Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The overall goal of this procedure is to generate tissue engineered vessel grafts that are functional for grafting into mice. This is accomplished by first reprogramming human fibroblasts into partially induced pluripotent stem cells in vitro, which are capable of differentiation into endothelial and smooth muscle cells. An aorta is then removed from a sacrificed mouse and decellularized with sodium doda sulfate or SDS solution to prepare the decellularized aorta grafts.

Next, the bioreactor flow circuit is set up for dual seeding of partially induced pluripotent stem cells. The final step is grafting of the double seeded, partially induced pluripotent stem cell graft into mice. Ultimately, the analysis of mice mortality rates three weeks post grafting is used to show the functionality of the tissue engineered vessel grafts.

The implications of this technique extend towards the therapy of cardiovascular diseases because it has been shown to successfully generate functional blood vessels using PIPS cells in a short period of time. In general, it's very important to keep the vessel graft intact and sterile during the whole procedure, especially for people new to this method. Begin this procedure with reprogramming of human fibroblasts into partially induced pluripotent stem cells or PIPS cells by transecting the cells with four micrograms of linearized OSKM plasmid using a human dermal fibroblasts nuclear affection kit.

Human fibroblasts become PIPS cells after four days of reprogramming. Following mouse sacrifice by cervical dislocation. Mix the mouse in a supine position under a dissection microscope.

Then cut through the sternum and around the rib cage to open the thoracic cavity. Remove the heart, lungs, and esophagus to expose the aorta, and then gently remove the peri aorta fat with forceps. Cut the aorta from the anterior end with scissors and gently hold the end with blunt forceps.

Then detach the aorta from the spine column behind by blunt dissection. Carefully close all the branching arteries from the aorta by ligation with a bipolar electro coagulator and cutting from the end of the ligation with scissors. Use a five milliliter syringe to flush the aorta lumen with three milliliters of saline solution containing 100 units of heparin.

To prevent the formation of blood clots, cut the posterior end of the aorta before it branches into the renal. Keeping the integrity of the aorta is very important during the whole procedure. Next, lush the aorta lumen with five milliliters of 0.075.

Sodium dool sulfate or SDS solution diluted in phosphate buffer saline or PBS soak that the rasic aorta in 0.075%SDS solution in a 10 centimeter petro dish on an orbital shaker for two hours at 150 RPM at room temperature. Then brush the aorta lumen with five milliliters of PBS. Next, soak the aorta in PBS in a 10 centimeter petro dish and place the petro dish on the orbital shaker at 150 RPM for two hours at room temperature.

Refreshing the PBS every 20 minutes after two hours. Blush the aorta lumen with five milliliters of PBS. To begin, assemble the bioreactor flow circuit as described in the text protocol.

Next, recondition the decellularized vessel with culture medium by soaking the aorta in differentiation media in a 10 centimeter Petri dish on the orbital shaker for one hour at 50 RPM at room temperature. Insert one centimeter length nylon tubes into both ends of the decellularized vessel under the microscope. Then tie the vessel and the tubes with eight zero silk sutures.

Assemble the decellularized aorta graft in the incubation chamber by connecting the nylon tubes with the inlet and outlook ports from the chamber wall. Maintain the incubation chamber with five milliliters of medium each time. Next, trypsin eyes the PIPS cells by adding prewarm trips into.

Cover the culture dish and gently rocking the dish 10 times. Discard the trips in and leave the dish in the incubator for two to three minutes. Then add prewarm culture medium in the dish and mix the medium with the cells.

Next, count the cell number using a hemo cytometer centrifuge. Two aliquots of cell suspensions containing half a million cells each for five minutes at 300 times G.After the supinate completely resuspend one pellet of the PIPS cell pellets. In 50 microliters of dm, inject the cell suspension into the decellularized vessel lumen.

Then resus suspend the other PIPS cell pellet in 100 microliters of matrigel. Carefully pipette the mixture onto the decellularized aorta graft. Wait for 10 to 15 minutes until the mixture turns into a gel-like state and evenly wraps around the vessel's external surface.

