T25 culture flasks

Take out the 10-day-old blue peacock embryos (Ruyi Ecological Agriculture Co., Ltd., Anhui, China) under a sterile operating table, remove the heads, the limbs, and the bones, wash the remaining tissue with PBS 3 times, cut into 1 cm³ tissue, and digest with 0.25% Trypsin-EDTA (25200072, Gibco, Carlsbad, CA) at 37°C under constant temperature shaker. After 15 min, terminate digestion and centrifugation at 1,000 g for 5 min; the supernatant was discarded, the DMEM supplemented with 10% FBS was pipetted to precipitate, and the peacock primary fibroblasts were inoculated into T25 culture flasks (NEST Biotechnology, Wuxi, China) and cells were incubated at 37°C in a 5% CO₂ incubator.

Human ESCC cell line KYSE-150 and primary human LECs were purchased from Procell (Wuhan, China). T25 culture flasks (707001, NEST) were coated with 0.1 mg/ml poly-L-lysine (PB180522, Procell) at 37°C for one hour. Human LEC were then seeded at a density of 0.5 million cells per flask. KYSE-150 was cultured with 10% FBS RPMI/F12 medium (32160801, Sigma-Aldrich) and human LECs were cultured with ECM medium (CM-H026, Procell) in a humidified incubator at 5% CO₂ and 37°C.