Development, Expansion, and Histological Characterization of Patient-Derived Liver Organoids for Drug Screening and Disease Modeling.

Organoids are self-organizing 3D tissues representing an innovative technology with interesting implications and potential for the study of tumor biology. They can be developed from fine-needle biopsies or resection material from healthy or tumor tissues. Patient-derived organoids are able to retain most of the histological characteristics, the expression profile, and the genomic landscape of the corresponding primary tissues, making them suitable for translational studies and for the identification of molecular alterations in the field of personalized medicine. Here, we describe a detailed protocol for the preparation and in vitro expansion of tumor and non-tumor organoids from surgical resections or needle biopsies of patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), enabling subsequent testing of small-molecule VDAC1 antagonists at different doses. In parallel, we developed a hepatic steatosis model by treating healthy liver organoids with oleic acid, recapitulating key features of lipid accumulation and metabolic dysfunction in vitro. This protocol enables the generation of patient-derived liver organoids that preserve the histological and molecular characteristics of their original tissue, providing a robust and versatile platform for translational studies, personalized drug testing, and the exploration of novel therapeutic strategies targeting tumor metabolism. Key features • Enables reliable establishment of liver organoids from small patient samples, including fine-needle biopsies, suitable for both tumoral and non-tumoral tissue. • Supports the generation of disease-relevant metabolic models, such as hepatic steatosis, enabling the controlled in vitro recapitulation of lipid accumulation and metabolic dysfunction. • Standardized and reproducible platform enabling longitudinal investigation of tumor biology and therapeutic response. • Adaptable system supporting drug screening and co-culture with stromal or immune cell components.