Fig. 2: Histological and functional characterization of BEL constructs.

a Representative photograph of a BEL seeded with HUVECs and porcine hepatocytes. b Hematoxylin and eosin staining of representative co-culture BEL tissue sections fixed 48 h after seeding hepatocytes. c–e Immunofluorescent staining of cell lineage markers in non-serial tissue sections 48 h after seeding hepatocytes: (c) CD31 & albumin; (d) CD31 & FAH; (e) CD31 and LYVE1. f Schematic depicting seeding and culture timeline for HUVEC only, hepatocyte only, and co-culture BEL constructs used in (g, h, j, k). g vWF production in grafts before and after hepatocyte seeding. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BEL constructs are shown. Error bars denote the mean and standard deviation at each time point. h 24-h average albumin production in co-culture grafts 48 h following hepatocyte seeding. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BELs are shown. Error bars denote the mean and standard deviation. i Schematic of in vitro ammonia clearance and urea production assay. Ammonium chloride is added to the bioreactor media at a concentration of 0.8 mM. Ammonia and urea levels are measured in media samples taken at t = 0, 1, 2 7, and 23 h following the addition of ammonium chloride. Error bars denote the mean and standard deviation each time point. j, k Ammonia clearance (j) and urea production kinetics (k) following the addition of ammonium chloride to the bioreactor perfusion media. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BELs, media only controls (n = 4) are shown. Error bars denote the mean and standard deviation each time point. BEL—bioengineered liver; FAH—fumarylacetoacetate hydrolase; CD31—cluster of differentiation 31; LYVE1—lymphatic vessel endothelial hyaluronan receptor 1; vWF—von Willebrand factor.