Three-dimensional-bioprinted cartilage-like tissue. (A and B) Histology sections of the 3D-bioprinted constructs. (A) At week 3 (blank - no cells), week 0, week 1, and week 2 of differentiation, which followed 2 weeks of proliferation in the iPSC maintenance medium (stained with Alcian blue-van Gieson for proteoglycans/glycosaminoglycans (GAGs) (blue) and nuclei (brown)) (the scale bar represents 100 μm). (B) The 3D-bioprinted chondrocyte-derived iPSCs (printed together with iChons, which had been diminished) at week 5 of differentiation, zoomed in (upper row) and whole section (lower row) images of sections stained for GAGs, Safranin O for cartilage (with nuclear counterstain), and hematoxylin and eosin (H&E) for extracellular matrix (with nuclear counterstain) (the scale bar represents 100 μm or 500 μm). (C) Label-free images of unstained sections (of areas corresponding to red and green boxes from the lower row of B) shows highly dense cell areas (cell autofluorescence, yellow) and collagen-like fibrils (second-harmonic generation, cyan). The highly dense cell area in the red box corresponded to higher GAG staining (the scale bar represents 50 μm). The number of cells per ml was calculated from the high-density (red square) and low-density (green square) areas. (D) Fluorescent image of an immunohistochemistry section (from the same 3D printed sample as B and C) stained for collagen type II (green) (with nuclear counterstain shown in blue), which shows the production of extracellular matrix collagen type II in a representative cell cluster (the scale bar represents 10 μm).