Fig. 5: 3D geometrical constrains imposed by hydrogel-in-hydrogel live bioprinting on organoid and organotypic cultures.

a, b Induction of polarization in human fetal hepatocyte organoids. a Bright field images showing two printing strategies: distant walls not touching the growing organoid (above) and adjacent pillars touching the growing organoids (below) after 7 days of culture. Scale bar 100 µm. b Immunofluorescent panels showing non-polarized organoid distant from the printed structures (above) and a polarized organoid in correspondence of the printed pillars (below). Integrin beta-4 (INTβ4) shown in green, multidrug resistance-associated protein 2 (MRP2) shown in red, zonula occludens-1 (ZO-1) shown in cyan. Nuclei are stained with Hoechst (blue). Scale bars, 50 µm (large images, left) and 10 µm (higher magnification, right). c–f Ex vivo culture of mesenchyme-free lung epithelium rudiments, isolated from embryonic mice at stage E12.5. c Schematic of isolated fetal lung tips ex vivo culture and guided branching morphogenesis following 2 P bioprinting of gelatin pillars (red circles) in Matrigel. d 5-h interval snapshots of time-lapse reconstruction of budding lung tip during guided morphogenesis around pillar (red circle, indicated by red arrow). See full time-lapse in Supplementary Video. 3. Scale bar 150 µm. e Plot showing angle of tip bifurcation vs time from tip-pillar contact time (time 0) to 24 h of culture. Data are shown as mean ± s.e.m. of 8 independent replicates. f Bright-field images and immunofluorescent panel showing lung tip branching in between two pillars (red circles) and inner (luminal) polarity maintained (F-actin, green). Nuclei are stained with Hoechst (blue). Scale bar 100 µm. g Immunofluorescent panel showing lung tip branching in between pillar (red circle) with downregulation of sox9 (red) in correspondence of the pillar. Nuclei are stained with Hoechst (blue). Scale bars 50 µm.