Decellularized Porcine Esophageal Scaffold Preparation: A Technique to Generate Acellular Scaffold from a Pig Esophagus

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Decellularized Esophageal Scaffold

1. Obtain intact porcine esophagi at an abattoir from freshly slaughtered Landrace pigs that are used for food production. One single esophagus will provide sufficient scaffolds for approximately 30 separate constructs.
   NOTE: Due to the extensive and time-consuming sterilization protocol required, it is normally worth obtaining and processing a minimum of 10 esophagi at one time.
2. Immediately place the freshly excised esophagus into a 180 mL sterile pot containing approximately 150 mL of 10% povidone-iodine solution for 5 min to reduce any contaminating microbial load. Transfer the esophagus to a second pot containing approximately 100 mL of sterile PBS containing 200 IU/mL penicillin, 200 µg/mL streptomycin, and 1.25 µg/mL amphotericin B.
   1. Replace the lid on the container and transport the esophagi back to the laboratory at room temperature. Two or three esophagi can be transported in each container, depending upon their size.
3. Once at the laboratory, handle the tissue in a laminar flow hood using aseptic technique to minimize microbial contamination. Sterilize tweezers, scissors, and scalpel blades by soaking for 5 min in 70% ethanol and rinsing in sterile PBS.
4. Handle the esophagus using sterile tweezers. Using the scissors, cut open the esophagus longitudinally. Rinse the tissue by placing the esophagus into a 180 mL sterile pot containing approximately 100 mL of sterile PBS and shaking gently to remove any debris.
5. Clean a cork dissection board by spraying liberally with 70% ethanol and allow to dry.
6. Remove the esophagus from the PBS, and using sterile needles, pin out the esophagus onto the board, mucosal surface uppermost. Grasp the mucosal surface at one end of the esophagus with sterile tweezers and pull away from the board. This will separate the mucosa from the underlying submucosa.
   1. Then, use a sterile scalpel blade to dissect the esophagus longitudinally along the lamina propria, removing pins as the dissection progresses to allow the mucosa to be lifted away.
7. Retain the dissected mucosa and discard the underlying submucosa.
8. Cut off and discard the most proximal and distal 2 cm of the mucosa as there is a greater number of submucosal glands and a thicker lamina propria in those areas, respectively.
9. Use a scalpel to cut the remaining tissue into 5 cm². Place all the cut tissue into a 180 mL sterile pot containing approximately 150 mL of sterile PBS and agitate gently to rinse the tissue. If processing more than five esophagi at one time, it may be necessary to use multiple pots for this and the three subsequent steps.
10. Using sterile tweezers, transfer the cut tissue into a 180 mL pot containing 150 mL of sterile 1 M NaCl, 200 IU/mL penicillin, 200 µg/mL streptomycin, and 1.25 µg/mL amphotericin B. Incubate for 72 h at 37 °C.
11. Place the tissue into a fresh 180 mL pot containing 150 mL sterile PBS. The epithelium will have begun to visibly separate from the underlying tissue. Gently peel the detaching epithelium from the pig esophagus using sterile forceps and discard.
    1. Place the remaining tissue into a fresh 180 mL pot and add 150 mL of sterile PBS. Agitate gently for 5 min to wash. Pour off the PBS and repeat a further two times with fresh PBS.
12. Place all the tissue into a 500 mL glass bottle containing 400 mL of sterile 80% (v/v) glycerol solution for 24 h at room temperature. Transfer to 400 mL of sterile 90% (v/v) glycerol solution for a further 24 h.
13. Store in 400 mL of sterile 100% glycerol solution at room temperature for a minimum of 4 months to dehydrate and sterilize the tissue while preserving the integrity of the basement membrane.