Napyruvate

Materials were obtained from the following suppliers: female rat non-cancerous type-II alveolar cells (CC-149, L2 cells) and fetal bovine serum (FBS), ATCC; low glucose DMEM, Hyclone. Phenol red-free low glucose DMEM, 17β-estradiol (E2) and 3,3′, 5-triiodo-L-thyronine sodium salt (T3), Sigma-Aldrich; charcoal-stripped FBS and 100X antibiotic-antimycotic, Invitrogen; Trypsin-EDTA .05%, VWR; Na-pyruvate, Cellgro; Hanks buffered saline solution (HBSS), Lonza; MTT, PGE2, and Apotox-Glo Triplex assay kits from Roche, Cayman Chemical, and Promega, respectively. Promega generously provided GSH/GSSG-Glo assay prior to public release.

MPM tumor specimens, MSTO-211H or LP9 cells were used to prepare MPM spheroids analogous to previous preparations [40 (link)] In brief, fresh MPM tumor specimens were minced in Dulbecco’s Modified Eagle’s medium (DMEM) (containing 10% FBS, 100 mmol/L Na pyruvate, Corning CellGro, Tewksbury, MA, USA), 100 U/mL collagenase type IV (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), and 15 mmol/L HEPES (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Visible red blood cells were removed using red blood cell lysis buffer (Boston Bio-Products, Ashland, MA, USA) and strained over 40 μm filters. Cells were maintained in ultralow-attachment tissue culture plates before injection into the culture chamber. Cell preparations or cell lines were pelleted and resuspended in type I rat tail collagen (2.5 mg/mL, Corning, Tewksbury, MA, USA) following the addition of 10× PBS with phenol red (pH 7.0–7.5). The spheroid–collagen mixture was then injected into the center gel region of the 3D microfluidic culture device. Collagen hydrogels were hydrated with media with or without indicated drugs or control treatments after 30 min at 37 °C. Microfluidic culture as well as live/dead staining and quantification of cells was performed as previously described [31 (link),40 (link)].