Begin by preparing all the reagents prior to the experiment and thaw the basement membrane matrix on ice. Then prepare the murine monolayer medium. Dilute the thawed basement membrane one to 20 with the cold murine monolayer medium, and keep it on ice.
Place a 0.4 micrometer transparent cell culture insert, using sterile forceps, into a single well of a 24 well tissue culture plate. Grab a corner prong of the insert and transfer it into the well. Repeat until the appropriate number of cell culture inserts are available for culture.
To cover the apical surface of each cell culture insert with diluted basement membrane matrix, place the tip of the P 200 pipette in the center of the insert and expel 150 microliters matrix. Then, transfer the plate to a sterile 37 degrees incubator with 5%carbon dioxide, for at least one hour. At the end of the incubation, rotate the 24 well plate at a 45 degree angle.
Place a single finger on the corner of the prong to stabilize the insert. Gently place the P 200 pipette tip along the bottom side of the insert and remove the basement matrix. Remove any excess residue by washing each cell culture insert with 150 microliters of sterile DPBS without calcium or magnesium ions and allow them to dry in a biological safety cabinet for a minimum of 10 minutes.
Once the inserts are dried, add 400 microliters of the murine monolayer medium to the bottom, and 75 microliters on top of the cell culture insert. Repeat until all the cell culture inserts are immersed. Leave the plate in the biological safety cabinet or the incubator until needed.
Then remove the 24 well plate of mature intestinal colonoids from the incubator and place it under the biological safety cabinet. Aspirate the medium using sterile tips and wash each well with one milliliter of sterile DPBS at room temperature. Using sterile tips, remove the DPBS without calcium or magnesium ions.
Digest each plug of the basement membrane matrix by adding 500 microliters of enzymatic dissociation reagent in each well to be passaged. To break up the plug, rotate the 24 well plate at a 45 degree angle. Place the tip of the pipette at the edge of the plug and gently dislodge it by pipetting each plug approximately six to 10 times.
Place the 24 well plate back in a sterile incubator at 37 degrees with 5%carbon dioxide, for three to four minutes. After incubation, pipette the trypsin up and down five to seven times for each well under a biological safety cabinet. Transfer the dissociated colonoids from each well into a sterile 15 milliliter conical tube and bring up to a volume of 10 milliliters with an ice cold mouse wash medium.
Next, centrifuge the partially digested colonoids at 300 G for five minutes at four degrees Celsius in a swinging bucket centrifuge. Under the biological safety cabinet, remove the supernatant from the pellet using a 10 milliliter pipette. Also, remove any remaining medium using a P 200 pipette.
Then, re-suspend the colonoid pellet by adding 75 microliters of murine monolayer medium. Dispense 75 microliters of the colonoid suspension to the center of each cell culture insert. Gently pipette the suspension once or twice before adding it to the well to ensure uniform platting.
Place the 24 well plate with cell culture inserts on a rotating platform for 10 minutes to allow for uniform dispersion across the cell culture insert, before placing the plate in a sterile 37 degree Celsius in 5%carbon dioxide incubator. At the end of the incubation, dislodged the unattached colonoids fragments by placing the tip alongside the inside of the cell culture and pipetting the medium three to five times with a P 200 pipette. Avoid disrupting the attached intestinal cells.
Remove the medium and colonoid mixture immediately. Repeat until all the cell culture inserts have been cleaned. Now add 150 microliters of pre-warmed murine monolayer medium to the apical side of each cell culture insert.
Add 400 microliters of warmed murine monolayer medium to each well of a new 24 well plate. Transfer the cell culture insert to the new plate by grabbing its prong using sterile forceps. Change the medium every other day until the desired confluency is attained.
The enzymatically disrupted colonoids plated on the cell culture inserts initially appeared in cellular clusters ranging from 15 to 150 cells. On day three, the cells flattened out and slowly covered the cell culture insert, generally reaching confluency. Over the next couple of days, monolayers continued to grow and formed a continuous barrier that can be quantitatively measured.
