BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 285

FIGURE 13.4
Laser-printed skin constructs: 10 days after printing, histological sections were prepared to investigate the formation
of intercellular junctions, which are essential for tissue function. Fluorescence microscopic images of 3D laser-printed
fibroblasts and keratinocytes are depicted as a qualitative analysis of adherens and gap junction formation. (a) pan-
cadherin-staining shows the adherens junctions consisting of cadherins, which are located between the membranes
of neighboring cells; (b) pancadherin-staining (green) and cell nuclei staining with Hoechst 33342 (blue); (c,d)
connexin-43 (Cx43) staining (green) and Hoechst 33342 nuclei staining (blue), connexins are the constituents of the
gap junctions; Picture (d) shows Cx43 distributed in a scattered, punctate fashion, which is a sign for the formation of
gap junctions; (e-g) gap junction coupling visualized in green with Lucifer yellow (e,g) after scrape-loading procedure,
the keratinocytes, transfected with mCherry, are depicted in red (f,g). All scale bars are 50 m m. Reprint from: Koch
et al. (2012) (Copyright © 2012 Wiley Periodicals, Inc.) A color version of this figure can be viewed online.
Immunostaining ( Figure 13.4 ) of connexin 43 (Cx43), the main connexin in human skin, shows its
expression in all cells and its localization within the cell membrane 10 days after the cell printing pro-
cedure ( Koch et al., 2012 ). As can be seen, Cx43 is distributed in a scattered, punctate fashion, which
is a sign for the formation of gap junctions ( Morritt, et al., 2007 ).
The gap junctions' functional capability for cell-cell communication was tested by a dye trans-
fer with a scrape loading method in vital 3D cell constructs. For this purpose, confluently grown
3D-printed keratinocyte cells were scratched with a razor blade and the dye Lucifer yellow (LY) was added,
which then could penetrate into the destroyed cells. Since LY molecules are small enough to pass the
gap junction channels, the penetration into channel-connected neighboring cells and the diffusion dis-
tance through further cells can be analyzed as a measure for gap junction coupling. Twenty layers of ke-
ratinocytes embedded in collagen were printed on a glass slide and cultivated for 10 days at 37°C (5%
CO 2 ). After washing with PBS, the slides were submerged in a DPBS solution including 0.25% LY.
With a razor blade, one scratch along the whole sample was set. After 5 min of incubation, the slides
were washed four times with DPBS for 5 min each. The diffusion distance of LY was documented via
fluorescence microscopy ( Figure 13.4 ). The LY dye penetrated from the scratched keratinocyte cells