At that point, build the incubation chamber with dm. Place the whole bioreactor setting in a 5%carbon dioxide incubator at 37 degrees Celsius. Then set the tubing connected between the media reservoir and the compliance chamber onto the peristaltic pump.

In the incubator. Manually rotate the decellularized graft 90 degrees around the longitudinal axis every half an hour in the first two hours after keeping the culture static for 12 hours to enable cell aian deliver DM through the lumen by the peristaltic pump to induce smooth muscle cell differentiation. Start the initial flow rate at five milliliters per minute.

Then apply a stepwise increase of 20 milliliters per minute over 24 hours after keeping the circulating medium flow rate at 20 milliliters per minute for 24 hours, stop the medium flow and move the bioreactor setting outta the incubator. Next, reede the lumen of the graft with PIPS cells by trypsin counting and centrif using 1 million PIPS cells. As before, re suspend the cells in 50 microliters of endothelial growth media.

Two containing 50 nanograms per milliliter of vascular endothelial growth factor. Then inject the cell mixture into the lumen of the graft. Change the medium in the incubation chamber and whole circulation to e GM two containing VEGF to induce endothelial differentiation.

After moving the bioreactor setting back to the incubator, manually rotate the graft 90 degrees every half an hour in the first two hours, and then keep a static culture for 12 hours. After 12 hours, start circulating flow from five milliliters per minute with stepwise increase to 35 milliliters per minute, and then keep the flow rate at 35 milliliters per minute for five days. Then obviously engineered graft by cutting the ends of the nylon tubes.

Base the graft on a Petri dish containing fresh e GM two media in preparation for grafting to the mouse following anesthetization of the recipient mouse. Fix the mouse in a supine position with its neck shaved and extended. Prepare a midline incision from the mandible to the sternum of the mouse under a dissecting microscope with five to 10 fold amplification.

Lift the right sali glands laterally and remove the right glider mastoid muscle To expose the right common carotid artery, gently remove attached tissues to mobilize the right common carotid artery from the distal end towards the proximal end. Ligate the middle portion of the common carotid artery twice with an eight zero silk suture and dissect between the two tires. Pass through a cuff made of autoclavable nylon tube in each vessel.

End and fix each end with micro hemostat clamps. Then remove suture ties on one end and apply a drop of heparin immediately after. Invert the distal end of the artery to cover the whole cuff body and fix the inverted vessel with an eight zero silk suture to the cuff.

After repeating the same procedure to the other portion of the artery, lush the artery with saline solution to remove blood clots. Next, implant a decellularized vessel graft between two ends of the carotid artery by sliding the decellularized vessel ends over the artery cuff and fixing it with eight zero silk sutures. Remove vascular clamps from either ends before evaluating pulsation of the graft, then lace the right siv gland back into its original position.

Close the skin on the surgical location with an interrupted suture using a six zero prolactin suture. Continue with post-surgical care as described in the text protocol representative. Morphology of human fibroblasts after four days of reprogramming into PIPS cells are shown here.

PIPS cells displayed a markedly distinct phenotype when compared to fibroblasts. As expected, all aorta decellularization was achieved by treatment with SDS for two hours. As a result, the decellularized aorta appeared as a translucent acellular scaffold when compared to a freshly harvested aorta.

Following the dual seeding of PIPS cells within decellularized aorta, the engineered vascular grafts display endothelial and smooth muscle cell properties unlike decellularized vessel grafts. This is visualized here through the green stain for endothelial cells and the red stain for smooth muscle cells. Mice grafted with PIPS cell vessel grafts exhibited a survival rate of 60%three weeks after transplantation as expected.

Mice grafted with decellularized vessels presented with markedly higher mortality rates as early as day one After its development. This technique paved a way for researchers in the field of cardiology to explore the use of PIPS cells and decellularized vessel grafts in the generation of functional tissue engineered vascular conduits. After watching this video, I hope you could get a good understanding about how to prepare the decellularized vessel graft, how to assemble the bioreactor, and how to implant the receded graft back into mice.