Co-culture increased iPSC density in the NFC/A 60/40 bioink after printing. (A) Confocal microscopy images of the NFC/A 60/40 co-culture samples at week 0 and week 1 stained for actin (green) and nuclei (blue) show cells evenly distributed with cluster formation after week 1, thus indicating proliferation (the scale bar represents 50 μm). (B) Label-free nonlinear microscopy (two-photon excitation fluorescence, with the autofluorescence of cells shown in yellow) images show similar distributions in the co-cultures compared to the confocal images. However, a decrease in the cell number was seen over time for the irradiated chondrocyte (iChons)-only prints as expected (co-culture prints were conducted with a 1:1 ratio of iPSC to iChons) (the scale bar represents 50 μm). (C) Fluorescence in situ hybridization (FISH) stained only the X chromosomes (X chromosomes, green; Y chromosomes, red) in the co-cultured cartilage-like tissue. Female line, iPSCs; male, iChons (the scale bar represents 10 μm).