Evos xl cell imaging system

Scratch wound assay was performed as follows: briefly, 2.5 × 10⁵ patient-derived primary cells were seeded using Culture-Insert 2 Well in µ-Dish 35 mm (Ibidi, Gräfelfing, Germany). After 24 h of cell attachment, the culture was exposed to DENO (Amgen Inc., Milan, Italy) LENVA (Eisai Ltd., Milan, Italy) and DENO + LENVA or vehicle for 72 h. Images were captured with EVOS XL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) at 0 and 72 h and the cell migration rate was obtained by observing the wound closure after 72 h treatment exposure and compared to untreated cells.

As similarly detailed in Azoidis et al. [31 (link)], after detachment with 0.05% trypsin/EDTA (Sigma-Aldrich), ADSC suspensions were mixed with a 1.0% aNFC hydrogel to achieve a desired hydrogel concentration of 0.1%, 0.2%, 0.4%, or 0.5% (w/v) aNFC with cell densities of 1, 2.5, 5 × 10⁴ or 1 × 10⁵ cells/ml, respectively. After 30 min of incubation at 37°C, standard culture medium was added to the top of the hydrogel. Bright-field microscopy images were acquired using an inverted microscope (EVOS XL Cell Imaging System, Thermo Fisher Scientific).
For XTT viability assays, ADSCs were cultured in a 0.2% or 0.4% aNFC hydrogel in standard cultivation medium for up to 7 days at 37°C and 10% CO₂. A 50% medium change was performed every 2-3 days.
Following 48 h incubation, retrieval of cells from aNFC was performed by enzymatic hydrolysis of the matrix with cellulase (600 μg/mg) (GrowDase®, UPM Biomedicals) for 6 h at 37°C. After 6 h incubation, the solution was mixed multiple times to facilitate cell liberation and then centrifuged at 300g for 10 min. The cell pellet was resuspended in (50 μl) media, and the cell count was performed using a haemocytometer and trypan blue (Sigma-Aldrich).

Cell migration was examined using scratch assays [30 (link)] in 6- and 24-well plates. Plates were prepared with 2 reference lines at the bottom side of each well to assure that the same sections were repeatedly recorded. At 80% confluence, standard starvation was initiated. Then, a small wound line was made in the cell monolayer—orthogonal to the reference lines—with the tip of a 200 µL pipette. The medium was removed, and the cells were rinsed with HEPES Buffered Saline Solution (Lonza, CC-5024) twice to remove loose cells, followed by incubation with sera at 1% (basal growth) and 5% (stimulated growth). For each well, four microscopic images were taken using transmitted-light microscopy (EVOS XL Cell Imaging System, Thermo Fisher Scientific) 48 h after scratching. The dimensions of the remaining scratch area were calculated planimetrically with ImageJ version 1.49u and the plugin MRI Wound Healing Tool (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool). Migration was calculated by subtracting the basal growth response from the stimulated growth response for each group, and data are given as arbitrary units (AU). Cell counts were set in relation to the healthy controls, as described previously [31 (link),32 (link)]. All experiments were performed twice [33 (link)].