Nunc 156367

AM were isolated by repeated flushing of lung resection tissue with PBS using a 19G needle. The collected flush was pelleted by centrifugation 300g, 5 min. AM pellets were resuspended in Xvivo10 (Lonza #BE04-743Q) supplemented with 2 mM l-glutamine, 1× penicillin–streptomycin, 1.25 μg/ml Amphotericin B (all from Gibco, Life technologies) and seeded at a density of 1 × 10⁶ total cells/ml in tissue culture treated 25 cm² flasks (Nunc #156367, Thermo Scientific). The macrophage fraction was purified by attachment to cell culture plastics for 1 h, followed by three repeated washes with 5 ml Xvivo10 media. Cultures were rested, or treated with 50 nM Dexamethasone or DMSO, overnight before stimulation with 100 ng/ml LPS for 2 h (RNA isolation), 6 h (measurement of soluble TNFα in media) or 24 h (conditioned media) at 37 °C and 5% CO₂. Cell viability prior to seeding were 92% (90–96), median (range). The purity of the macrophage culture after purification by attachment was monitored by differential staining during the setup of the method (> 95%, n = 3). During the study, the purity of the washed cultures was monitored by visual inspection of cell morphology.

Human osteosarcoma cells (MG-63) obtained from Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan) are used as the model cell line in the experiments. The growth medium is composed of Minimum Essential Medium (MEM) (Gibco 41090, Invitrogen Co., Carlsbad, CA) with 10% (v/v) fetal bovine serum (FBS) (Heat Inactivated FBS, Gibco 10082, Invitrogen), 1% (v/v) antibiotic-antimitotic (Gibco 15240, Invitrogen), 1% (v/v) sodium pyruvate (Gibco 11360, Invitrogen) and 1% (v/v) non-essential amino acids (Gibco 11140, Invitrogen). The stocks are maintained in T-75 cell culture flasks (Nunc 156367, Thermo Scientific Inc., USA) in a 37°C humidified incubator with 5% CO₂, TrypLE^™ Express Enzyme (1X) (Gibco 12604, Invitrogen) is used to dissociate and subculture the cells. In the experiments, the 3D cell spheroid culture and conventional monolayer cell culture models are established to study their responses under different cellular stresses as shown in Fig 2.

To demonstrate the device’s capabilities for tumor spheroid formation and oxygen consumption analysis, human osteosarcoma cells (MG-63) obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) are utilized for the cell experiments in this study. The stocks of MG-63 cells are cultured in growth medium composed of Minimum Essential Media (MEM) (Gibco 41090-036, Invitrogen Co., Carlsbad, CA, USA) with 10% v/v heat-inactivated fetal bovine serum (Gibco 10082, Invitrogen), 1% Antibiotic-Antimycotic (Gibco 15240, Invitrogen), 1% sodium pyruvate (Gibco 11360, Invitrogen), and 1% non-essential amino acids (Gibco 11140, Invitrogen). The cells are maintained under 5% CO₂ in T25 cell culture flasks (Nunc 156367, Thermo Scientific Inc., Rochester, NY, USA), and passaged by dissociation with 0.25% trypsin–EDTA (Gibco 25200, Invitrogen) every three days. Cell suspensions for the experiments are made by centrifugation of the dissociated cells at 1000 rpm (209× g) for 5 min at room temperature. The culture medium is changed every other day during the experiments.