The overall goal of this procedure is to evaluate the effects of intestinal ischemia on intestinal epithelial stem cells. This method can help answer key questions in the field of stem cell biology, such as how stem cells resist injury and contribute to repair following ischemic injury. The main advantage of this technique is that it is a large animal model that can be used for the study of clinical intestinal disease and repair.
Demonstrating the procedure will be Amy Stieler Stewart, a graduate student, and John Freund, a research specialist from my laboratory. Begin by using a scalpel blade to make an eight to 10 centimeter ventral midline incision on the abdomen centered at the umbilicus of an eight to 10 week old Yorkshire crossbred pig. Locate the jejunum, approximately 40 centimeters oral to the ileocecal junction.
Circumferentially ligate the bowel two times to delineate 10 centimeter long loops of jejunum with one centimeter between each ligature. Create two loops per time point of ischemia adjacent to each other, one for ischemia and one for ischemia with an additional one hour of reperfusion. To induce reversible complete ischemia, use bulldog vascular clamps or hemostats to obstruct approximately three mesenteric vessels per clamp for the appropriate experimental time period.
Keep the abdomen covered throughout the ischemic period. At the end of the experiment, use Metzenbaum scissors to first collect a control piece of normal jejunum at least five to 10 centimeters proximal to the last ischemic loop, followed by collection of the rest of the loops. Store the loops from each injury time point in small containers of ice-cold PBS until crypt isolation.
To isolate the crypt stem cells, use a 20-gauge wire and tissue forceps to invert each loop of small intestine so that the mucosal surface is exposed. Suture the top and bottom of each loop securely to the wire, and rinse each inverted loop in ice-cold PBS. When all the luminal debris has been removed, immediately place the samples into individual 50 milliliter conical tubes containing 30 milliliters of dissociation reagent number one on ice for 30 minutes with shaking and inversion every five minutes.
At the end of the incubation period, transfer the samples into corresponding 50 milliliter tubes containing 30 milliliters of dissociation reagent number two and pellet the two to four hour ischemic loop samples by centrifugation. Resuspend the pellets in five milliliters of PBS and use a 50 microliter aliquot from each sample to check the degree of crypt association and amount of debris by light microscopy. Next, place the samples in a 37 degree celsius water bath for 10 minutes with shaking or inversion every five minutes.
Then transfer the samples directly into 25 milliliters of ice-cold PBS on ice in an orbital shaker set to 60 RPM for two to five minutes of shaking with additional manual shaking or inversion every 30 seconds. At the end of the shaking incubation, check the degree of crypt association and amount of debris and transfer the samples into new 50 milliliter conical tubes containing 25 milliliters of cold PBS for shaking until intact crypts are isolated with minimal debris and villi. When the loops have been fully dissociated, remove the remaining tissue, pellet the samples by centrifugation, and resuspend the remaining crypt pellets in five milliliters of fresh PBS.
Then aliquot 150 crypts per tube into individual microcentrifuge tubes and pellet the crypts by microcentrifugation. Using a pre-cooled pipette, gently resuspend the pellets with 50 microliters of master mix per well followed by rapid pipetting 15 times to mix. Next, dispense a 50 microliter crypt droplet into the center of each well of a pre-wormed and gridded, 24 well plate and place the plate in a 37 degree celsius incubator.
After 30 minutes, overlay each matrix patty with 500 microliters per well of intestinal epithelial stem cell medium adding 500 microliters of sterile PBS to any unused wells to maintain humidity and count the number of plated, day zero crypts. Add growth factors to each well every 48 hours replacing the supernatant every 96 hours with 500 microliters of fresh growth factor-supplemented medium. Complete intestinal ischemia is created in small intestinal loops by utilizing vascular occlusion with sutures or clamps, as shown.
If performed correctly, ischemic injury will begin at the tip of the intestinal villi and migrate down within the crypt as the duration of ischemia increases. One common mistake with the surgical technique can occur when the blood vessels and not ligated or clamped evenly, resulting in a hemorrhagic ischemia in which the thin-walled vein collapses before the artery, allowing additional blood to infiltrate the tissues. Following removal of the ischemic intestinal loops, the intestinal crypts can be successfully isolated following the dissociation protocol as just demonstrated.
Crypts from more severely damaged time points are often broken and contain more background cellular debris compared to those that undergo no or mild damage. When normal and mildly damaged intestinal crypts are plated in culture, enterospheres form within 24 to 48 hours. With severe ischemic damage, the intestinal crypts survive but are damaged resulting in the formation of much smaller spheres initially.
By 72 to 120 hours, enteroids from all of the crypts become more complex with obvious central lumens and budding structures, and with an overall decreased crypt growth efficiency, as well as decreased size in the enteroids derived from the severely damaged intestinal tissue. Once mastered, the crypt isolating plating procedures can be completed in about two to three hours if they are performed properly. It is best that the crypts collected for plating are derived from washes not from disassociation fractions due to the increased likelihood of culture contamination in the disassociation fraction.
This method can also be used to test the efficacy of drugs for treating epithelial injuries resulting from ischemia plus or minus reperfusion. After its development, this technique paved the way for researchers in the field of gastrointestinal biology to explore the impact of ischemia on the epithelium specifically on intestinal stem cells in a translational, clinically relevant large animal model. After watching this video, you should have a good understanding of how to create intestinal ischemia with or without reperfusion as well as to isolate intestinal crypts for 3D culture following in vivo injury.
