The overall goal of this procedure is to create a space where tissue can grow and expand concomitantly with the development of a robust blood supply, ultimately leading to the generation of a vascularized, large, three-dimensional block of tissue. This method can help answer key questions in the engineering of vascularized tissue for reconstructive purposes as well as for organ replacement and repair. The main advantage of this technique is that the newly generated tissue is intrinsically supplied by its own vascular network without having to rely on diffusion from the environment for survival.

Demonstrating the procedure will be Sue McKay and Liliana Pepe. Anesthetize a rat weighing at least 250 grams, and assess the depth of anesthesia using a toe pinch. If the rat is unresponsive, maintain its sedation using 2%isoflurane throughout the procedure.

Next, apply epsomic ointment to the eyes. Then, with the rat supine, use electric razors to shave the groin and remove the hair with moistened gauze. Then, clean the surgical sites with a chlorhexidine solution containing 70%ethanol.

Finally, drape the animal with sterile towels and proceed with the surgery. Using a number 15 blade, make a four centimeter long skin incision into the left groin parallel to the inguinal ligament. Next, cut through the exposed inguinal fat pad circumferentially with scissors, leaving it attached to its vascular pedicle, based on the epigastric vessels.

Now, with micro-scissors, free the filmy connective tissue adhesions between the abdominal wall and underlying femoral vessels. Then, place a retractor on the abdominal wall and pull it medially to expose the inguinal ligament and the whole length of the femoral vessels. Next, using micro-forceps and curved scissors, dissect the epigastric vein and isolate it from the surrounding fat.

This vein will function as a tether. Proceed by opening the perivascular sheath containing the femoral vessels and nerve. Open it all the way from the inguinal ligament to its bifurcation distal to the epigastric branch.

At this point, and throughout the whole procedure, gentle handling of the femoral vessels is critical. Their damage can result in thrombosis and failure of the whole experiment. Then, using micro-forceps, pick up the femoral vein by its adventitia and gently separate it from the surrounding tissues and the accompanying artery.

During this step, do not grasp the whole thickness of the vein wall, which could cause trauma to the intima, thus making the vein prone to thrombosis. Also during this step, ligate the side branches with 10-0 nylon suture, or coagulate them with a bipolar coagulator. With the femoral vein completely free, ligate it proximally and distally with 4-0 silk sutures to obtain a vein graft that is at least 10 millimeters long, which includes about half a centimeter length of the epigastric branch.

The branch works as a guy rope tether to hold the loop open in the chamber. Next, trim the adventitia from the graft's ends. If necessary, this step may be postponed to before the micro-surgical anastomoses.

Finish preparing the vein graft by flushing it with heparinized saline solution and leaving it to rest loaded with heparinized saline solution. Then, close the wound using continuous running 4-0 silk suture and two or three additional simple interrupted stitches. To begin, expose the inguinal ligament and the whole length of the femoral vessel of the contralateral limb as previously demonstrated.

Then, using micro-forceps, dissect and isolate both the epigastric artery and vein from the surrounding fat pad. Be gentle when pulling the tissue away from the vessels. Continue by picking up the femoral artery by its adventitia and freeing it from the surrounding tissues.

In this process, ligate or coagulate the artery side branches. Then, place a clamp proximally on both the femoral artery and vein. Now, using 4-0 silk suture, ligate the femoral artery and vein distal to the emergence of the epigastric vessels.

Now place a sterile plastic contrast background under the vessels and use a sharp, straight micro-scissor to cut each vessel transversely. Once cut, flush the vessels out vigorously with generous amounts of heparinized saline until all the blood has been removed from the lumen. Now bring the vein graft into the operative field.

Position the vein graft so that the proximal end of the vein graft is ready to be anastomosed to the recipient femoral vein, and the distal end to the recipient femoral artery. This will allow the blood to flow from the arterial to the venous side without resistance from the valves inside the vein graft. Make sure the femoral vessels and the vein graft rest in their natural position without any twists.

Using 10-0 nylon suture, perform surgical anastomosis at both sides. A good and atraumatic micro-surgical anastomosis is key. Damage to the vessel intima, dessication of the vessels, or incorrectly placed stitches make the anastomotic site prone to thrombus formation.

Then, check for leaks at both anastomotic sites. Resolve small leaks, which look like non-pulsating blood coming out of the anastomotic site, by placing a small piece of fat or a sponge on top and gently compressing for five to 10 minutes. Larger pulsating leaks that rapidly flood the entire field will need additional stitches.

Next, check the patency of the arteriovenous loop. Gentle occlusion of the femoral artery should make it shrink. Conversely, occlusion of the femoral vein should engorge the loop.

Now place the base of the tissue engineering chamber under the arteriovenous loop. Make sure that the loop rests in its natural position without any twists or kinks. Then, with 6-0 nylon sutures, secure the base of the chamber to the inguinal ligament and the underlying muscle fascia.

Next, place the lid over the base of the chamber. Pinch the epigastric branches under the lid to hold the loop in position. Then, close the wound using continuous running 4-0 silk suture, plus two or three additional simple interrupted stitches.

Finally, allow the animal to recover from anesthesia in an individual cage, and administer a single dose of carprofen subcutaneously as an analgesic. Prepare the animal for surgery as before. Two chambers can be implanted into a single rat, one in each groin.

Then, isolate the femoral vessel as previously described. With both the artery and vein completely freed of surrounding tissues and their branches ligated, bring the chamber into the operative field. Then, place each of the intact femoral vessels into the corresponding slits of the chamber base.

Make sure that neither is twisted or kinked. Next, close the chamber by attaching the lid to the base. Then close the wound and allow the animal to recover as previously described.

The micro-surgical creation of tissue engineering chambers was performed as described. Various tissue types were successfully engineered using the in vivo vascularized chamber, which include cardiac tissue with neonatal rat cardiomyocytes, muscle tissue with rat skeletal myoblasts, and adipose tissue with hydrogel derived from adipose tissue extracellular matrix. The stereology system allows for unbiased quantification of specific tissue volume.

For example, lectin-stained endothelial cells in transverse sections were used to estimate vascular volumes. Following in vivo implantation of cells labeled with fluorescent dyes, their fate can be tracked. For example, neonatal rat cardiomyocytes labeled with dye i were detected in the tissue constructs three days post-implantation.

Alternatively, species-specific antibodies can be used to identify implanted cells in xenotransplanation studies. For example, human-induced pluripotent stem cells implanted in immuno-compromised rats could be identified in the tissue harvested from the chamber. Once mastered, the flow-through chamber and the arteriovenous loop can be implanted in approximately 20 and 90 minutes, respectively.

While attempting this procedure, it's important to maintain sterility at all times and take the necessary measures should contamination occur. Don't forget that gentle handling of the blood vessels and atraumatic micro-surgical anastomosis are key to ensure success. In this sense, we do recommend basic micro-surgical training before attempting this procedure, particularly for the construction of the arteriovenous loop.