Why is mucosal separation critical in scaffold preparation?

Mucosal separation isolates the epithelial layer from submucosal tissue, enabling decellularization of a defined mucosal scaffold while preserving the underlying extracellular matrix architecture for tissue engineering applications.

How does hypertonic saline treatment contribute to decellularization?

Hypertonic saline induces cellular dehydration and shrinkage, facilitating detachment of cells from the extracellular matrix and enhancing the efficiency of subsequent decellularization steps.

What quantitative measurements confirm successful decellularization?

Successful decellularization is indicated by visible epithelial detachment, absence of cellular debris after washing, and preservation of structural integrity and biochemical composition of the scaffold.

Why are replication requirements important for scaffold validation?

Replication ensures consistent scaffold quality across preparations, supporting reliable data generation for cross-functional teams evaluating tissue-engineered constructs in preclinical models.

What statistical analysis is needed before implementing this scaffold in screening workflows?

Basic comparative analysis of scaffold integrity, decellularization efficiency, and storage stability is required to establish reproducibility and suitability for high-throughput screening applications.

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

JoVE Encyclopedia of Experiments

Cancer Research

0 views • 4:33 min • April 30th, 2023

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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.

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

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