The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The overall goal of this procedure is to use ex vivo cultured mouse intestinal tissue to study the role of pathogen associated molecular patterns and the pattern recognition receptors for intestinal antimicrobial host defense responses.This method can help answer how innate immune pathways regulate intestinal microbiota via regulation of antimicrobial peptides.The main advantage of this technique is that the intestinal architecture remains intact while measuring antimicrobial peptides ex vivo.After euthanizing a mouse according to the text protocol, place the animal dorsal side down on a pinning board and pin the limbs.Then use 70%ethanol to spray the mouse.With presterilized dissecting scissors and forceps, make a midline incision in the peritoneum of the abdomen.Then use forceps to open the abdomen by folding the peritoneum.With dissecting forceps and scissors, separate the colon from the small intestine by cutting at the bottom of the cecum, and the other end at the rectum.Place the colon in sterile Petri dish containing ice cold PBS, and then place it on a paper towel.Use the ice cold buffer and a 20 milliliter syringe, holding a 20g needle, or an oral gavage needle, to flush the contents of the lumen of the colon until the stool is completely removed.Using scissors, cut the colon longitudinally.Then wash the tissue by placing it into ice cold PBS in a sterile Petri dish and vigorously shaking.Repeat this wash three times.With a sterile scalpel or scissors, cut the colon tissue into pieces approximately one centimeter long, then record the weight of the colon pieces.To culture colon tissue, place a cell strainer on a six well cell culture plate.Then transfer all of the colon pieces collected from one mouse into the cell strainer.Add 5 milliliters of DMEM F12 medium containing 5%FBS, one x penicillin streptomycin, and 20 micrograms per milliliter of gentamycin, completely covering the colon pieces with the medium.Incubate the tissue at 37 degrees Celsius with 5%CO2 and 95%air for two hours.Then lift the cell strainer and aspirate the medium.Next, place the cell strainer back on the well and add five milliliters of fresh medium without antibiotics.Then lift the cell strainer and aspirate the medium.Repeat the wash two more times to remove all of the residual antibiotics.Transfer the colon pieces from a single cell strainer into a single well of a sterile 12 well cell culture plate.Add one milliliter of DMEM F12 medium containing 5%FBS per one milligram of tissue, then incubate the tissue at 37 degrees Celsius, injecting 5%CO2 and 95%air for 12 hours.Following the incubation, collect the culture supernatant in a sterile 1.5 milliliter tube.Centrifuge the sample at 12, 000 times g and four degrees Celsius for five minutes.Separate the supernatant into a new 1.5 milliliter tube for the use in the bacteria killing assay and/or other immune assays.Store the supernatant at minus 80 degrees Celsius until ready to perform the assay.To carry out an e.coli killing assay, inoculate e.coli into give milliliters of LB in a 15 milliliter tube.While keeping the cap on the culture tube slightly loose incubate the cell culture at 37 degrees C while shaking at 200 rpm overnight.Centrifuge the bacterial culture tube at 12, 000 times g and four degrees Celsius for 10 minutes.Then remove the supernatant and resuspend the bacterial pellet in five milliliters of ice cold PBS.Transfer one milliliter of the bacterial suspension into a cuvette and measure the optical density at 600 nanometers.Use a predetermined standard curve to calculate the colony forming units.In this experiment, assume that one optical density unit equals two times 10 to the ninth CFU per milliliter.Next, dilute the bacterial suspension to make a stock suspension of one times 10 to the fifth colony forming units per milliliter.Then transfer the previously collected colon organ culture supernatant and control media into duplicate wells of a 24 well cell culture plate.Add 10 microliters of e.coli culture into one well of the supernatant and control.Leave the duplicate wells without e.coli to confirm the colon organ culture supernatant is not contaminated.Incubate the culture at 37 degrees Celsius for one hour.After collecting colons from mice as demonstrated earlier in this video, place the colon on a sterile paper towel and use scissors to cut the colon longitudinally into three parts.Use ice cold PBS to wash each part of the colon.Then cut each part of the organ into small pieces and weigh.Transfer the colon pieces into three separate cell strainers placed on three wells of a six well cell culture plate.Add five milliliters of DMEM F12 medium containing 5%FBS, one x penicillin streptomycin, and 20 micrograms per milliliter gentamycin.Incubate the tissue at 37 degrees Celsius and 5%CO2 for two hours.After washing the colon pieces as demonstrated earlier in this video, transfer the tissue from a single cell strainer into a single well of a sterile 12 well cell culture plate.Designate the three wells as untreated, IL-1