<i>In situ</i> differentiation and generation of functional liver organoids from human iPSCs in a 3D perfusable chip system

TLDR

Liver organoids derived from human pluripotent stem cells offer a novel in‑vitro model for studying liver development, disease mechanisms, and drug testing, yet achieving functional organoids in a controlled microenvironment remains challenging. This study introduces a strategy to engineer hiPSC‑derived liver organoids within a 3D perfusable chip by integrating stem cell biology with microengineering. The approach involves forming hiPSC‑based embryoid bodies, inducing in‑situ hepatic differentiation, and maintaining long‑term 3D culture in a perfusable micropillar chip. The resulting organoids displayed heterogeneous hepatocyte and cholangiocyte populations, enhanced viability, up‑regulated endodermal and mature hepatic genes, superior albumin and urea production, metabolic activity, and dose‑dependent hepatotoxic responses to acetaminophen, demonstrating that fluid flow promotes liver‑specific functions and validating the platform for regenerative medicine, disease modeling, and drug testing.

Abstract

Liver organoids derived from human pluripotent stem cells (PSCs) represent a new type of in vitro liver model for understanding organ development, disease mechanism and drug testing. However, engineering liver organoids with favorable functions in a controlled cellular microenvironment remains challenging. In this work, we present a new strategy for engineering liver organoids derived from human induced PSCs (hiPSCs) in a 3D perfusable chip system by combining stem cell biology with microengineering technology. This approach enabled formation of hiPSC-based embryoid bodies (EBs), in situ hepatic differentiation, long-term 3D culture and generation of liver organoids in a perfusable micropillar chip. The generated liver organoids exhibited favorable growth and differentiation of hepatocytes and cholangiocytes, recapitulating the key features of human liver formation with cellular heterogeneity. The liver organoids in perfused cultures displayed improved cell viability and higher expression of endodermal genes (SOX17 and FOXA2) and mature hepatic genes (ALB and CYP3A4) under perfused culture conditions. In addition, the liver organoids showed a marked enhancement of hepatic-specific functions, including albumin and urea production and metabolic capabilities, indicating the role of mechanical fluid flow in promoting the functions of the liver organoids. Moreover, the liver organoids exhibited hepatotoxic response after exposure to acetaminophen (APAP) in a dose- and time-dependent manner. The established liver organoid-on-a-chip system may provide a promising platform for engineering stem cell-based organoids with applications in regenerative medicine, disease modeling and drug testing.