Primary human umbilical vein endothelial cells huvecs

Human intestinal epithelial Caco2 cells from ATCC (New York, United States) were cultured in Dulbecco’s modified Eagle’s medium containing 4.5 g*L⁻¹ glucose medium (DMEM/HG) supplemented with 10% (w/v) fetal bovine serum (FBS). Primary human umbilical vein endothelial cells (HUVECs) (Lonza, Basel, Switzerland) were cultured in ECM medium (DMEM/F12) containing 5% (w/v) FBS. Primary human macrophage cells (OriCells, Shenyang, China) were cultured in RPMI 1640 medium containing 10% (w/v) FBS. Penicillin (100 units/ml) and streptomycin (100 μg/ml) were added to all aforementioned media. All cells were cultured in a cell incubator with 5% CO₂ at 37°C. Antibiotics were removed from the culture media for coculture of human cells with living intestinal microbes. All cell culture media were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Caco2 cells were used in generations 2–10, and HUVECs were used in generations 2–6 and macrophages were used in the first passage.

The human bladder carcinoma cell lines, 5637 and T24, were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO₂ humidified incubator. The 5637 cells were referred to as MGH-U1 cells. Additionally, primary human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Walkersville, MD, USA). Cells were grown on plates coated with 0.1% gelatin (Sigma, San Diego, CA, USA) in endothelial basic medium (EBM) and cultured in endothelial growth medium-2 (EGM^TM 2) Bulletkit^TM (Lonza) at 37 °C in a 5% CO₂ humidified incubator. All experiments were performed between passages 2 and 5.