2.2. Cell preparation and culture
We used human gut epithelial cell line (Caco-2; ATCC, USA, HTB-37), murine brain endothelial cell line (bEnd.3; ATCC, USA, CRL-2299), and primary human brain microvascular endothelial cells (hBMECs; Cell Systems, USA, ACBRI 376). The two cell lines were cultured using Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA, 11965118) supplemented with 10% fetal bovine serum (FBS; ScienCell, USA, 0500) and 1% Penicillin/Streptomycin (P/S; ScienCell, USA, 0513). The hBEMCs were cultured using endothelial cell medium (ECM; ScienCell, USA, 1001) supplemented with 5% FBS, 1% P/S and 1% endothelial cell growth supplement (ECGS; ScienCell, USA, 1052). All cell types were incubated in humidified atmosphere of 5% CO2 at 37 ℃ during flask culture and chip experiment. Cell culture media were changed every 2-3 days.