To begin, place the euthanized rat onto the dissection surface with the ventral side up. Pinch the skin layer with sterile forceps and make cuts at the surface level, avoiding damage to internal organs. Using large sharp dissection scissors, make a longitudinal surface level cut in the center of the abdomen.
Then stemming from that cut, make two shorter horizontal cuts one on each side. Using forceps, peel the skin away to expose the abdominal cavity. Cut through the peritoneal membrane to fully expose the internal organs in the abdominal cavity with ready access to the intestine.
Using scissors and forceps, locate the stomach and identify the duodenum, approximately two to three centimeters distal to it which appears as a yellowish segment. The proximal jejunum is located approximately four to five centimeters distal to the ligament of Treitz. A landmark between the duodenum and jejunum.
Place the isolated intestinal fragment into a 10 centimeter Petri dish. Flush the desired intestinal segment of mesentery with 10 milliliters of ice cold PBS until cleared of luminal contents. On a paper towel, cut the intestinal segment into two centimeter long pieces.
Then open each intestinal piece longitudinally to expose the epithelium. Scrape the exposed luminal surface using a glass microscope slide to remove villi. Place the intestinal pieces into the EDTA solution on ice and rotate it four degrees Celsius for 30 minutes on a tube revolver set to 10 RPM.
At the dissecting microscope, pour the contents of the tube into a 10 centimeter Petri dish. Add in an additional five milliliters of ice cold PBS. Using fine forceps, hold an intestinal segment and shake vigorously to observe the epithelium release into the PBS.
Initially, the PBS will contain mostly villi. Shake the intestinal fragments continuously, periodically discard the PBS containing villi, and add 10 milliliters of fresh PBS to the intestinal fragments. Continue to shake the fragments and repeat the PBS washing step until villi are no longer released into the PBS.
But instead, the PBS primarily contains crypts. Discard the remaining intestinal fragments and enrich the remaining PBS in the Petri dish for intestinal crypts. In a tissue culture hood, collect the PBS containing crypts and filter through a 70 micron cells drainer.
Centrifuge the filtrate at 250 x g for five minutes. Then remove their supernatant and resuspend the pellet in five millimeters of advanced DMEM plus. Centrifuge again at 250 x g for five minutes.
And remove the supernatant, leaving 50 microliters of media with the pellet. Resuspend the pellet in the remaining media and add it to the aliquot of the extracellular matrix extract or EME on ice. Gently pipet up and down to suspend the crypts evenly throughout the EME avoiding making bubbles.
Place 50 microliters of the EME domes into a 35 millimeter tissue culture dish. An incubated 37 degrees Celsius with 5%carbon dioxide for 20 minutes. After incubation, add a two milliliters of rat intestinal organoid media, or RIOM, containing 10 micromolar each of Y27632 and CHIR99021.
Next, to passage rat intestinal organoids, aspirate the medium from a plate containing organoids and add one milliliter of dissociation reagent to release the organoids from the EME domes. Transfer the dissociation reagent with fragmented organoids to a 15 milliliter conical tube. Wash the culture plate with two milliliters of advanced DMEM plus and add it to the 15 milliliter conical tube containing the fragmented organoids.
Using a glass pasture pipette, gently pipette up and down 15 to 20 times to fragment the organoids. Centrifuge the organoids at 350 x g for two minutes and add 50 microliters of solution from the bottom of the tube into the EME. After mixing the solution, plate 50 microliters of the EME domes into a 35 millimeter tissue culture dish and incubate at 37 degrees Celsius with 5%carbon dioxide for 20 minutes.
After incubation, add two milliliters of RIOM containing 10 micromolar each of Y27632 and CHIR99021. Representative images of villi fragments and crypts are shown. Crypts are smaller in size compared to villi.
Plated crypts expand over the next few days and begin to bud and differentiate by days four to seven. After two days of thawing, a healthy organoid culture showed the presence of both spheroids and budded organoids. The same organoid line after passaging showed the presence of single crypt-like domains.
