Glutamine

The bone marrow was harvested from purebred healthy dogs (n = 3). Donor 1—male, German Shepherd, 35 kg, 3 years old. Donor 2—female, Cane Corso, 70 kg, 4 years old. Donor 3—male, German Shorthaired Pointer, 32 kg. Before bone marrow collection, all donors were examined (clinical examination, biochemical, and haematological parameters were evaluated). Harvesting was performed under general anaesthesia. The place of the collection was prepared according to all principles of sterility and asepsis as during surgery. We selected the proximal part of the humerus as the sampling site. The collections were performed with an 18 G injection needle and a 10 mL syringe. After penetrating the bone marrow cavity, the bone marrow was aspirated with a 10 mL syringe containing 3 mL of flushing medium. Flushing medium composition: Dulbecco’s modified Eagle’s medium/Nutrient Mixture F12 (DMEM-F12), 10% foetal bovine serum (FBS), 1% antibiotic–antimycotic (ATB+ATM; penicillin–streptomycin–amphotericin B) and 1% glutamine (all Biowest). The aspirate was then centrifuged twice for 10 min at 400× g. Cells were plated in cultivation flask T25, at a seeding density of 10⁶ cells/flask, and were cultured in media containing DMEM-F12 + 10% FBS + 2% ATB + ATM at 37 °C and 5% CO₂. After 48 h of incubation, nonadherent cells were removed.

For cell culture studies, lung cancer epithelial cells A549, kindly provided by Valentina Monica, from the Department of Oncology, University of Torino, AOU San Luigi Gonzaga, were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (all from Sigma Aldrich) and 2 mM glutamine (Biowest). When needed, cell morphologies were assessed with a phase-contrast microscope (DMi1, Leica Microsystems GmbH).

Primary hMSCs from trabecular bone were isolated as described previously [31 (link)]. Briefly, the bone biopsies from the femoral head were digested in 1 mg/mL collagenase (Roche, Basel, Switzerland) at 37 °C for 3 h. The digested tissue suspension was filtered through a 70 µm nylon strainer (Corning, New York, NY, USA). The cells were seeded in 1.0 mL MSC expansion medium (Kit XF, human; Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 2% penicillin and streptomycin (100× stock; 8.5 g/L sodium chloride, 0.025 g/L amphotericin B, 6.028 g/L penicillin G sodium salt, 10 g/L streptomycin sulphate; Biowest, Nuaillé, France), and 2 mM glutamine (all Biowest, Nuaillé, France), and maintained at 37 °C in a 5%/5% humidified CO₂/O₂ atmosphere. The cells were culture expanded, and cells at passages 3 to 5 were used for all of the experiments. The MSC-like phenotype of these primary hMSCs was confirmed prior to the experiments, according to the International Society for Cellular Therapy [32 (link)], as previously reported [31 (link)].

Six probiotic strains were chosen as stimulators on PBMCs. The six strains are L. paracasei BRAP01 (Bio Ray Biotech), L. acidophilus AD300 (Biena), B. longum BA100 (Biena), E. faecium BR0085 (Synbio), L. rhamnosus AD500 (Biena), and L. reuteri BR101 (Bio Ray Biotech). Ficoll-Paque (GE) separation method was conducted to isolate the PBMCs from the blood. Briefly, 10 mL of blood was diluted to 14 mL with PBS and then the diluted blood was carefully layered on top of the Ficoll-Paque solution. The tubes were centrifuged at 500 g for 20 minutes in RT. After centrifugation, the PBMCs were separated from the blood cells and stored in PBS after one-time wash by PBS. The purified PBMCs were cultured with 6 different probiotics in RPMI-1640 (GE) medium with 10% FBS (Gibco), 1X Glutamine (Biowest), and PSN (Gibco) in a 96-well culture plate (SPL). The plate was put in a CO₂ incubator for 40 hours (37°C, 5% CO₂). Finally, the plate was centrifuged at 250 g and the supernatant was collected and stored in −20°C.