2.3 Cell culture
HepG2 (JCRB1054, JCRB Cell Bank, Japan) was used as a liver model cell for metabolic functions, whereas A549 (RCB0098, RIKEN BRC, Japan) was used as a cancer model cell. The cells were cultured at 37 ˚C in an incubator in a humidified atmosphere containing 5% CO2. Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS, Bio West, Japan), 1% non-essential amino acid solution (11140–050, Thermo Fisher, USA), and 1% antibiotic antimycotic solution (161–23181, FUJIFILM Wako, Japan) was used as culture media for both cells.
Cell inoculation and cell culturing were performed within the MO–MPS according to the following procedure. The cell culture chambers in the MO–MPS were coated with collagen type I-P (634-00663, Nitta Gelatin Inc., Japan) as an extracellular matrix (ECM). The collagen was chemically bonded to PDMS through 3-aminopropyltriethoxysilane (aminosilane, KBE-903, Shin-Etsu Chemical Co., Ltd., Japan) and glutaraldehyde (GAD, 17026-32, KANTO CHEMICAL CO., INC., Japan) that were coated to the bottom surface of the cell culture chambers.(Chuah et al., (2015)) After the permanent bonding of PDMS chips, the cell culture chambers were coated with aminosilane by immediately removing the aminosilane after its introduction and allowing to stand at 54 ℃ for 2 h. Then, 2.5% glutaraldehyde was introduced to the microchannel and allowed to stand at room temperature for 1 h. Finally, the microchannel was filled with 0.1 mg/mL collagen type I-P and allowed to stand at 4 ℃ for 12 h. The microchannel was washed by ultra-pure water after coating with aminosilane and glutaraldehyde and by phosphate-buffered saline (PBS) after collagen coating. HepG2 cells were seeded into the liver part chamber at 1.7–2.0 ×105cells/cm2. After prior culture for 48 h, the A549 cells were seeded into the lung cancer part chamber at 1.7–2.0×104 cells/cm2 and cultured for 24 h. The culture medium in the MO–MPS was exchanged daily.