Culture medium

HCC cells (1×10⁵) were added to Transwell chambers with culture medium (8 µm, Corning, New York, USA). The bottom layer of the membrane was suspended in a medium supplemented with FBS. After 12-24 h, cells were fixed and stained with crystal violet. The number of transmembraned cells was determined using ImageJ software.

PBMCs were cultured in a serum-free X-VIVO 15 medium with the addition of 1% of penicillin/streptomycin (P/S) (Lonza, Allendale, NJ, USA) and IFN-γ (Boehringer Ingelheim, Vienna, Austria) at a final concentration of 1000 U/mL (day 0) as previously described [9 (link)]. Briefly, 30 × 10⁶ of those cells were seeded in non-treated flasks in 15 mL of culture medium (Corning Incorporated, Corning, NY, USA) with vented cap. The flasks were maintained in a vertical position to ensure a better oxygenation of the cellular suspension, a reduction of the culturing medium distribution surface and an appropriate culturing medium volume to re-suspend the cells homogeneously.
The following day (day + 1), Proleukin, which is an IL-2 cytokine (Novartis, Origgio, VA, Italy), and CD3 Pure, which is an IgG anti-CD3 (OKT-3) (Miltenyi Biotec, Bergisch Gladbach, Germany), were added to a final concentration of 300 U/mL and 50 ng/mL, respectively.
Every 48 or 72 h, a cell count was performed to monitor their expansion and, on the basis of cell growth, fresh medium with IL-2 (300 U/mL) was added to obtain a cellular concentration of between 1 and 1.5 × 10⁶ cells/mL. The culture was carried out for approximately 21 ± 3 days and the QCs were performed. The release criteria are shown in Supplementary Materials: Table S1.

G-MSSCs were allowed to attach to and grow on 24-well tissue culture plates (50 000 cells well^-1) containing culture medium (Costar, Corning, USA). After 24 h of incubation, the medium was discarded, washed with PBS, and replaced with new medium without Pen/Strep. cells that were pretreated with L. rhamnosus (MOI 1:100) for 12 h at 37°C and 5% CO₂. Subsequently, the culture wells were washed with PBS and then stimulated with 20 ng mL-1 IFN-γ (Invitrogen, USA) or P. gingivalis (MOI: 1:100) for another 12 h. At the end of the experiment, culture supernatants were harvested to determine their cytokine levels, and the cells were prepared for detection of TLR expression by flow cytometer.