Collagen coated 24 well plates

The iPSC derived human hepatocytes (Definigen, Cambridge, England) were obtained as a cryopreserved sample and cells were recovered for 12 days following the manufacturer’s instructions. Briefly, cells were thawed on collagen-coated 24 well plates (Greiner Bio-One, Frickenhausen, Germany) at density of 5 × 10⁵ cells per well using minimum essential medium, supplemented with 2% non-essential amino acids, 2% chemical defined lipid concentrate (Thermo Fisher Scientific, Waltham, MA), 0.1% insulin (Sigma, Saint Louis, MO) and manufacturer’s cytokines. Cells were cultured in Hepatozyme medium (Invitrogen, Waltham, MA), supplemented with 2% non-essential amino acids, 2% chemical defined lipid concentrate (Thermo Fisher Scientific, Waltham, MA), 0.1% insulin (Sigma, Saint Louis, MO) and manufacturer’s cytokines, and were maintained at 37 °C with 20% O₂.

hCMEC/D3 were trypsinized and cultured in collagen-coated 24 well plates (Greiner Bio-One) at a density of 25 × 10³ cells/cm². After reaching near confluency (90%), cells were replenished with experimental medium as described before. One day later, cells were left untreated or were treated with TNF-α (100 ng/mL) and IFN-γ (10 ng/mL, both from Peprotech) for 24 hours in reduced medium as described before. After 24 hours, hCMEC/D3 cells were washed once and put on fresh reduced medium. After another 24 hours, supernatant were collected and stored at –80°C. To look at cytokine production by Tregs and Teff, the supernatants of suppression assays were collected after 5 days and stored at –80°C. Samples from BBB-ECs and cocultures were thawed and used undiluted in LEGENDplex Human Anti-Virus Response Panel (BioLegend) according to manufacturer instructions to measure different cytokines. Samples were acquired using the LSRFortessa (BD Biosciences) and analyzed using LEGENDplex Data Analysis Software (BioLegend). Concentrations were calculated by the use of the standard curves following the software guidelines.

Viral kinetics in HRECs were performed by seeding 1 × 10⁵ cells per well in collagen-coated 24-well plates (Greiner Bio-one) using growth medium #2; for staining 96-well clear flat bottom black polystyrene surface-treated microplate (CellBIND Costar; Corning) was used, as described previously [15 (link)]. After 24 h of incubation, cells were washed twice with 500 μL of LHC base medium (Gibco, Thermo Fisher Scientific), and inoculated at MOI-2 and MOI-1 in triplicate with HCoV-NL63 or SARS-CoV-2 or mock inoculated with infection medium #2 (airway epithelial cell basal medium [PCS-300-030; ATCC] supplemented with 2% Ultroser G [Sartorius, Göttingen Germany], 1% MEM nonessential amino acids solution [Gibco, Thermo Fisher Scientific], 1% HEPES [Gibco, Thermo Fisher Scientific], 1% GlutaMax [Gibco, Thermo Fisher Scientific], 100 IU/mL penicillin, and 100 μg/mL streptomycin) and incubated at 37 °C with 5% CO₂ for 2 h. Cells were rinsed with LHC basal medium, fresh infection medium #2 was added to each well, and plates were incubated at 37 °C with 5% CO₂ for 96 h.