Hpa001636

ALI were harvested on day 14, then fixed in formalin, paraffin-embedded and serially sectioned. Slides were deparaffinized in xylene and rehydrated thru a series of ethanol washes. Histology slides were stained in hematoxylin, rinsed, then stained in eosin (Azer Scientific) on a Shandon Gemini automated stainer (ThermoFisher). Immunohistochemistry slides were incubated with E1 (Leica Biosystems) immunohistochemistry antigen retrieval solution for 20min. Claudin-1 (1:50; LS Bio LS-C415827; 1hour primary incubation) and ZO-1 (1:50; Sigma HPA001636; 1hour primary incubation) antibodies were used for staining with Bond Refine polymer staining kit on the Bond-Max automated staining system (Leica Biosystems).
All stained slides were dehydrated in ascending ethanol and xylene washes before coverslipping with cytoseal (Fisher Scientific, USA). Stained slides were digitally scanned at 20x magnification on an Aperio CS-O slide scanner (Leica Biosystems). ALI membrane thickness, number of basolateral nuclei per 100 μm, percent dilated intracellular space and percent strong 3,3′-Diaminobenzidine (DAB) staining intensity were calculated using Aperio imaging software algorithms.

2D co-culture with human intestinal fibroblasts was required to develop tight-junctions (determined with anti-TJP1, Sigma #HPA001636) in hIECs. On Day 0, a polycarbonate membrane (Whatman #111707) that was adhering to two silicone rings was coated with 50 μg/ml collagen-I for 1 hour and rinsed 3 times with PBS. Then 1 × 10⁴ human colon epithelial cells were seeded onto the polycarbonate membrane (5.0 × 10³ cells/cm²) and cultured with hIEC medium at 5% CO₂ and 37°C for 4–5 days in the insert portion of transwell plates (VWR, PA #3412), then hIEC medium was added, and the cells were allowed to grow for 6 days. The medium was refreshed every other day. On Day 6, 2–3 × 10⁴ human colonic fibroblasts were then seeded in the bottom wells (1.0 × 10⁴ cells/cm²) where hIEC cells were growing on the membrane in the insert portion of the transwells. The transwell plates were maintained in a 50:50 mixture of hIEC medium and fibroblast medium in a humidified incubator at 5% CO₂ and 37°C. These transwell plates were fed every other day for 14 more days until assembled into the device on Day 20 or for 22 more days until used in a drug permeability study on Day 28.