Fig 1: Functional validation of hepatic organoids and establishment of a platform for toxicity testing. (A) Schematic overview of the experimental workflow for hepatic organoid generation and chemical toxicity testing. (B) Representative bright-field images showing morphological transitions across culture stages in HM, EM, and DM. Scale bar = 500 µm. Images of differentiated organoids uniformly seeded into U-bottom 96-well plates for subsequent chemical treatment. Scale Bar = 1,000 µm. (C) Representative three-dimensional holotomography imaging of morphological differences before and after differentiation. Scale bar = 50 μm, 100 µm. Functional analyses comparing organoids before and after differentiation included (D) ALB expression, (E) AAT expression, and (F) mRNA expression of hepatocyte markers and drug-metabolizing genes. Data are presented as the mean ± standard error of the mean (n = 3) and were analyzed using Student’s t-test (*p < 0.05, **p < 0.01 versus HM). (G) Representative image of ICG uptake and release. Scale bar = 500 µm. (H) IF staining images of liver-specific markers for ALB (green) with nuclei counterstained using 4′,6-diamidino-2-phenylindole (blue). IF images were acquired at ×200 magnification with a scale bar of 500 µm. (I) IF staining images of HNF4α (red) and nuclei (blue). Scale bar = 500 µm. HM, hepatic medium; EM, expansion medium; DM, differentiation medium; ALB, albumin; AAT, α1-antitrypsin; IF, immunofluorescence; ICG, indocyanine green.