at two to eight degrees Celsius in the dark. The next day, proceed for mounting stained apical out enteroids by spinning the tubes and removing 100 microliters of supernatant. Resuspend the cells in the remaining 100 microliters of supernatant and transfer the cell suspension to 500 microliter tubes.

Centrifuge the tubes in a mini centrifuge for 20 seconds. Remove the supernatant and resuspend the pellets in 100 microliters of room temperature far red nucleic acid stain. After 20 minutes of incubation, centrifuge the cells and resuspend the pellets in 100 microliters of PBS.

After the second wash and centrifugation remove 70 microliters of the supernatant and resuspend the pellets in the remaining volume of PBS. Then transfer the cell suspension to a labeled 24 by 60 millimeter cover slip. Apply 75 microliters of mountant directly onto the specimen and remove any bubbles with a pipette tip.

After overnight curing in the dark, apply a thin layer of glycerol to the cover slips, then mount the cover slips onto the glass slide and gently press it into place. Tap to remove any bubbles between the cover slip and the slide. Allow the cover slip to set at room temperature in the dark for two hours before imaging the enteroids at 20 times magnification with a confocal microscope.

For enteroid treatments, establish an apical out necrotizing enterocolitis in a dish model for 24 hours as described in the text manuscript. Before performing the assay, remove 100 microliters of media from each well leaving the remaining 400 microliters. Add 400 microliters of cell viability assay reagent to each well.

Vigorously mix the contents on a plate shaker for five minutes at 200 RPM to induce cell lysis. Next, transfer 200 microliters to a single well of a 96 well clear bottomed plate. Repeat for the remaining 600 microliters creating four technical replicates per well.

Using a plate reader capable of luminescence, record values at 0.25 milliseconds integration and compare the relative values among treatments. The representative immunofluorescence staining of control confirmed the apical out localization of nuclei toward the lumen and the detection of villain at the outer edge of the epithelium. Compared to the control, exposure to lipo polysaccharide, tumor necrosis alpha, or hypoxia alone did not induce overt changes in gross cell morphology E-cadherin or villain localization or fluorescent intensity.

Treatment with lipo polysaccharide or tumor necrosis alpha in combination with hypoxia disrupted epithelial architecture and demonstrated a significant loss of adherence junction and brush border protein expression revealed by the loss of ECA hearing and villain staining. The apical out cell viability was determined under normoxic or hypoxic conditions. Under normoxic conditions compared to the control lipo polysaccharide or tumor necrosis alpha did not significantly affect the cell viability of enteroids.

However, significant reductions in viability were observed in lipo polysaccharide or tumor necrosis alpha in combination with hypoxia compared to hypoxia alone. While establishing apical out in-a-dish model. Remember to keep EDTA in the cell suspension ice cold.

This will enhance polarity reversal and ensure all ECM is properly liquified and removed. Other downstream applications such as QPCR, RNA-seq, western blotting or functional evaluations of barrier permeability can be performed further to characterize the response to inflammatory signaling in hypoxia.