Vico confocal

Immunofluorescence assay on in vitro 3D constructs was performed to evaluate morphologically the differentiation of C2C12 cells laden into different bioinks at 7, 14, 21 and 28 days of culture in DM. 3D constructs were blocked with PAT (PBS containing 1% [w/v] bovine serum albumin (BSA) and 0.02% [v/v] Tween 20) solution for 1 h at RT. Subsequently, samples were incubated with MF20 primary antibody (Myosin Heavy Chain Antibody, diluted in PBS‐Tween 0.1% and in BSA 1% 1:20) for 1 h at RT. After several washes with buffer solution, sections were incubated with a secondary antibody diluted in 0.1% PBS‐Tween and 1% BSA (1:1000), and in diluted Phalloidine (1:40). Samples were counterstained with DAPI to detect nuclei, washed three times with a washing buffer, and ultimately mounted. Finally, sections were observed with a semi‐confocal microscope (ViCo confocal, Nikon), supported by the ImageJ PRO 6.2 software.

To obtain cultures with visibly separable cells, NHDF and HSF were seeded in Falcon 35 mm cell culture dishes (Avantor VWR, Milan, Italy) at a density of 2000 cells/cm² in complete media. Cells were grown for 24 h before the treatment with Biofiber PF for 6 days; untreated cells were used as controls. The samples were collected by fixing cultures in 4% paraformaldehyde in PBS 1× and storing in PBS 1× at 4 °C until all samples were ready to be stained. Fixed cells were then permeabilized via 10 min incubation in 0.1% Triton X-100 (Sigma Aldrich, Milan, Italy) in PBS 1×, then rinsed with PBS before blocking for 1 h in 5% normal donkey serum in PBT (PBS with 0.1% Tween 20; Sigma Aldrich, Milan, Italy). The cells were incubated for 1.5 h with the primary antibody anti-α-SMA (Sigma Aldrich, Milan, Italy), diluted at 1:50 in blocking buffer. Cells were rinsed with PBT before 1 h of incubation with Alexa Fluor 594-labeled goat anti-mouse secondary antibody (1:500; Immunological Science, Milan, Italy), Alexa Fluor™ 488 phalloidin (1:40; Immunological Science, Milan, Italy) and nuclear label 4′,6-diamidino-2-phenylindole 1:1000 (DAPI; Sigma Aldrich, Milan, Italy) for 10 min. Fluorescent image detection was performed using a semi-confocal microscope (ViCo confocal, Nikon, Amstelveen, The Netherlands), [34 (link)]. The experiment was carried out in three replicates.

live/dead staining provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live (green) and dead (red) cells with two probes. This study used calcein and ethidium homodimer (EthD-1), optimal dyes for this application. Calcein is well retained within live cells, producing an intense, uniform green fluorescence. EthD-1 enters cells with damaged membranes and undergoes a 40-fold fluorescence enhancement upon binding to nucleic acids, producing a bright red fluorescence in dead cells. Therefore, live/dead staining (Invitrogen) was used to assess and monitor cell viability throughout the biological experiment. For this reason, it was performed at four different time points (1, 7, 14, and 21 days of cell culture). According to the protocol, a solution was prepared consisting of 1.5 mL of phosphate-buffered saline (PBS), 3 μL of ethidium homodimer-1 (EthD-1), and 1.5 μL of calcein. Three-dimensional constructs were covered with 500 μL of this solution and incubated for 45 min in the dark, and then the solution was removed. The image acquisition was performed by a semi-confocal microscope (ViCo confocal, Nikon).