Collagen r solution

Caco2 cells cultured in T75 flask were suspended and subsequently seeded into 48-well plate (5 * 10⁴ cells/well) in DMEM complemented with 20% (vol/vol) FCS and 100 IU/mL penicillin-streptomycin. After 2–3 days of culture, culture medium was removed when the cell confluence was about 80%. The cell layers were then washed twice with 500 μL PBS. Serum-free DMEM medium (100 μL) containing 5 μg/mL of trypsin (Gibco, Paisley, UK) and SA11 RV were added and incubated for 60 min at 37 °C with 5% CO₂ to allow efficient infection, followed by four times washing with PBS (500 μL each) to remove free virus particles. Subsequently, culture medium containing 5 μg/mL of trypsin (and indicated treatments) were added to the infected cells and incubated for 24 or 48 hours at 37 °C with 5% CO₂.
For organoid infection, SA11 RV (contain 5000 genome copies) was first activated with 5 μg/mL of trypsin at 37 °C with 5% CO₂ for 30 minutes. Subsequently, organoids were infected with the activated SA11 for 60 minutes at 37 °C with 5% CO₂, followed by four times washing with PBS to discard the free viruses. Organoids were then aliquoted into 48-well plates that have been coated with 20% (vol/vol) Collagen R Solution (SERVA, Heidelberg, Germany) and maintained in culture medium containing indicated treatments at 37 °C with 5% CO₂.

The protocols of inoculation of Caco2 cells with SA11 and patient-derived rotavirus were described previously.²³ (link) Briefly, cell monolayers of Caco2 cells were incubated with SA11 rotavirus at 37°C with 5% CO₂ for 60 min for infection, followed by removing free virus particles. Subsequently, cells were added with culture medium (FCS free) containing 5 µg/mL of trypsin and indicative drugs, followed by incubation at 37°C in a humidified 5% CO₂ incubator.
Organoids were inoculated with activated virus (5,000 genome copies) for 60 min at 37°C with 5% CO₂, followed by removing free viral particles. Then, organoids were aliquoted in wells of a 48-well plate coated with 20% (vol/vol) Collagen R Solution (SERVA, Heidelberg, Germany) and culture medium containing drugs of interest was added. Subsequently, the 48-well plate containing organoids was spin down at 500 g for 5 minutes to promote the adherence on the bottom of the wells, followed by maintaining them at 37°C with 5% CO₂.
Preparation of patient-derived rotavirus was performed as described earlier,²³ (link) and protocols of viral inoculation and treatment of drugs are similar to SA11 rotavirus. 48 h post-infection was used to determine viral replication levels, since it is the optimal time point for viral replication without lysis of the cells.

Collagen gel cultures were prepared essentially as published before (Trachslin et al., 1999 (link)). All stock solutions were stored at +4°C and kept on ice whilst mixing the working gel solution. For 7 ml collagen gel (volume for 16 wells of a 24-well culture dish), we sequentially added to 5.25 ml Collagen R solution (2 mg/ml sterile rat tail collagen type I; SERVA Electrophoresis GmbH, Heidelberg, Germany) the following: 0.7 ml 10× minimum essential medium (MEM; Gibco) containing 5× antibiotics/antimycotics (Gibco), 0.7 ml 10× (0.44 M) NaHCO₃ (sterile filtered) containing 5× antibiotics/antimycotics, 50 μl 1.5 N NaOH (sterile filtered) and 0.35 ml FN-depleted FCS (diluted to 60% with PBS, resulting in a final concentration of 3% FCS in the gel). The solution was rapidly vortexed. At the end, the generated foam was dissolved by quick run centrifugation at 3000 rpm. To a 24-well culture dish, 400 μl collagen gel solution (final concentration 1.5 mg/ml) was added per well. The dish was incubated for 30 min at 37°C in a humidified CO₂-incubator to allow gelation of the collagen.