FIG. 3.

Generation of organotypic raft cultures from HEOs. Day 27 HEOs (21 days post embedding) are collected into a conical tube and dissociated to single cell using enzyme-based dissociation and serial passaging through pipette tips and syringe needles and re-cultured as monolayer for 5–6 days for keratinocyte selection. Then cells are re-dissociated and cultured on top of a collagen/mice fibroblasts plugs in trans-well inserts. After 5 days of culture, medium is removed from top compartment to generate air-liquid interface (ALI). Cells continue to proliferate and differentiate for up to 2 weeks to generate esophageal organotypic raft cultures. For complete and detailed protocol and medium composition turn to Section 5.3 in the text.