A full understanding of the role the M cells play in intestinal homeostasis and immune defense is lacking, due to the rarity of M cells in the intestine. Induced differentiation of M cells in human intestinal stem cell derived cultures will allow for the study of M cell development and functions. To coat the Transwell membranes, place the desired number of Transwells in a 24 well plate, creating a two chamber system.
Dilute the extracellular matrix, or ECM, 25 fold in cold sterile phosphate buffered saline. Then, add 100 microliters of cold diluted solution into each upper chamber onto the membrane. Cover the 24 well plate with a lid, and place the plate into a tissue culture incubator at 37 degrees Celsius for two hours to permit the ECM solidification on the membrane.
After two hours, remove the plate from the incubator, and place in a tissue culture hood. Using sterile tweezers, invert each Transwell to gently remove remaining solution. Allow the membranes to air dry in the hood with the lid open while cells are being collected.
Remove the plate of ileal enteroids from the incubator, and gently remove the culture media from each well by vacuum aspiration or with a pipette. To break up the ECM, add 500 microliters of ice cold 0.5 millimolar EDTA to each well containing ileal enteroids suspended in ECM. Pipette up and down vigorously with a P1000 pipetter set at 500 microliters to break up ECM, thereby releasing ileal enteroids into the solution.
Collect the solution from each well into 15 milliliter conical tubes. Pellet the cells in a centrifuge at 140G and four degrees Celsius for five minutes. Pellet should be visible, but can be easily dislodged, so slowly remove the supernatant by vacuum aspiration or with a pipette.
To digest tight junction linkages and break up the ileal enteroids into single cells, resuspend the pellet in 500 microliters of room temperature trypsin per every five wells collected. Using a P1000 pipetter, pipette up and down to disaggregate the clumps. Incubate the tubes in a 37 degree Celsius water bath for five minutes or less.
Add one milliliter of advanced DMEM F-12 with 10%FPS per 500 microliters of trypsin to inactivate the trypsin. Pipette up and down with a P1000 set at 500 microliters at least 50 times against the side of the conical tube to further disaggregate remaining clumps into single cells. Place a 40 micron cell strainer over a 50 milliliter conical tube, and add one milliliter of advanced DMEM F-12 with 10%FPS to wet the cell strainer.
Pipette the single cell suspension from the 15 milliliter conical onto the strainer. Wash the strainer with one milliliter of advanced DMEM F-12 with 10%FPS. Transfer the cells that went through the cell strainer from the 50 milliliter conical tube into a new 15 milliliter conical tube.
Count the cells using a hemocytometer. Centrifuge the cells in the new 15 milliliter tube at 400G for five minutes at room temperature. The cell pellet should be visible.
Carefully remove the supernatant with a pipette, again saving the supernatant in case the pellet becomes dislodged. Resuspend the cell pellet in MCMGF plus with 10 micromolar Y27632. Ensure that the ECM coded membranes have fully dried as assessed by eye.
Wash the upper chamber with 200 microliters of MCMGF plus. Then, add 200 microliters of cell solution into each upper chamber. Add 700 microliters of MCMGF plus with 10 micromolar Y27632 to each lower chamber.
Place the plate in a 37 degrees Celsius tissue culture incubator with 5%CO2. After one day of growth, remove the media from the upper chamber, and replace with 200 microliters of fresh MCMGF plus to prevent growth of multiple cell layers. Once monolayers are approximately 80%confluent, usually between days one to three post seeding, replace basilateral media with differentiation media for control wells, or with M cell media for M cell induction wells.
Replace the media in the upper chamber with differentiation media for both conditions. To verify M cell differentiation by QRTPCR, first remove the media from upper and bottom chambers. Gently wash the upper chamber twice with 300 microliters of room temperature PPS.
Add 300 microliters of TRIzol to each upper chamber, and incubate at room temperature for five minutes. Meanwhile, label micro centrifuge tubes for each well, and add 700 microliters of TRIzol to each tube. Collect cell homogenate by gently pipetting up and down three times with a P1000, and transfer the contents into corresponding micro centrifuge tubes.
Vortex for five seconds to mix. Keep the samples at room temperature for an additional three minutes, and then continue with standard QRTPCR protocols, or store samples at negative 80 degrees Celsius. To verify M cell differentiation by immunofluorescence, first, remove the media from the upper chamber.
Gently wash two times with 300 microliters of room temperature PBS. Add 100 microliters of room temperature 4%PFA in PBS to the upper chamber. Cover the plate with foil and let stand for 25 minutes at room temperature.
After removing the PFA, wash the upper chamber three times with 300 microliters of room temperature PBS. Incubate the monolayers with 100 microliters of 5%BSA dissolved in PBS for 30 minutes in the dark at room temperature to block the monolayers before removing the solution. Prepare GP2 primary antibody solution in one percent BSA in PBS at a dilution of one to 100, and add 100 microliters per well.
Stain for one hour at room temperature in the dark, before removing the solution. Wash the upper chamber three times with 300 microliters of room temperature PBS. Prepare a secondary stain solution of fluorescently tagged goat anti-mouse IgG phalloidin and DAPI, and add 100 microliters per well.
Stain for 30 minutes at room temperature in the dark. Wash the wells three times with 300 microliters of PBS. Then, place a five microliter drop of mounting solution on a glass slide.
Remove the well from the 24 well plate and invert. During removal of the membrane from the Transwell, the membrane must remain completely flat. Bumps in the membrane prevent a firm seal to the glass slide, and can greatly affect image quality.
To ensure success, carefully cut the membrane from the well using a sharp scalpel. Place the membrane with the cells facing up onto the droplet of mounting solution on the glass slide. Add 10 microliters of mounting solution onto the top and center of the membrane, and place a cover slip on top to seal the membrane between the glass slide and cover slip.
Dry the slides at room temperature in the dark for 24 hours. M cells are detected by surface expression of GP2 by immunofluorescents. Typically, in a confluent monolayer, one to five M cells are observed in a given microscope field at 40X magnification by days six through eight post seeding in samples treated with RANKL and TNF alpha.
No GP2 expression is seen in the untreated samples. The orthogonal view of the XZ plane overlaid with a phalloidin probe shows actin structures surrounding each cell, and GP2 expression on the apical surface of M cells. This model recapitulates the low frequency of M cells found in the human intestine.
To determine if the M cells developed in this model are able to bind to IgA, the presence of IgA on M cells is visualized using a fluor conjugated secondary antibody that recognizes the heavy chain of human serum IgA. M cells treated with IgA for one hour have IgA bound to the apical surface, whereas M cells in control wells that were only treated with the secondary antibody to IgA, have no detectable signal. Further, IgA specifically binds to the apical surface of M cells, and is not found bound to any cells lacking GP2 surface stain.
In addition, M cells have characteristically shorter dense actin on their apical surface. Switching to RANKL TNF alpha supplemented differentiation media when the monolayers are around 80%confluent, is key to achieving good M cell differentiation. This technique will allow researchers to explore questions related to M cell development, as well as host pathogen interactions and drug transport studies involving M cells.
