Biology
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Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts
Chapters
Summary February 6th, 2021
In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.
Transcript
Our protocol provides a fast, reproducible, and reliable method to generate direct primary intestinal epithelial monolayers on different surfaces with minimal debris. The main advantages of this protocol are low consumption of media and low cost maintenance of cell culture. It's a quick method to perform functional tests and fast downstream applications.
This protocol will provide insight in the study of wound repair, permeability barrier, and transepithelial migration of different cell types. Also, this model can be useful in host-pathogen interaction, damaged epithelial, and drug discovery. To begin, add 200 microliters of coating solution to each well of a 48-well plate and chamber slide.
Pre-incubate both in a 5%carbon dioxide incubator at 37 degrees Celsius for two hours. Coat 0.4 micrometer cell membrane inserts with 200 microliters of collagen solution. Incubate the plate with the membrane inserts at four degrees Celsius for 30 minutes, then transfer it to a 5%carbon dioxide incubator at 37 degrees Celsius for two hours.
Disinfect a euthanized mouse with 70%ethanol, then dissect out the colon from rectum to cecum with the help of scissors and forceps. Gently flush out the feces with ice cold PBS in a 10 milliliter syringe fitted with a 20 gauge feeding tube. Remove the proximal colon and slide the distal colon gently onto the 20 gauge feeding tube.
Tie the colon at the end of the tube with 4-0 silk suture thread. Invert the colon inside out over the tied end and tie the other end with thread, then cut the colon below the tip of the feeding tube using surgical scissors. Gently open the untied end of the inverted colon onto the tip of a 1.25 milliliter repeat syringe using its plunger.
Slide the untied end of the inverted colon onto the syringe and tie it tightly with a thread. Insert the plunger into the syringe and inflate the colon until it looks turgent with no visible wrinkles. Place the inflated colon sausage in a 15 milliliter tube containing five milliliters of cell recovery solution on ice for 20 minutes.
Inflate and deflate the colon once every five minutes. Tie off the inflated colon below the tip of the repeat syringe using 4-0 silk suture thread. Cut the colon sausage off the repeat syringe and place it in a 15 milliliter tube containing 10 milliliters of 50 millimolar EDTA for 40 minutes.
Rotate the tube at four degrees Celsius. Replace the EDTA solution with five milliliters of shake buffer and shake the sausage manually in the vertical position for two minutes. Decant the shaking solution into a new 15 milliliter tube and repeat the shaking step until a total of 10 milliliters of crypts and shaking buffer is collected.
Count the number of crypts in 20 microliters of the suspension under a microscope and dilute the sample to obtain the concentration of five crypts per microliter. Centrifuge the crypt sample at 400 times G for 10 minutes at four degrees Celsius. In the meantime, remove the 48-well plate and membrane inserts from the incubator and place it in the biosafety cabinet.
Aspirate the coating solution using a P200 pipette and leave the plate with the lid slightly offset until the cells are ready to be plated. After centrifugation of the crypt suspension, remove the shaking buffer and resuspend the intact pellet in three milliliters of LWRN complete media. Add 200 microliters of crypts to each well of the pre-coated 48-well plate and chamber slide and incubate both in a 5%carbon dioxide incubator at 37 degrees Celsius.
On the next day, aspirate the media and add fresh media. Generally, the cells become confluent in 24 to 48 hours. For cell culture membrane inserts, add 200 microliters of crypts to the top of the inserts and 600 microliters of complete LWRN media to the bottom.
On the next day, aspirate the media and add fresh media only to the top chamber. Incubate the plate in a 5%carbon dioxide incubator at 37 degrees Celsius. Measure transepithelial electrical resistance every day using an epithelial volt/ohm meter.
After the isolating the crypts from a murine colon and verifying the concentration, the crypt suspension was concentrated to five crypts per microliter. 48-well plate wells were subjected to a scratch wound assay upon reaching the cell confluence. After 24 hours, wound repair was observed indicating that the culture was healthy and viable.
Higher tier values were achieved when media was changed from LWRN to differentiation media. Also, a decrease of ISC markers and an increase in differentiation markers were observed indicating that the differentiated monolayers were generated successfully. In addition, the appearance of subtypes of differentiated epithelial cells grown in different conditions was shown by immunofluorescence.
When attempting this protocol, it is important to make sure that the colon is not ruptured at any point during the prep and stays inflated to release crypts in extremely clean preparation. Once the 2d intestinal epithelial cell monolayers form, we can perform immunofluorescence, scratch wound and permeability assays and other downstream applications. This will provide knowledge in wound healing, migration of different cell types, and epithelial repair and damage studies.
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