Characterization of patient-derived liver organoids

Methods

Organoids were isolated from tumor and matched non tumor liver tissue, expanded, and differentiated by removing stemness/progenitor-maintening and adding hepatocyte promoting factors. They were characterized by immunohistochemistry and whole mount immunofluorescence, by gene expression via qPCR and bulk RNA seq, and were assessed for their mitochondrial and glycolytic function using Seahorse assays. Four differentiation media were tested side by side.

Matrix free culture was performed in a Cero 3D bioreactor. Starvation was modeled by exposure to nutrient depleted medium. Results: Organoids formed epithelial monolayers with lumens, expressed both hepatocyte and cholangiocyte markers to various extent.

Differentiation yielded more compact structures, and upregulated hepatocyte associated genes, while downregulating stemness and proliferation-relevant genes. RNA seq confirmed clear separation between expansion and differentiation states, with enrichment of bile secretion and xenobiotic/drug metabolism pathways and reduced proliferation signatures. Culture in four differentiation media resulted in broadly similar transcriptional profiles, with one medium supporting the strongest metabolic activity.

Matrix free culture produced smaller, denser organoids with multiple lumens, while maintaining the bipotential state of the organoids. