Penicillin streptomycine

GripTite 293 MSR cells (further referred to as HEK293 cells, Invitrogen), a modified HEK293 cell line expressing human macrophage scavenger receptor for stronger adhesion, were maintained at 37°C, 5% CO₂ and 95% humidity in DMEM/F-12 medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Sigma), 4 mM L-glutamine (PAA) and penicillin/streptomycine (PAA). HepG2 cells (Clontech) were cultivated under the same conditions in DMEM medium with all supplements listed above, except for L-glutamine. HEK293 cells were transiently transfected with human NTCP and monkey Ntcp constructs for all transport and peptide binding assays. Transfection was performed with Lipofectamine 2000 (Thermo Fisher Scientific). NTCP/Ntcp-transfected HepG2 cells were used for all HBV/HDV infection experiments as reported before [4 (link)]. They were transfected using X-tremeGENE (Sigma-Aldrich).

We used several glioma cell models including a classical glioblastoma culture (U87) and three stem cell models of glioblastoma (BTSC233, JHH520, NCH644). These models were kindly provided by colleagues: BTSC233 (M.S. Carro, Freiburg University, Freiburg im Breisgau, Germany); JHH520 (G. Riggins, Johns Hopkins, Baltimore, MD, USA); NCH644 (C. Herold-Mende, Heidelberg University, Heidelberg, Germany); U87 (A. Weyerbrock, Department General Neurosurgery, Medical Center Freiburg, Germany).
All cells were cultivated as neurospheres (Figure A1, in Appendix A) in DMEM media (high-glucose, no pyruvate, Thermo Fisher, Waltham, MA, USA), containing F12 (3:1) and 1X B27 supplements (both Thermo Fisher, Waltham, MA, USA), 20 ng/mL human EGF, 20 ng/mL human VGF (both Peprotech, Hamburg, Germany), 5 µg/mL heparin (Sigma-Aldrich, Taufkirchen, Germany), 1X penicillin/streptomycine (Sigma-Aldrich, Taufkirchen, Germany) in standard conditions (humidified 37 °C, 5% CO₂) [28 (link)]. For all assays, volume-adjusted medium was used as mock treatment. Ethical approval to conduct the in vitro studies was granted by the ethics commission of the Medical Faculty of the Heinrich-Heine University (study ID 5841R).

Cells were routinely maintained in RPMI 1640 (Invitrogen Gibco) supplemented with 10% FBS (Invitrogen Gibco) and penicillin-streptomycine (Sigma-Aldrich, 100 U/mL) at 37.0 °C in a humidified atmosphere containing 5% CO₂.