Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The overall goal of this procedure is to produce a 3D tissue engineered model of the human esophageal epithelium for use as an experimental platform. This is accomplished by first isolating normal human esophageal fibroblasts and epithelial cells from human esophageal tissue samples. In the second step, the fibroblasts are seeded onto a de epithelial acellular, pore sign, esophageal scaffold, and cultured for seven days.

Next, the scaffold is inverted and the isolated human esophageal epithelial cells are added to the culture for four days. In the final step, the model is moved to the air liquid interface for a further five days of culture, allowing a mature stratified epithelium to develop. Ultimately, immunohistochemistry is used to characterize key markers of proliferation and differentiation expressed by the model epithelial cells.

The main advantages of this technique over conventional 2D cell culture is that it allows cells to have the cell, cell and cell matrix interactions that they would have in vivo. And using this technique, we can produce a mature stratified squamous epithelium similar to the normal esophagus. Generally, individuals new to this method will struggle for three reasons.

One, it can be a challenge to ensure that the majority of epithelial cells do not differentiate before being included in the scaffold. Two, care must be taken to ensure that the correct air liquid interface is provided during the period when the epithelium is maturing. And three, it can be a challenge to maintain ity throughout the culture period.

To isolate human esophageal epithelial cells, place the harvested esophageal squamous mucosa tissue in a sterile Petri dish and use an ethanol sterilized scalpel to cut the tissue into 0.5 centimeter strips. Then incubate the tissue in 0.1%tryin covered at 37 degrees Celsius, stopping the digestion after an hour with five milliliters of FCS. Next, holding the strips with forceps, gently scrape the epithelial surface with the scalpel blade for one to two minutes per strip to remove the epithelial cells into the surrounding medium when all of the strips have been scraped, transfer the medium containing the detached epithelial cells into a 15 milliliter centrifuge tube.

After spinning down the cells, discard the senna and resuspend the pellet in 12 milliliters of epithelial medium. Then discard the medium from a fetter cell culture and use a sterile pipette to plate the epithelial cells on the feeder cells to isolate the human esophageal fibroblasts. Transfer the scraped tissue strips into a sterile Petri dish, and finally mince the tissue with the scalpel.

Digest the pieces in 10 milliliters of 0.5%Collagenase a solution covered at 37 degrees Celsius and 5%carbon dioxide with a humidified atmosphere overnight. The next day, transfer the digest into a 50 milliliter centrifuge tube and spin down the cells. Resus suspending the palate in 10 milliliters of fibroblast culture Medium.

The culture in a T 75 flask in a cell culture incubator. 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 day debris while the tissue is being rinsed.

Spray a cork dissection board with 70%ethanol when the cork is dry in 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 lanar 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 five 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 one molar sodium chloride solution.

Supplemented with antibiotics at 37 degrees. 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, begin the preparation of the esophageal mucosa model by rehydrating pieces of porcine scaffold that have been sterilized in glycerol for at least four months. In 100 milliliters of sterile PBS for five, 10 minute agitations. Replacing the supinate with fresh PBS for each wash.

After the fifth agitation test the scaffold sterility with an overnight incubation in 100 milliliters of DMEM at 37 degrees Celsius. The next morning, transfer the scaffolds into individual wells of a six well plate submucosal side up. Then place a sterile medical grade stainless steel ring onto the center of each scaffold and use sterile forceps to gently press down on the rings to ensure an adequate seal with the tissue at five times.

Center the five human esophageal fibroblasts in 0.2 milliliters of fibroblasts medium into the center of each ring. Then flood the area around the outside of the ring with at least two more milliliters of fibroblasts medium and incubate the scaffolds in a cell culture incubator. After 24 hours, remove the rings and replace the medium in each well with at least five milliliters of fresh fibroblasts.

Medium, taking care that the scaffolds are totally immersed. After one week, discard the medium and use forceps to invert the scaffolds mucosal surface face up. Place the rings back onto the scaffolds, and then add one time center of the six epithelial cells in 0.2 milliliters of composite medium, one into the center of each ring.

Plug the area outside of the ring with approximately two milliliters of composite medium one and return the constructs to the incubator. After 24 hours, remove the rings and replace the medium in each well with at least five milliliters of fresh composite medium, one, ensuring that the scaffolds are fully submerged. After another 24 hours, replace the medium with at least five milliliters of composite medium.Two.

On day four, place sterile medical grade stainless steel mesh grids into the wells of a new six Well plate and transfer the constructs to the grids mucosal side up. Finally, add enough composite medium three to reach the underside of the composite while leaving the surface exposed to the air, ensuring that the sample is maintained at an air liquid interface. The trickiest part of the procedure is setting up the air liquid interface.

If it's not clear whether the grid is in liquid touch with a sterile to pipe tip, as it's easier to see if the pipee is submerged. Alternatively, you could try rocking the plate gently as it's easier to see liquid when it's moving and check that there is liquid on the grid and not covering your sample. This, the logical assessment of the epithelium presents a mature multilayered stratified squamous epithelium, similar but thinner to that observed with the normal human esophagus with the cells becoming progressively flatter and ultimately a nuclear as they migrate towards the surface.

Immunohistochemical characterization of key markers of proliferation and differentiation demonstrates that the microanatomy of the model epithelium is similar to that of the normal human esophageal epithelium. For example, comparable cytokeratin 14 expression is observed in both the native esophagus and the model epithelium with staining restricted to the cells in the basal layer. This model has been successfully modified to incorporate tumor cells by replacing the primary epithelial cells with carcinoma cells demonstrating the flexibility of the model.

For example, squamous carcinoma cells produce an epithelium visible on the construct as a defined yellow region with large clefts within the epithelium, likely reflecting the presence of dysfunctional cellia molecules, including esophageal adenocarcinoma cells within the model results in a large amount of scaffold degradation visible to the eye as a thinning of the scaffold. After two weeks of growth at the air liquid interface and confirmed in h and e analysis as an obvious reduction in the thickness of the scaffold in the region below the cells Following this procedure, other methods of gene expression like northern or western blotting can be applied to the same tissue to answer questions like, how does the epithelium respond to environmental stresses? So this provides a really valuable resource for looking at more questions in greater depth After its development.

This technique paved the way for researchers interested in Barrett's Metaplasia to study the effect of exposure to specific reflux components on gene expression in the normal human esophagus.