Mass generation and long-term expansion of hepatocyte organoids from adult primary hepatocytes

Adult primary human hepatocytes (PHHs) are the gold standard in ex vivo toxicological studies and possess the clinical potential to treat patients with liver disease as advanced therapy medicinal products (ATMPs). However, the utility of this valuable cell type has been limited by short-term functionality and limited expansion potential in vitro. While notable advances have been made in the long-term maintenance of primary hepatocytes, there has been limited success in driving the efficient generation and expansion of adult PHH-derived organoids which recapitulate both liver tissue architecture and function, hampering in vitro studies and regenerative medicine applications. Here we describe the mass generation and long-term expansion of hepatocyte organoids with functionally interconnected hepatic and biliary-like structures from adult primary human hepatocytes. Hepatocyte organoids retain the expression of lineage and functional markers, closely resembling PHH, while also acquiring the expression of regeneration, fetal and biliary markers. Organoids perform key hepatocyte functions while proliferating and can be matured to enhance their functionality. As a proof-of-principle, we demonstrate that hepatocyte organoids can recapitulate hallmarks of cholestasis and steatosis in vitro. Moreover, we show that hepatocytes can be transfected, transduced and gene edited in 3D prior to organoid generation, facilitating a wide range of applications. Our novel hepatocyte organoid system bridges the gap between short-term functionality of primary human hepatocytes and the need for scalable, long-term organoid models of the adult liver, offering immense potential for drug testing, disease modeling, and advanced therapeutic applications. Overall design: Primary adult human hepatocytes were used to form spheroids in hepatocyte spheroid medium, which were then embedded in Matrigel and expanded into hepatocyte organoids (HOs) using a defined hepatocyte expansion medium. These organoids were matured in hepatocyte maturation medium and cultured for several weeks with periodic passaging. Bulk RNA-sequencing was performed on the primary hepatocytes, spheroids and hepato-biliary organoids using the Kapa mRNA HyperPrep Kit, followed by sequencing on Illumina NextSeq2000 and analysis with DESeq2, incorporating donor batch correction and functional annotation of differentially expressed genes.