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.