Emem medium

Virus viability was assessed only in samples where the result differed between the antigen test and RT-PCR. Testing was performed in monolayer CV-1 cells (African green monkey kidney fibroblasts) cultured at 37 °C in the E-MEM medium (Sigma-Aldrich, St. Louis, MO, USA) in Leighton tubes. Cells were inoculated with 300 µl of the sample used for the RT-PCR testing or blanks. The cultures were examined daily under the microscope (100–200× magnification) for changes indicating a cytopathic effect of the virus. After 7 days (or sooner if the cytopathic effect was observed in approx. 75% of cells), the cells were passaged (1:6) and cultured for an additional 7 days. If no cytopathic effect (virus action) was observed over the next 7 days, the sample was declared free of viable virus. Where cytopathic effect was observed, SARS-CoV-2 presence was verified by RT-PCR.
The sensitivity of the method was verified through serial dilution of the virus stock suspension prepared by cultivation and measured by PCR (3 × 10¹¹ RNA copies/mL), both directly and after freezing at −80 °C. The cytopathic effect was observed from approx. 10⁴ RNA copies/mL, which, with the infectious particles:RNA ratio in the tissues ranging typically between 1:1000 and 1:10,000 [10 (link),11 (link)] corresponds to 1–10 infectious particles/mL.

The human Caco-2 cell line (ATCC^® HTB-37, Basel, Switzerland) was derived from the colorectal adenocarcinoma of a 72-year old Caucasian male patient, while the SK-CO1 cell line (ATCC^® HTB-39, Basel, Switzerland) was derived from the metastatic site ascites, from a colorectal adenocarcinoma of a 65-year old Caucasian male patient. The Caco-2 cell line was grown and sub-cultured in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA), while the SK-CO-1 cell line was grown and sub-cultured in EMEM medium (Sigma-Aldrich, Germany), both containing L-glutamine (Sigma-Aldrich), 10% v/v fetal bovine serum, and antibiotics (50 U/mL penicillin; 50 µg/mL streptomycin; Euroclone, Milan, Italy). All cells were maintained in an incubator at 37 °C in a 5% CO₂ humidified atmosphere. Decitabine (5-Aza-2′-deoxycytidine, Sigma-Aldrich) treatments were performed at a final concentration of 5 µM, 96 h before harvesting the cells. For each condition, at least three independent experiments were conducted. After each experiment, cells were cultured in serum-free media for 24 h before medium collection and cell harvesting.