Transwell cell culture inserts

Transwell^® cell culture inserts (6.5 mm, 3.0 μm pore size, VWR) were coated with 100 μL of 20 μg/mL collagen type I (rat tail, Ibidi) in 0.01% (v/v) acetic acid for 1 h at room temperature (RT), and washed with PBS before use. Human fetal organoids were expanded for one passage and subsequently collected at day 3. Single cell suspension was obtained by treatment with TryplE™ (Gibco, Thermo Fischer Scientific) for 10 min at 37°C. Cells were diluted to 1·10⁶ cells/mL and 100 μL of cell suspension per insert was seeded (=1·10⁵ cells per insert) unless stated otherwise.
Cells were cultured in 100 μL apical and 600 μL basolateral HIOGM for the first week, containing 10 μM Y-27632 (Stemcell™ Technologies) for the first 3 days. After 7 days, to promote cell differentiation as described previously (Noel et al., 2017 (link)), medium was replaced by a 1:1 mixture, referred hereon as HIOGM-diff, of HIOGM component A and advanced DMEM/F12 supplemented with 100 U-mg/mL penicillin-streptomycin, 7.5 mM HEPES and 0.5x Glutamax, which was refreshed every 3-4 days.

The fetal intestinal epithelial monolayers were cultured on Transwell cell culture inserts (6.5 mm, 3.0 μm pore size; VWR) as described previously (Roodsant et al, 2020 (link)). Briefly, the inserts were coated with 100 μl of 20 μg/ml collagen type I (rat tail, Ibidi) in 0.01% (vol/vol) acetic acid (Sigma-Aldrich) for 1 h at RT, and washed with PBS (Lonza) before use. Human fetal enteroids were collected, and a single cell suspension was obtained by treatment with TrypLE (Gibco, Thermo Fisher Scientific) for 10 min at 37°C. Cells were diluted to 10⁶ cells/ml and 100 μl of cell suspension per insert was seeded (10⁵ cells per insert). The cells were then cultured and differentiated over a period of 14 d as described previously (Roodsant et al, 2020 (link)).