This article discusses the preparation of decellularized scaffolds from porcine esophagus, which mimic the native extracellular microenvironment. These scaffolds are promising for tissue engineering applications.
Decellularized scaffolds derived from the extracellular matrix of specific tissue mimic their native extracellular microenvironment. These scaffolds retain their structural integrity and biochemical composition and serve as promising platforms for tissue engineering.
To prepare a decellularized porcine esophageal scaffold, begin with a freshly excised porcine esophagus. Bisect the esophagus longitudinally and rinse it with a suitable buffer to remove any debris. Next, secure the esophagus with its mucosal surface, the innermost layer, facing up.
Separate the mucosa from the underlying submucosa at one end of the esophagus. Dissect the esophagus longitudinally along the loose connective tissue of the mucosa called the lamina propria to fully separate the mucosal surface from the underlying submucosa.
Further, excise the proximal and distal regions of the mucosa, which contain a higher density of submucosal glands and a thicker lamina propria layer. Cut the mucosal tissue into uniformly sized pieces.
Treat the tissue with a hypertonic saline solution supplemented with antibiotics. Antibiotics help prevent bacterial growth. At high solute concentration, water moves out from the cells and causes their shrinkage. This step further aids in cellular death and detachment from the extracellular matrix.
Now, gently detach the loosened epithelium from the underlying tissue. Rinse the tissue with buffer to remove cellular debris. Finally, incubate the tissue in glycerol, which further facilitates tissue decellularization by dehydrating and lysing cells while preserving the integrity of the basement membrane.
To prepare the decellularized pig esophageal scaffolds, begin by using scissors and a pair of tweezers to cut open a pig esophagus longitudinally. Next, wash the esophagus in a 180-milliliter sterile pot containing 100 milliliters of sterile PBS for 10 minutes with gentle shaking to remove any debris.
While the tissue is being rinsed, spray a cork dissection board with 70% ethanol. When the cork is dry, pin the esophagus onto the board with the mucosal side up. Now, use sterile tweezers to grasp the mucosal surface at one end of the esophagus and pull the tissue away from the board to separate the mucosa from the underlying submucosa.
Then, using a sterile scalpel blade, dissect the esophagus longitudinally along the lamina propria, removing the pins as the dissection progresses to allow the mucosa to be lifted away. Discard the underlying submucosa, and use a scalpel to cut the remaining mucosa into 5-centimeter square pieces. Wash the pieces in 150 milliliters of sterile PBS with gentle agitation as just demonstrated.
After five minutes, transfer the tissue into a 180-milliliter pot containing 150 milliliters of 1 molar sodium chloride solution supplemented with antibiotics at 37 degrees Celsius. After 72 hours, transfer the tissue into 150 milliliters of sterile PBS, at which point the epithelium will have begun to visibly separate from the underlying tissue.
Use sterile forceps and a scalpel blade to remove the detaching epithelium, and place the remaining tissue in a fresh pot of 150 milliliters of sterile PBS with three five-minute washes with gentle agitation. The tissue must now be stored in glycerol for a minimum of four months to fully sterilize the scaffold.
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