Hi, I am Zu from the laboratory of Douglas Cowan in the Department of Anesthesiology at Children's Hospital and Harvard Medical School. Today we will show you a procedure for implanting engineered tissue in the red heart. We use this procedure in our laboratory to create an alternative atrial ventricular electrical conduction pathway.
So let's get started. This video describes a surgical implantation of a myoblast containing engineered tissue construct. This proof of principle experimental protocol provides evidence that tissue casts can be fabricated in vitro and transplanted in vivo into recipients requiring reparation of a non-functional organ.
And accompanying video describes how cells were cast into the mold that fits the AV groove of the heart of an adult rat. The protocol begins by anesthetizing a 200 to 250 gram Lewis rat with a ketamine and aine cocktail injected through a 30 gauge needle into the intraperitoneal cavity. Once the rat is fully anesthetized and does not respond to a foot pinch, prepare for the surgical procedure by clipping off the hair of the upper abdomen and right chest.
Then the incision side is thoroughly cleansed with 70%ethanol, followed by a povidone iodine, topical antiseptic solution. Using a 16 gauge intravenous catheter, the rat is intubated and Dora lead and connected to a small animal respirator. The respirator is connected to a vaporizer set to supply 0.5 to 1%ISO fluorine mixed with 100%oxygen to maintain general anesthesia.
The respiration rate and tidal volume is adjusted according to the body weight of the rat. To maintain body temperature, the intubated rat is kept on a heating pad During the surgical procedure as a final precaution to monitor heart rate and oxygen saturation throughout the surgical procedure, a pulse oximeter device is clipped onto one of the back limbs. The surgical site is prepared with sterile surgical draping that is held in place with towel clamps.
This surgery begins with an incision from the anterior lateral to the lateral wall of the right chest, just above the fifth intercostal space. The interior of the chest is accessed through the ribs. In a small retractor is position to maximize the view.
The pericardium is cut just above the appendix of the right atrium and right ventricle from the base toward the apex of the heart. Afterward, the AV groove is identified in the epicardial layer adjacent to the aorta. Atrial ventricular triangle is gently teased apart.
A seven oh prolene suture is placed through the basal shoulder of the right ventricle from the engineered tissue, dissect out a two millimeter cube of tissue. The two millimeter tissue cube is positioned within the AV groove and gently tied down with the proline suture Before the rat is closed up. Any air in the space around the lungs is removed by expanding the lungs with a ventilator.
The surgical field is checked for bleeding and two four oh Vicryl sutures are placed between the upper and lower intercostal space for closure of the ribcage. Finally, to close the chest, the muscular plane is brought together with a running suture and the skin is closed. When an intra cutaneous placed running suture while the animal recovers from the surgery, the incision side is infiltrated with five milliliters, a 0.25%bupivocaine.
The animal is extubated following weaning from the respirator and is returned to a cage with fresh bedding. Once it is fully awake and ambulatory, the rat is subsequently administered 0.01 milligram per milliliter of buprenorphine and analgesia subcutaneously every 12 hours for the next three days if performed properly. The described surgical implantation of engineered tissue in the AV groove of the heart results in greater than 90%recovery of the animals.
Subsequent experiments have shown that donor cells from implanted tissue functionally integrate into the host myocardium. We've just shown you how to implant an engineered tissue construct into our red heart. When doing this procedure, it is important to eliminate any pneumothorax to allow the lungs to fully expand and to reduce atelectasis.
So that's it. Thanks for watching and good luck with your experiments.I.
