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

1. Production of the Human Esophageal Mucosa Model

1. Perform all work in a laminar flow hood using aseptic technique to maintain a sterile environment. Sterilize all tools by soaking in 70% ethanol for 5 min, before rinsing in sterile PBS.
2. The day before it is required, rehydrate the acellular porcine esophageal scaffold. One piece of scaffold is required for each construct to be prepared.
   1. To rehydrate the tissue, place sufficient pieces of porcine scaffold into a pot containing 100 mL of sterile PBS, agitate and soak for 10 min. Decant the PBS and replace it with fresh PBS. Repeat the washing process five times to ensure removal of all glycerol traces.
3. Test the scaffold sterility by incubating the rehydrated tissue overnight in 100 mL of DMEM at 37 °C. If there is no change to the color or turbidity of the culture medium at the end of the incubation period, the scaffold is assumed to be sterile. Once sterility has been confirmed, place the 5 cm² acellular scaffold into a 6-well plate, submucosal side uppermost.
4. Place a sterile medical grade stainless steel ring (internal diameter 10 mm, external diameter 20 mm) onto the center of each scaffold and gently press down using sterile forceps to ensure an adequate seal with the tissue.
5. Harvest the fibroblasts using trypsin-EDTA to produce a cell suspension. Count the cells using a hemocytometer. Centrifuge the cell suspension (10 min, 200 x g). Resuspend the cell pellet into an appropriate volume of fibroblast medium to produce a final cell count of 2.5 x 10⁶ cells per mL.
6. Add 0.2 mL of fibroblast medium, containing 5 x 10⁵ human esophageal fibroblasts, within each ring. Flood the area around the outside of the ring with approximately 2 mL of fibroblast medium. Incubate at 37 °C in a 5% CO₂, humidified atmosphere.
7. After 24 h, remove the rings and fibroblast medium and add at least 5 mL of fresh fibroblast medium to each well, ensuring that the scaffold is totally immersed in the medium. Culture for 1 week at 37 °C in a 5% CO₂, humidified atmosphere, replacing the medium every 2 to 3 days.
8. After 1 week remove the 6-well plates containing the scaffolds to a laminar flow hood, remove the medium and invert each scaffold using sterile forceps, placing the mucosal surface uppermost. This time point is referred to as Day 0.
9. Place the steel rings on the mucosal surface in the center of the tissue.
10. Prepare the T75 flasks of epithelial cells for trypsinization. After the PBS wash and prior to the addition of trypsin-EDTA solution, add 2 mL of sterile 0.02% (w/v) EDTA solution to each flask. Incubate for 2 min at 37 °C. Tap the flask gently to selectively detach the i3T3 feeder layer while leaving the epithelial cells attached.
    NOTE: It is generally not practical or necessary to patient match the epithelial cells to the fibroblasts already incorporated in the model the previous week.
11. Pour off the EDTA solution containing the feeder layer and add trypsin-EDTA solution to harvest the epithelial cells. Count the cells using a hemocytometer. Centrifuge the cell suspension (10 min, 200 x g). Resuspend the cell pellet into an appropriate volume of Composite Medium 1 to produce a final cell count of 5 x 10⁶ cells per mL.
12. Add 0.2 mL of Composite Medium I, containing 1 x 10⁶ epithelial cells, within the ring. Flood the area around the outside of the ring with approximately 2 mL of Composite Medium I and place the constructs into the incubator at 37 °C in a 5% CO₂, humidified atmosphere.
13. After 24 h (Day 1), remove the rings and medium and add at least 5 mL of fresh Composite Medium I, ensuring the scaffolds are fully submerged and return to the incubator. After further 24 h (Day 2), remove the medium and replace it with at least 5 mL of Composite Medium II, again ensuring the scaffolds are fully submerged and return to the incubator.
14. On Day 4, place sterile medical grade stainless steel mesh grids (2 cm wide x 2 cm long x 0.5 cm high) into fresh 6 well plates, one per construct. Use sterile tweezers to transfer the constructs onto the top of the steel grids, mucosal surface uppermost.
    1. Add sufficient Composite Medium III so that the liquid reaches the underside of the composite, but the surface is exposed to the air, this ensures that the sample is maintained at an air-liquid interface. The exact volume of medium required will vary depending upon the thickness of the scaffold, the volume of the well, and the precise height of the stainless steel grid.
15. Maintain the composites at an air-liquid interface for between 10 and 20 days (Day 14 to Day 24), depending upon the requirements of the experiment. Replace the Composite Medium III with fresh medium every 2 to 3 days (Monday, Wednesday, Friday is a user-friendly regime).
    NOTE: After 5 days, at the air-liquid interface (Day 9), the constructs have a sufficiently mature esophageal epithelium to be used in experiments of the impact of environmental factors on the esophageal epithelium.
16. At the end of the experiment, fix the constructs for histological analysis and immunohistochemical staining, or extract protein or RNA from the epithelium for further analysis. If required, retain the conditioned culture medium for analysis of extracellular signaling molecules.