Fig. 3.

Endothelial cells penetrate hepatic microtissues forming lunemized, perfusable microvasculature. a Maximum intensity projection of hepatocyte spheroid co-cultured with microvessels for 7 days and stained against albumin (green) and CD31 (red). Scale bar: 200 µm. b Maximum intensity projection of hepatocyte organoids co-cultured with microvessels for 21 days and stained against albumin (green) and CD31 (red). Scale bar: 200 µm. c Single-plane confocal images of hepatocyte spheroid co-cultured with microvessels for 7 days and stained against albumin (green) and CD31 (red). Lumenized spaces are observable in the vessels connecting the main tubes to the hepatocyte spheroid (I, white arrow) and in close proximity of hepatocytes (ii, white arrow). Scale bar: 200 µm (i) and 50 µm (ii). d Cryo-sections of spheroids co-cultured for 1 (i), 4 (ii), and 7 (iii) days on microvascular beds stained against albumin (green), CD31 (red), and nuclei (blue). White arrow indicates penetration of CD31 + endothelial cells inside the hepatocyte spheroid. Scale bar: 200 µm. e EM images of sections of hepatocyte organoids co-cultured with microvessels for 7 days indicating endothelial cells (EC) with distinct lumen (i) and vascular-hepatic contact point (ii) with intracellular pinocytic vesicles (iii, white arrow). Scale bar: 10 µm. f Schematic depiction of flow through the chip to assess perfusability of vascularized liver cultures using 150-KDa FITC-labeled dextran. g Over-time fluorescence microscopy images of 150-KDa FITC-labeled dextran perfusing a hepatocyte spheroid co-cultured for 7 days with microvessels. Dotted ellipse indicates FITC-labeled dextran emerging from the spheroid and flowing in the lateral perfusion channel