Figure 4

Bioprinted RHE constructs were achieved using distinct populations of NHKs on a same sample in order to pattern the concentric design. Morphological analysis of the printed epidermal substitutes and validation of the pattern using H&E staining and fluorescence analysis were done after 14 days of culture at air–liquid interface. (A) GFP staining (Green) on the entire skin substitute with nuclei counterstain with DAPI (blue) where we can observe both conserved parts of the epidermal sample, Z1, Z2 and Z3 are for zone 1, zone 2 and zone 3 represent the areas illustrated in (B); a macroscopic picture of the semi-circle sample has been represented with the orientation of sample slices for the analysis; (B) H&E staining and Green fluorescence analysis on the WT part of the sample (Z1), the transition between the WT and the WTGFP parts (Z2) and the WTGFP part (Z3) proving that pattern is conserved after 14 days of culture and that both NHK populations form at the end of the culture a unique sample. Sc stratum corneum, epi epidermis, mbrne polycarbonate membrane support, WT reconstructed epidermal part from native phenotypic keratinocytes, WTGFP reconstructed skin substitute from GFP-transduced native phenotypic keratinocytes, Transition specific region of the patterned reconstructed skin substitute where both WT and WTGFP are in contact (n = 11).