Cardiopulmonary Complex Decellularization: A Technique for Decellularizing Heart and Lungs from Murine Model
Transcrição
Begin by shaving the thorax, abdomen, and back of the euthanized mouse with the hair clipper. Disinfect the area with 70% ethanol. Then, pin the mouse to a polystyrene tray, extending its fore- and hindlimbs, as well as its head and tail. Place it under the microsurgery microscope.
Using a Mayo straight pattern scissors, make a cutaneous incision running from the submandibular region to the lower abdomen and dissect subcutaneously to expose the thoracic wall and peritoneum. Then, use microsurgical scissors to cut the pectoralis major and pectoralis minor muscles along the sixth intercostal space on both sides of the thoracic wall.
Cut the sternum along the previous incisions with straight pattern scissors. Then, complete a sternotomy by cutting the sternum along its long axis. Elevate and pin both sides of the thoracic wall to expose the cardiopulmonary complex. Use round-tipped micro-forceps to excise the thymus and surrounding adipose tissue by delicately pulling them off their attachments and cut the esophagus with micro-scissors.
Separate the brachiocephalic veins and brachiocephalic artery with sharp micro-forceps. Then, separate the left common carotid and left subclavian arteries from the underlying tissue to facilitate ligation and cauterization. Use a micro-needle holder and sharp micro-forceps and a 9-0 suture to place stitches above the emergence of the brachiocephalic left common carotid and left subclavian arteries. Cauterize the brachiocephalic veins.
Using micro-scissors, open an entrance by sectioning the ligament. Introduce a 27-gauge catheter into the trachea and delicately push until the trachea branches into the bronchi, taking care to not disrupt the bronchi. Using a 6-0 suture, place three stitches around the trachea to secure the catheter.
Section the mouse at the height of the 12th thoracic vertebra. The descending aorta runs anteriorly to the spine and should be sectioned here along with the spine. Retrogradely catheterize the aorta and push the catheter until it reaches the aortic arch. Using a suture, place four stitches around the aorta beginning 5 millimeters below the catheter tip.
Connect the mouse to a pump system using silicone tubing and lower connectors. Perfuse with deionized water at 200 microliters per minute for 15 minutes. Then, change the perfusion agent to 0.5% DOC diluted in deionized water and perfuse overnight. On the next day, change the perfusion agent to 0.1% SDS diluted in deionized water and perfuse for 8 hours. Then, perfuse with deionized water for 24 hours to wash away the SDS and DOC.
Resect the decellularized heart and lungs by sectioning its attachments to the thorax. Store the tissue in a sterile cryotube in deionized water with 1% penicillin-streptomycin and 0.3 micromolar sodium azide at 4 degrees Celsius.
