Fig. 1.
Characterization and re-population of decellularized scaffolds with hMAB and mFB. a Macroscopic appearance (left) of native and decellularized rat oesophagus after 2 cycles of DET. DAPI staining (centre) of native and decellularized scaffold sections to identify nuclei. Scale bar: 100 µm. Hematoxylin and eosin (right) of sections of native oesophagus and decellularized scaffold. Scale bar: 100 µm. b DNA quantification in samples of native and decellularized oesophagi. Data: mean ± SD (n = 3; **p = 0.0011; t-test). c Characterization of hMAB isolated from paediatric skeletal muscle biopsies and expanded for up to 10 passages in culture: immunofluorescence staining for Ki67 and phalloidin, colorimetric staining for alkaline phosphatase, immunofluorescence staining for αSMA, NG2 and PDGFRβ. Nuclei were stained with DAPI. Scale bar: 100 µm. d Characterization of mFB isolated from murine skeletal muscles and expanded for up to 7 passages in culture: immunofluorescence staining for vimentin and Tcf4. Nuclei were stained with DAPI. Scale bar: 100 µm. e, f Top row: cell distribution maps showing single cells (purple) and ECM scaffold (green) in sections of 3 representative scaffolds seeded with hMAB only (e) or hMAB + mFB (f) and cultured for 6 to 9 days in static conditions. Cells were identified using DAPI staining. Middle row: polar distribution maps obtained from the correspondent cell distribution maps (in the top row) assuming a perfectly circular section of the scaffolds. Bottom row: cell density maps obtained from the maps above showing cell density in seeded scaffolds from the lumen (top) to the external surface (bottom). g Total number of cells per area in scaffolds seeded with hMAB only or hMAB + mFB and cultured for 6 to 9 days expressed as 25th to 75th percentile, median and min to max whiskers (n = 6). h Immunostaining for hNuclei and DAPI used to discriminate between hMAB and mFB. Scale bar: 100 µm. i Proportion of hMAB and mFB in co-seeded scaffolds after culture. Data: mean ± SEM (n = 3; ***p = 0.0002 hMAB vs mFB; t-test). j Representative images of MTT colorimetric assay on scaffolds seeded with 8.5 × 105 hMAB or 1 × 106 hMAB with/without mFB (ratio 85:15) and cultured in static for 6 days. Scale bar: 1 mm. Viable cells are coloured in purple. k Graph shows the measure of the maximum distance in mm covered by the cells from the centre of the cell cluster to the empty edge of the scaffolds. Data: 25th to 75th percentile, median and min to max whiskers (n = 6; **p = 0.0049; ANOVA)