Hoechst blue dye

To distinguish EK and DF, the cytoplasm of EK was stained with green cell tracker CMFDA (Invitrogen), while the nucleus of DF was stained with Hoechst Blue Dye (Invitrogen) according to the manufacturer’s instructions at a final concentration of 5 µM. Forty thousand (4 × 10⁴) EK and an equal amount of DF were seeded on the hydrogel followed by 3D confocal imaging on day  0, 3 and 7 using a Nikon A1R confocal microscope (Nikon).

The potential of cell migration activity of HDFs was evaluated according to the 3D model design encapsulated with HDFs. In brief, green cell tracker CMFDA (Invitrogen) was used to stain the cytoplasm of HDFs, and Hoechst Blue Dye (Invitrogen) was used to stain the nucleus at a concentration of 5 μM, as recommended by the manufacturer. The hydrogels were printed with two layers without cells followed by one layer of bioink with the encapsulated green stained cells on top of the hydrogels. Using a Nikon AIR confocal microscope for 3D confocal imaging, the cell migration activity throughout the hydrogels was assessed on day 1 and day 7.