Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model

A human esophageal mucosa model comprises human fibroblasts and epithelial cells grown on an animal-derived esophageal scaffold.

To prepare a 3D raft model of a human esophagus, begin by taking a sterile esophageal scaffold pre-treated to remove its native cells. Place the scaffold with its submucosal side up in a culture well.

Next, set a stainless-steel ring onto the scaffold. Within the ring, seed a human-fibroblast cell suspension. Subsequently, dispense a fibroblast medium around the ring and incubate.The ring confines the fibroblasts onto the submucosal surface, facilitating their adhesion.

Now, remove the ring, add fresh media to immerse the scaffold, and incubate. Fibroblasts proliferate to form a multilayer cell sheet that secretes essential cofactors.

Next, invert the scaffold, mucosal side up, and place the stainless-steel ring over it. Seed a human-epithelial cell suspension within the ring and add the culture medium. Incubate to allow the confined epithelial cells to adhere to the mucosal surface.

Then, remove the ring and add fresh media. Incubate to diffuse the fibroblast-produced cofactors through the scaffold and promote epithelial cell proliferation.

Now, using a metal grid, raise the scaffold to expose the proliferating epithelial cells to the air-liquid interface. This allows for the complete maturation of the culture into a human-esophageal model.

Begin the preparation of an 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 supernatant with fresh PBS for each wash.

After the fifth agitation, test the scaffold's 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 6-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.

Add 5 times 10 to the fifth human esophageal fibroblasts in 0.2 milliliters of fibroblast medium into the center of each ring. Then, flood the area around the outside of the ring with at least two more milliliters of fibroblast 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 5 milliliters of fresh fibroblast 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 1 times 10 to the sixth epithelial cells in 0.2 milliliters of Composite Medium I into the center of each ring.

Flood the area outside of the ring with approximately 2 milliliters of Composite Medium I and return the constructs to the incubator. After 24 hours, remove the rings and replace the medium in each well with at least 5 milliliters of fresh Composite Medium I, ensuring that the scaffolds are fully submerged. After another 24 hours, replace the medium with at least 5 milliliters of Composite Medium II.

On day four, place sterile medical-grade stainless steel mesh grids into the wells of a new 6-well plate and transfer the constructs to the grids, mucosal side up. Finally, add enough Composite Medium III 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.