3D intestinal organoid culture limits the study of host epithelial-pathogen interaction because the lumen is not directly accessible to bacteria or virulence factors. Additionally, secreted materials such as small molecules, proteins, or mucus cannot be easily sampled from the 3D culture for downstream analysis. To address these limitations, we present a modified protocol for converting 3D human enteroids or colonoids to a monolayer.
Colonoid monolayers secrete a thick, apical mucus layer, allowing for ex vivo studies of pathogen-mucus interaction. This method aims to provide a tractable model to study the earliest intestinal host-pathogen interactions upon infection. Colonoid monolayers will allow for a new way to study mucus biology, which is important in host-pathogen interaction, microbiome studies, and inflammatory bowel disease.
This method can be applied to other mucus-secreting organs such as the upper GI tract, respiratory system, and female reproductive system. Visual demonstration will help the user with mucus preservation during monolayer growth and during fixation for immunostaining. Demonstrating the procedure will be Karol Dokladny, a staff scientist from our laboratory.
After incubated the coated cell culture, aspirate the culture medium from the 24-well plate and replace with one milliliter ice-cold harvesting solution per well. Use a mini cell scraper to dislodge and break up the basement membrane matrix pellet. Pay particular attention to any material near the well edges.
Then, agitate the plate on an orbital shaker at approximately 200 revolutions per minute at four degrees Celsius for 30 to 45 minutes. Use a P200 single channel pipette fitted with sterile filter tips to triturate the cell suspension. Deliver the cell suspension in a 15-milliliter conical vial.
Use multiple vials if total suspension volume is greater than six milliliters. After that, add into the vial an equal volume of Advanced DMEM/F12 medium containing 10 millimolar HEPES, L-alanyl-L-glutamine dipeptide, and penicillin-streptomycin. This is the wash medium.
Invert the vial three to four times to mix. Centrifuge at 300 times g for 10 minutes at four degrees Celsius. Prior to cell plating, equilibrate the coated inserts in a tissue culture incubator for at least 30 minutes.
For each insert to be plated, warm one milliliter of expansion medium in a 37-degree-Celsius water bath and add two inhibitors. Then, add the mixture to the wells. Aspirate the wash medium from the tube containing the cell pellet and replace with expansion medium with a volume sufficient to yield at least 100 microliters per insert and resuspend.
Then, aspirate the collagen IV solution from each insert and wash twice with 150 microliters of the wash medium per insert. Pipette 600 microliters of expansion medium into the space beneath each insert. Pipette 100 microliters of cell suspension from the vial into each insert.
Return the plate to the tissue culture incubator and leave undisturbed for at least 12 hours. Avoid shaking or sharply tilting the plate. After incubating, place the plate on a phase contrast light microscope with a 2.5 times to 10 times objective lens.
Refresh with culture medium without inhibitors to discontinue inhibition treatment. Continue to refresh the medium every two to three days until confluent. Lift the cell culture inserts from the 24-well plate with fine-tipped forceps or tweezers.
Carefully invert on a laboratory tissue paper to remove the apical medium and wipe away external liquid clinging to the insert. In a new 24-well plate, immerse the inserts in a one to three solution of glacial acetic acid and absolute ethanol for 10 minutes at room temperature. Then, on the bench, invert the insert onto tissue paper to remove the fixative.
Place the inserts in a plastic container filled with 1X PBS for 10 minutes to rehydrate the cells before proceeding with standard immunostaining protocols. To preserve, use a razor blade to cut around the perimeter of the insert membrane. Using forceps, transfer the membrane to a glass microscope slide.
Then, add the mounting medium and affix a cover glass. In this experiment, human enteroid and colonoid cultures are grown as 3D structures, then dissociated and fragmented for plating on human-collagen-IV-coated cell culture inserts. The progress of monolayer formation is monitored on a daily basis via bright field microscopy and immunofluorescence staining.
Colonoid fragments seeded onto human-collagen-IV-coated filters form a multiple monolayer islands two to four days post-seeding. Both maximum-intensity projection and confocal optical Z-section with the corresponding orthogonal projections show that cell-free areas are identifiable by the absence of both nuclear and apical F-actin staining. A confluent colonoid monolayer with continuous apical surface was detected by F-actin immunostaining approximately one week post-seeding.
High magnification of a representative maximum-intensity projection and confocal optical section with the corresponding orthogonal projections show that cells in confluent colonoid monolayers form the F-actin perijunctional rings and an immature apical brush border. EdU incorporation demonstrates a progressive loss of proliferation during jejunal monolayer differentiation. Undifferentiated jejunal monolayers have broader, shorter cells, and a less mature apical actin-based brush border compared with jejunal monolayers after five days of differentiation.
Fragmenting the colonoids is critical. If the fragments are too large, they will not attach to the coated filter, but will instead reform into a 3D colonoid. We presented a modified method to study mucus biology on colonoid monolayers.
Other host-pathogen studies can be performed to look at the interaction between the pathogen and the intestinal apical surface.
