Ultroser g

For virus titration assays, 20,000 (Vero-E6/ HRECs/ PRECs) cells were seeded per well in a 96-well plate. In both PRECs and HRECs, before the day of infection, the cells were washed once with LHC media and pre-incubated with an infection medium containing ATCC airway epithelial cell basal medium, 2% Ultroser-G (Sartorius Stedim Biotech GmbH, Goettingen, Germany), 1×4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher Scientific), 1X MEM non-essential amino acids (Thermo Fisher Scientific), 1X Glutamax (Thermo Fisher Scientific), Pen-Strep and AmpB for 24 h. The cells were washed once with LHC medium on the day of infection and replaced with infection media containing different doses of SARS-CoV-2 (MOI 5.0, 5.0 × 10⁻¹, 5.0 × 10⁻², 5.0 × 10⁻³, 5.0 × 10⁻⁴, 5.0 × 10⁻⁵, 5.0 × 10⁻⁶, 5.0 × 10⁻⁷) and mock inoculum. A volume of 100 µL of viral stock/supernatants with along with 100 µL of inoculation media was used as inoculum. After 2 h incubation at 37 °C and 5% CO₂, the virus and mock inoculum were removed, cells were washed once with LHC medium and replaced with fresh infection media and incubated for 2-, 12, 24, 48, 72, 96, or 120 hpi, respectively. Following infection, the cells on plates were either fixed in 4% paraformaldehyde for imaging or lysed in Trizol for RNA isolation, while the supernatants were directly collected into Trizol for viral RNA extraction.

The protocol for obtaining and culturing human airway epithelial cells was approved by the University of Iowa Institutional Review Board. Airway epithelial cells from non-CF donors were isolated from trachea or bronchi from postmortem lungs deemed not suitable for transplant, whereas CF tissues were obtained after lung transplant. The genotypes of the CF donors were c.[1521_1523delCTT];[1521_1523delCTT] (ΔF508/ΔF508, n = 7) and c.[1521_1523delCTT ];[1585-1G>A] (Δ508/1717-1G->A, n = 2). Cell suspensions were prepared in USG culture medium, a 1:1 mixture of DMEM/F12 supplemented with 2% Ultroser G (Sartorius Stedim). The cell suspension was transduced with an HIV-based lentivirus (MOI = 4) and hexadimethrine bromide (Polybrene) at a final concentration of 2 μg/mL. A VSV-G lentivirus expressing GFP served as a control. The cell-virus mixture was then seeded onto collagen-coated, semipermeable membranes (0.33 cm², no. 3413 polycarbonate, Corning Costar Transwell Permeable Supports) and differentiated at the ALI as previously described (35 (link)). Epithelial cells were studied at least 21 days after seeding. Initial transepithelial conductance (G_t) values are reported throughout this paper and corresponding transepithelial resistance (R_t) measurements are available in the Supporting Data Values XLS file.

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.