The overall goal of this procedure is to create a natural acellular scaffold for intestinal tissue engineering. This is accomplished by first cannulating, a rat superior mesenteric artery or SMA, and harvesting the small intestine along with the vascular access. In the second step, the intraluminal debris is washed out and the intraluminal and vascular cannulas are connected to a peristaltic pump.
Next, the small intestine is perfused with deionized water for 24 hours at four degrees Celsius. In the final step, the small intestine is perfused with sodium deoxy, coate, and DNAs at room temperature. Ultimately, the complete decellularization of the small intestine, as well as its architecture and components can be visualized by histology and electron microscopy respectively.
We are currently faced with the clinical problem of intestinal failure, devastating condition that affects both children and adults. Parental nutrition and conventional transplantation do not provide a complete cure. The production of three-dimensional vascularized intestinal scaffold as demonstrated in the following video, extend towards the therapy of intestinal failure.
What is remarkable about this technique is that the use of gentle digitalization chemicals preserves intestinal micro architecture as evident by the electron microscope results. Additionally, the maintenance of hierarchical vascular network following the civilization allows seeding of a structure that can be reconnected to the host. Blood supply Begin by fitting a peristaltic pump with two tubes, then run 70%ethanol through the tubes of the peristaltic pump for 15 minutes at maximum speed, followed by freshly prepared PBS AA for another 15 minutes.
After flushing the pump spray and wipe the abdomen of a euthanized 250 to 350 gram spra dolly rat with 70%ethanol. Then using autoclave sterilized surgical instruments, perform a XO pubic laparotomy incision on the abdomen to ensure a clear surgical field eviscerate the small intestine to the right side of the animal onto a paper towel sprayed with 70%ethanol. Take care to continuously wet the intestine with P-B-S-A-A to avoid dehydration of the tissue and denaturation of the extracellular matrix.
Next, locate the SMA, which arises at a 90 degree angle from the aorta towards the intestine. Place a suture around the SMA and prepare a knot. Use a 27 gauge cannula to enter the SMA from the aorta, and then quickly withdraw the needle to avoid tearing the wall of the vessel.
Now advance the plastic tube of the cannula into the SMA, securing the tubing with Vicryl five oh sutures, and then inject 10 milliliters of P-B-S-A-A to confirm cannulation. Next, use scissors to incise the aorta proximal and distal to the SMA to release the cannula to avoid leakage of feces when dissecting out the large intestine, place one silk five oh suture knot on the proximal end of the ileocecal junction and one at the distal end of the sigmoid colon. Then cut the terminal ileum and colon between the two knots and remove the large intestine.
Now cut the small intestine at the Juno duodenal junction and free the intestine from any connective tissue connections it might have with the abdomen. Transfer the small intestine into a Petri dish containing PBS aa. Then use a plastic posterior pipette to cannulate the proximal section of the intestine, securing it in place with sutures.
Finally, cut the pipette so it will fit the lure lock of a 60 milliliter syringe, and then flush the intestinal lumen with two washes of 60 milliliters of P-B-S-A-A to remove any feces and debris Before beginning the decellularization process, ensure no bubbles are present within the intestinal lumen and vascular cannulas. Then connect a three-way stop cock to the vascular cannula and place the Petri dish in a container to collect any overflowing solution. Next, start running deionized water through the master flex LS variable speed roller pump at 0.6 milliliters per hour.
Handle the tissue carefully to ensure the bubbles exit from the distal end. Once all the bubbles have been removed from the cannulas, transfer the apparatus to the cold room. Then start the decellularization process by perfusing both the intestinal lumen and the vascular tree with running deionized water for 24 hours at 0.6 milliliters per hour during the first hour.
Check the apparatus regularly to ensure that all the connections are secure. The leakage is minimal and no bubbles are present within the intestine. After 24 hours, move the decellularization apparatus back to room temperature.
The color of the intestine should have changed to a light pink under a suction hood. Weigh enough sodium deoxy coate to make a 4%solution in 300 milliliters of deionized water. Use a vortex to mix the solution until it is clear.
Now, replace the deionized solution with the sodium deoxy collate. Check the connections. Remove any bubbles and perfuse the small intestine at 0.6 milliliters per hour for four hours.
After the sodium deoxy collate step, the tissue becomes transparent. The maintenance of the vascular network also becomes apparent after adding PBS AA to the pump. Wash the intestine with PBS AA for 30 to avoid any interactions between the sodium deoxy, coate and DNA one.
Then perfuse the tissue with freshly prepared DNAs, one at room temperature at a speed of 0.6 milliliters per hour. After three hours, transfer the scaffold into a tissue culture hood and using a septic technique, change the plate. Re sterilize the tubing of the peristaltic pump by running ethanol through.
Wash the intestine one last time with P-B-S-A-A for 30 minutes to ensure all remnants of the decellularization solutions are removed. Finally, transfer the scaffold to a UV crosslinker and run two sterilization cycles. Then store the scaffold in P-B-S-A-A.
Changing the solution every three days. Upon successful decellularization, the scaffold should have a macroscopic appearance similar to that of the native intestine, but with translucent walls. The preservation of macro architecture should also be evident by the patency of the vascular tree.
When dye is injected through the SMA cannula, it should distribute evenly throughout the scaffold and reach the capillary tree. The scaffold should be free of nuclear and cytoplasmic material, something that can be assessed. Using a DNA quantification kit and simple histological analysis, hematin and eoin staining should demonstrate an absence of nuclear and cytoplasmic material while preserving the small intestinal layers as demonstrated in this image.
In particular, the maintenance of the villi and crips on the luminal side should be evident following scanning electron microscopy. Further conservation of the extracellular matrix components such as collagen, elastin, and glyco Amin glycans should also be expected as shown here. For example, Massen Tri CHRO staining allows demonstration of the intact connective tissue in the scaffold.
After watching this video, you should have a good understanding of how to create the natural cellular intestinal scaffold.
