Tgfβ inhibitor a83 01

PDX-derived primary ESCC cells were prepared as described previously.^{52 (link),53 (link)} Briefly, PDX was dissected from mouse and gently minced to small fragments at about 2 mm in diameter. Tissue sample was digested in RPMI-1640 medium (Invitrogen) containing collagenase IV (Gibco) and hyaluronidase (Sigma-Aldrich) for 30 min at 37 °C. Cell suspension was filtered through a 70-μm cell strainer, briefly centrifuged for 5 min and the resultant cell pellet was resuspended in advanced DMEM/F12 (Gibco, 12634010) supplemented with B-27 (17504-044), HEPES (15630080), GlutaMAX™ (35050061), epithelial growth factor (PMG8043), 10 µM Rho-associated coiled-coil-containing kinase inhibitor Y-27632 (Selleck Chemicals, S1049) and 5 µM TGFβ inhibitor A83-01 (Tocris Bioscience, 2939). The cells were kept in a humidified 5% CO₂ incubator at 37 °C.

Colons were extracted from 8-week-old Klf5^ΔIND female mice. Crypt cells were isolated as previously described (27 (link), 55 (link)). All crypts were embedded in Matrigel (Corning). Colonoid culture media was prepared with L-WRN cells (ATCC) as previously described, and TGF-β inhibitor A83-01 (500 nM; Tocris Biosciences) and antibiotic cocktail Primocin (100 μg/mL; Thermo Fisher Scientific) were supplemented. GSK3β inhibitor CHIR99021 (10 μM; Tocris) and ROCK inhibitor Y-27632 (10 μM; MilliporeSigma) were added only on the first 2 days of culture. After the first 2 days, CHIR99021 and Y-27632 were removed, and PBS or 4-OHT (10 μM) was added. For rIL-22 cultures, PBS/4-OHT was added with PBS/rIL-22 (100 ng/mL).