Trypsin edta 1

Olive pomace (OP), obtained after a three-phase process (Taggiasca cultivar), was supplied by a local producer (Liguria, Italy). The fresh OP was collected and dried in a laboratory oven at 50 °C until there was constant moisture, and then stored in dark conditions at room temperature.
Ethanol, mEthanol, acetonitrile, acetic acid, and sodium carbonate were purchased from Carlo Erba Reagents (Cornaredo, Milan, Italy), while Folin–Ciocalteu’s reagents included caffeic acid, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate, 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), ionophore A23187, NMDA, and neutral red solution from Merck-Sigma-Aldrich (Milan, Italy). Calcium Green™-1AM, Neurobasal™ medium, B-27 supplement, Glutamax^®, penicillin, and streptomycin solution were purchased from Life Technologies Italia (Milan, Italy). DMEM, FBS, MEM non-essential amino acids 100×, and trypsin-EDTA 1× solutions were from Euroclone (Milan, Italy).

Alg/HAp scaffolds underwent two cycles of sterilization under an ultraviolet (UV) light (15 W) for 1 h each, and they were rehydrated and conditioned in complete MEM-α overnight as previously reported [13 (link)]. After being expanded up to the sixth passage, DPSCs were trypsinized (trypsin/EDTA 1×, EuroClone, Milan, Italy) and collected by centrifugation (1200 rpm for 10 min at room temperature). DPSCs were then counted, and 5 × 10⁴ cells were resuspended in 130 μL of complete medium and afterward used for seeding drop by drop on each scaffold. Samples were immediately placed at 37 °C and 5% CO₂ for 3 h to allow cell adhesion and interpolation onto/into scaffolds. Next, complete medium or differentiation medium (DM) was added to each sample (named in figures as Alg/HAp and Alg/HAp DM, respectively). Complete DM was supplemented as reported previously [13 (link)], with 100 μM ascorbic acid, 10 nM dexamethasone, 5 mM β-glycerol phosphate disodium salt pentahydrate (all purchased from Sigma Aldrich, MI, USA), and 1.8 mM potassium phosphate (Alfa Aesar Chemicals, Haverhill, MA, USA). DPSCs onto Alg/HAp scaffolds with or without DM were incubated for 1, 3, 7, 14, 21, and 28 days, and medium was refreshed every three days.

For cell viability assay, human GM03816 FRDA fibroblasts were cultured in 12-well plates by seeding 0.6 × 10⁵ cells per well. Cells were left untreated or treated with IFN-γ. After incubation in presence of 800 μM hydrogen peroxide for 5 h, fibroblasts from each incubation point were collected as follows: floating cells in culture supernatant were recovered into a centrifuge tube and pooled to the same tube with adherent cells detached by incubation at 37 °C in Trypsin-EDTA 1× (Euroclone ECB3052). After centrifugation at 800× g for 10 min (4 °C), cell pellet was resuspended in DPBS and cell death was analysed by Trypan Blue assay using a Countess Automated Cell Counter (Thermo Fisher Scientific).