Transepithelial electrical resistance (TER) was measured by an epithelial volt/ohm meter (World Precision Instruments, EVOM2 300523) with colon cancer cell lines and healthy human colonoid-derived cells grown as monolayers on Transwell permeable supports (Costar, 3470). To grow colonoid-derived cells on the Transwell cell culture insert, established colonoids were dislodged and resuspended in TrypLE (Invitrogen, 12605010) for 5–10 minutes in a 37oC water bath to disrupt colonoid formation and generate single cells. The cell suspension was supplemented with 10% v/v of FBS and pipetted using a 100 μL pipette tip by gentle strokes of approximately 50 times. Cells were counted using an automated Corning cell counter. Next, 100 μL of single-cell suspension containing 2 × 105 cells in growth medium were plated on 0.4 μm pore, 12-mm diameter polycarbonate membrane inserts (Corning, 3413) coated with 33.3 μg/mL human collagen IV (Millipore, C954D93). After 24 hours, the lower chamber medium was replaced with differentiation medium (growth medium supplemented with 2 μg/mL hBMP4 [MilliporeSigma]) and the upper chamber medium was supplemented with 100 μL of differentiation medium. Fresh media were replaced every 48 hours. Cells were maintained at 37°C, 5% CO2. TER was evaluated when filter-grown monolayers reached full resistance (at least 800 Ω-cm2 of TER).

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