We purpose a normal strategy that involves employing a decellularized spleen matrix size and alternative scaffold by artificial levels, which shows a detailed procedure of harvesting red spleen, and preparing decellularized, the spleen matrix that preserves the macro structures, and the components of the extracellular metric. Our protocol provides a readily available spleen metric size, and alternative to scale donor levels. Our protocol present method for the preparation of a decellularized spleen metric is demonstrating efficiency, stability, and minimal invasiveness.
This method maintains the spleen's inherent architecture composition, and the natural vascular network. It offers the scaffold, full cell implantation, and a three dimensional dynamic culture, thereby a establishing a basis for advancing investigations in tissue engineered liver. To begin, place an anesthetized shaved rat in a supine position on a surgical table.
Inject two milliliter of heparinized saline into the penile dorsal vein for systemic anticoagulation. Disinfect the shaved abdominal skin with povidone iodine solution, and cover with a sterile draping cloth. With a pair of surgical scissors, make a cruciform incision in the abdomen.
Stretching the abdominal cavity with the hemostatic forceps, flip the liver towards the diaphragm. Exteriorize the gastrointestinal tract to the right side of the abdomen, and cover it with moist gauze. Carefully separate and cut the splenogastric ligament to expose the splenic hilum.
Gradually dissect along the splenic hilum to separate and expose the common hepatic artery, gastroduodenal artery, and splenic artery. Ligate and cut the gastroduodenal artery and common hepatic artery, while progressively dissociating the surrounding tissue. Flip the spleen towards the right side to expose the abdominal aorta.
Gently perform blunt dissection with cotton swabs to expose the abdominal aorta and celiac trunk. Place a three zero silk suture approximately three centimeters in length, above and below the branches of the celiac trunk. Then, place a 10 centimeter long six zero silk suture at the branch of the celiac trunk.
Ligate the abdominal aorta below and above the branches of the celiac trunk. Then, make a small incision in the arterial branch. Gently lift the six zero silk suture, and insert a 24 gauge venous catheter into the splenic artery along the celiac trunk.
Ligate the catheter to secure it in place. Use a syringe pump to infuse 50 milliliters of heparinized normal saline, at a rate of four milliliters per minute. Simultaneously, sever the portal vein as an outflow channel, to allow the infused fluid to flow out of the spleen.
Carefully dissect the surrounding tissue of the spleen, avoiding damage to the pancreas, while preserving the major accessory vessels. Check for any leakage around the spleen. Then, remove the spleen and pancreas, and rinse them in normal saline.
Transfer the spleen into a 50 milliliter centrifuge tube filled with normal saline. And store it in a minus 80 degrees Celsius freezer. To begin, freeze and thaw a frozen rat spleen three times to lyce the spleen cells.
On a clean bench, set up a peristaltic pump, a two liter reservoir, a silicone tube with an inner diameter of 2.4 millimeters, and a bubble trap. Fill the perfusion system with deionized water. Keep it running for 10 minutes.
Carefully transfer the harvested spleen to the deionized water filled container. Next, connect the silicone tube to the venous catheter that has been inserted into the splenic artery. Perform profusion with deionized water, 0.1%SDS, 1%Triton X-100, and PBS solutions.
Fill a 50 milliliter centrifuge tube with PBS, containing 10%penicillin streptomycin. Transfer the decellularized spleen matrix into the PBS. Store it at minus 20 degrees Celsius until further experimentation.
The complete decellularization of the spleen was achieved in approximately 11 hours. The color gradually transitioned from deep red, to a modeled light red, and eventually a white translucent appearance, with overall morphology remaining intact.
