In this study, we established a rat intestinal organoid model that is robust in long-term cell culture. We also demonstrate the first successful genetic manipulations using both lentiviral infection and transient transfection. This model is an excellent tool for manipulating intestinal biology in the rat, where the genetic tools available in vivo are limited.
Rat intestinal organoids have previously been difficult to culture long-term. By generating a robust genetically manipulatable rat organoid model, we are providing researchers additional flexibility and choice to select an organoid model best suited for their particular circumstances. Mouse intestinal organoids are used to study stem cell behavior, cell fate, and physiology, but they can be a suboptimal model since there are key differences between mouse and human biology.
On the other hand, human intestinal organoids can be difficult to obtain and have complex cell culture requirements. The rat organoid model we describe here retains key physiological relevance to human biology while being considerably easier to access and care for than human intestinal organoids. Developing and optimizing the rat intestinal organoid model allows for genetic manipulation, pharmacological treatments, and higher throughput studies of intestinal biology in an accessible model with key physiological relevance to humans.
Additionally, these rat organoids will allow for the study of species-specific physiology, cell fates, and phenotypic variations in disease models in the intestine. 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 mesentary 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 at 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 the tissue culture hood, collect the PBS-containing crypts and filter through a 70 micron cells drainer. Centrifuge the filtrate at 250G for five minutes.
Then remove their supernatant and resuspend the pellet in five millimeters of Advanced DMEM Plus. Centrifuge again at 250G 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. And incubate at 37 degrees Celsius with 5%carbon dioxide for 20 minutes.
After incubation, add two milliliters of rat intestinal organoid media or RIOM containing 10 micromolar each of Y27632 and CHIRA99021. 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 Pasteur pipette, gently pipette up and down 15 to 20 times to fragment the organoids. Centrifuge the organoids at 350G 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 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 CHIRA99021. Representative images of villar fragments and crypts are shown.
Crypts are smaller in size compared to villi. Plated grips 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 packaging showed the presence of single crypt-like domains. To coat the plate with EME, dilute EME one to 20 in cold Advanced DMEM Plus. For coating with collagen, dilute five milligrams per milliliter of collagen one in Advanced DMEM Plus to 100 micrograms per milliliter.
Coat the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely, and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids. Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME.
Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids, and transfer them to the same conical tube. Centrifuge the sample at 350G for two minutes and remove the supernatant, including any EME residue.
Then add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM Plus to neutralize trypsin. Centrifuge at 350 G for five minutes and aspirate the supernatant before resuspending the pellet in 4.8 milliliters of RIOM 2D.
Remove excess EME or collagen in Advanced DMEM Plus from the wells. Then add 200 microliters of organoids in RIOM 2D and 10 micromolar Y27632 to each pre-coated well. After four to 16 hours, collect the media and centrifuge at 1000G for one minute.
Transfer the supernatant to a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged RIOM 2D to each well. Change the RIOM 2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells.
In contrast, a collagen one substrate was insufficient to reform 3D structures.
