M 795 inverted microscope

Cells were isolated from the dental pulp of extracted third molars from healthy donors (N = 4, age 18–20) according to the Helsinki Declaration after the donor’s informed consent and approval by the local ethical committee of the Hospital Ruzinov, Bratislava, Slovakia. The teeth were removed due to orthodontic therapy (not because of the experiment), and no pathological alterations were observed.
Pulp tissues were washed thoroughly in PBS containing antibiotics and cut into 1–2 mm² pieces. Small pieces were placed in 60 mm culture dishes in a random pattern. Drops of foetal bovine serum (FBS, Sigma Aldrich, Taufkirchen, Germany) were applied on the tissues, sufficient to cover them entirely, and maintained at 37 °C in a humidified incubator with 5% CO₂. After 2 h incubation, explants were maintained in low-glucose DMEM (Dulbecco’s modified Eagle medium, Sigma Aldrich, Germany) enriched with 10% FBS, penicillin (100 IU/mL, Sigma Aldrich, Germany), and streptomycin (100 µg/mL, Sigma Aldrich, Germany). The culture medium was changed every 2–3 days, and the cell outgrowth was monitored regularly with an M-795 inverted microscope (OPTIKA S.R.L., Ponteranica, Italy). The outgrown cells at 70–80% confluence were detached using 0.25% Trypsin-EDTA solution and transferred to a T-75 flask. Between 4 and 6 passages were performed for each experiment.

To confirm the phenotype of isolated cells, every hDPSC population at the 3rd passage was identified by flow cytometry (MACS Quant Analyzer, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the phenotypic signature described by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) using the MSC Phenotyping kit (Miltenyi Biotec, Germany) according to manufacturer’s instructions. Cell viability was assessed with propidium iodide (PI, Miltenyi Biotec, Germany). The morphology of DPSCs was evaluated with microscopic observations using the M-795 inverted microscope (OPTIKA S.R.L., Italy). For the experiment, 0.5 × 10⁶ cells of the required passage were transferred to a cell culture plate. After pharmacological treatment, DPSCs were evaluated by flow cytometry, as described above. Medians with 25–75% percentiles were calculated using two experiments with cells from different donors (N = 4).

Cell morphology and characterization of BM-MSCs were evaluated before and after atorvastatin treatment. Imaging of BM-MSCs was performed using an M-795 inverted microscope (OPTIKA S.R.L., Ponteranica, Italy). Expression of surface antigens in BM-MSCs was quantified by flow cytometry (MACS Quant Analyzer, Miltenyi Biotec, Bergisch Gladbach, Germany; with MACSQuantify software 2.13.3) using the MSC Phenotyping kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s instructions (≥95% of the MSC population must express CD73, CD90 and CD105, and these cells must lack expression (≤2%) of CD14, CD20, CD34 and CD45). Cell viability was assessed with propidium iodide (PI, Miltenyi Biotec, Bergisch Gladbach, Germany).