Nu serum

The placental choriocarcinoma cell line BeWo (ATCC^® CCL-98™, passages 195 to 207) was cultured in F-12K Medium (ATCC^® 30-2004) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific). Human Brain Microvascular Endothelial Cells (HBMEC), passages 3 to 20, were cultured in RPMI-1640 medium with L-glutamine (Gibco, Thermo Fisher Scientific) supplemented with 1mM Na-Pyruvate (Sigma-Aldrich), 1% MEM non-essential amino acids (Sigma-Aldrich), 1% MEM Vitamin Solution x100 (Sigma-Aldrich), 10% Nu-Serum (Corning^®, VWR, Switzerland) and 10% FBS (Gibco, Thermo Fisher Scientific), adapted from Silwedel et al. (2018) (link). Both cell lines were cultivated in 75 cm² culture flasks (Bioswisstech, Switzerland) at 37°C and 5% CO₂ in a humid atmosphere. To maintain the culture monolayer cell cultures were split when 70 to 90% confluence was reached.

Wild-type human brain microvascular endothelial cells (hBMVEC) are an immortalized cell line, a gracious gift from Dr. Supriya Mahajan (University at Buffalo). Verification of proper cell behavior is provided ^{26 (link),27 (link)}. hBMVEC were cultured in RPMI, 10% FBS (Gibco) and 10% Nuserum (Corning), passaged at confluency and used between passage numbers 7 and 15 in experimentation. shRNA-integrated cells are continued in culture under selective pressure at 7.5ug/ml puromycin in RPMI until plated for an experiment, at which all cells are in normal growth media. Cells were housed in a 37°C incubator, 5% CO₂ injection, and constant humidity.

A549 cells were obtained from ATCC (CCL-185) and cultured in DMEM, supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich), 1% (v/v) penicillin/streptomycin (Invitrogen), and 1% (v/v) glutamax (Invitrogen). Jeg-3 cells were obtained from Dr Coyne (Duke University) and cultured in minimum essential medium (MEM) with Earle's salts supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. HBMECs were obtained from Dr Coyne (Duke University) and cultured in RPMI-1640 (Corning), supplemented with 10% (v/v) FBS, 10% (v/v) Nu-serum (Corning), 1% (v/v) penicillin–streptomycin, 1× MEM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 10 µg/mL endothelial cell growth supplement (Fisher Scientific). All cell lines were maintained at 37°C and 5% CO₂. Rubella RA27/3 vaccine strain was obtained from the Centers for Disease Control and Prevention (CDC). Rubella RA27/3 was generated in Vero CCL81 cells (ATCC) from supernatants and viral titer was determined by TCID₅₀ assay.

Two human ACC cell lines, SW13 and NCI-H295R, were purchased from the American Type Culture Collection™ (Cat # CCL-105, CRL-2128; Manassas, VA, USA) and cultured in 5% CO₂ atmosphere at 37 °C in Dulbecco's Modified Eagle Medium (Cat # 11195–065, Thermo Fisher Scientific, MA, USA) supplemented with 2.5% Nu-Serum (Cat # 355100, Corning, MA, USA) and 0.1% Insulin-Transferrin-Selenium (Cat # 41400045, Thermo Fisher Scientific, MA, USA). Cell lines were authenticated by short tandem repeat profiling. We routinely subcultured every 3–5 days, depending on the degree of cell confluence.
NCI-H295R cells used to generate human ACC xenograft were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector (9PIE132, Promega) encoding the luciferase reporter gene luc2 (Photinus pyralis) and maintained in the above medium with up to 500 μg/mL of G-418 antibiotic (11811–023, Gibco, MA, USA) for selection.

DMEM/Ham’s F12, FBS, HEPES, penicillin/streptomycin and amphotericin B were purchased from Merck Millipore, while other reagents were obtained from Sigma unless otherwise indicated. Media used in this culture procedure are modifications of a protocol reported for mouse tracheal cells [10]. The basic medium consisted of DMEM/Ham’s F12, 15 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. The proliferative medium (M1) for d 0–d 7 was basic medium supplemented with 5% FBS, 10 μg/ml insulin, 5 μg/ml transferrin, 0.1 μg/ml cholera toxin, 25 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract. Differentiation medium (M2, from d 7 onwards) for ALI-MOEC and -POEC (M2a) was serum free: basic medium supplemented with 1 mg/ml BSA, 5 μg/ml insulin, 5 μg/ml transferrin, 0.025 μg/ml cholera toxin, 5 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract. For ALI-BOEC the differentiation medium (M2b) consisted of basic medium with 3% FBS and 2% Nuserum (Corning). All culture media were freshly added with 0.05 μM retinoic acid directly before use.