Inverted light microscopy

Primary cultures of human gingival fibroblast (hGFs) were established by the explant method previously described (Mammana et al., 2019 (link)). Fragments of healthy gingival tissue were rinsed three times in Phosphate Buffered Saline (PBS, Lonza, Basel, Switzerland) solution, cut into small tissue pieces and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) supplemented with 10% Fetal Bovine Serum (FBS, Lonza) and 0.1% gentamicin [10 mg/ml; Euroclone, Pero (MI), Italy] at 37°C in 5% CO₂ atmosphere. The gingival tissue biopsies were cultured until hGFs spontaneously migrated (about 4 weeks; Cavalcanti et al., 2015 (link)). All cells were incubated in standard conditions [37°C in a humidified atmosphere of 5% (v/v) CO₂]. Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy), as previously described (Libro et al., 2016 (link)). All the experiments were performed with cells processed between 4 and 8 passages and each assay were performed in triplicate.

The cultured cells of both MSC-lung and MSCs-CPAM were seeded in a 6-multiwell plate with a density of 8 × 10⁴ for each well. After seeding cells were maintained for 24 h in the standard medium MSCBM (Lonza) in incubator at 37°C with a humidified atmosphere at 5% CO₂. Then cultured cells of MSC-lung and MSCs-CPAM were subjected to hypoxic treatment by inserting the plates into the ProOx Model P110 (BioSpherix, New York, NY, United States) hypoxia chamber for 24 h at 0.2% of hypoxia. Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy) to evaluate the morphological features in normoxic and hypoxic conditions. Cultured cells of MSC-lung and MSCs-CPAM were maintained in incubator with normoxic condition and used as control cells.

Primary cultures of human gingival fibroblasts (hGFs) were established by the explant method as previously described [26 (link)]. Fragments of healthy gingival tissue were rinsed three times in Phosphate Buffered Saline (PBS, Lonza, Basel, Switzerland) solution, cut into small tissue pieces and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza) supplemented with 10% Fetal Bovine Serum (FBS, Lonza) and 0.1% gentamicin (10 mg/mL; Euroclone, Milan, Italy) at 37 °C in 5% CO₂ atmosphere. The gingival tissue biopsies were cultured until hGFs spontaneously migrated (about 4 weeks; [27 (link)]). Cells were incubated in standard conditions (37 °C in a humidified atmosphere of 5% (v/v) CO₂). Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy) as previously described [28 (link)]. All the experiments were performed with cells processed between 4 and 8 passages and each assay was performed in triplicate.