Vybrant mtt colorimetric assay

The Vybrant MTT colorimetric assay (Life Technologies, Thermo Fisher) was used to determine viable cell migration. Tubular scaffolds seeded with either 8.5 × 10⁵ hMAB or 1 × 10⁶ hMAB with/without mFB (ratio 85:15, n ≥ 3) were cultured in static conditions for 6 days and then incubated with MTT diluted 1:10 in proliferative medium for 4 h at 37 °C. Scaffolds were washed with PBS, opened longitudinally and placed at the bottom of a multiwell plate for imaging with a stereomicroscope. Formazan-positive cells were visible within the scaffolds. Images of flat open scaffolds were analysed for cell migration measuring the colour intensity along 8 random lines drawn radially from the centre of cell clusters to the edge of the scaffold using ImageJ software. The Gray Value graph obtained from the lines was used to measure the distance in mm covered by the cells, calculated between the centre of the cluster and the most distant cell. Analyses and calculations were performed by three independent operators in blind on 6 scaffolds per condition and in 2 experimental replicates. The average cell migration distance in seeded scaffolds was determined.

The Vybrant MTT colorimetric assay (Life Technologies, Thermo Fisher) was used to assess cell viability and proliferation in 2D cultures, to determine cell engraftment and localization in 3D cultures and was performed according to manufacturer's instructions. Two-dimensional and 3D-cultured cells were incubated with MTT diluted 1:10 in proliferative medium for 4 h at 37°C. In 2D cultures, the reaction product, formazan, was solubilized in 100 μL of SDS and its concentration was determined by optical density at 550 nm. In 3D cultures, scaffolds were washed with PBS and imaged with a stereomicroscope.^{17 (link)} Formazan-positive hMABs were visible within the scaffolds.