Dermal fibroblast culture and analysis

Human adult dermal fibroblasts were used between passages 3 and 5. Collagen sponges were sterilised prior to seeding with UV for 2 h. The cells were seeded onto collagen sponges at a density of 30,000 cells per cm² in 48 well plates. The cells were cultured for 3, 5 and 7 days in Dulbecco’s Modified Eagle Medium, supplemented with 1% penicillin streptomycin and 10% foetal bovine serum at 37 °C and 5% CO₂. Media were changed every 2 days. Cell proliferation was assessed using PicoGreen^® dsDNA assay kit after 3, 5 and 7 days in culture, according to manufacturer’s protocol. Metabolic activity was assessed using the alamarBlue^® assay (Thermo Fisher Scientific, UK) after 3, 5 and 7 days in culture, according to manufacturer’s protocol. Cell viability was assessed with calcein AM (Thermo Fisher Scientific, UK) and ethidium homodimer I (Thermo Fisher Scientific, UK) staining after 3, 5 and 7 days in culture, according to the manufacturer’s protocol. The cells were visualised under Andor Revolution Spinning Disk Confocal Microscope (Olympus IX81, Japan). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), whilst cytoskeleton was stained with rhodamine phalloidin based on established protocols. Briefly, media were removed and sponges were washed three times with Hank’s Balanced Salt Solution prior to staining. Cells were fixed with 2% paraformaldehyde, permeabilised with 0.2% Triton X-100 and then stained with DAPI and rhodamine. The sponges were imaged using Andor Revolution Spinning Disk Confocal Microscope (Olympus IX81, Japan). Cell morphometric analysis was conducted with ImageJ software (National Institutes of Health, USA). The total area and aspect ratio (the ratio of the major axis divided by the minor axis of each nuclei based on a fitted ellipse) of the nuclei were assessed. For cell viability and morphometric analysis, for each experimental group, cells on three sponges were analysed by taking five images per sponge (15 images in total were analysed per experimental group).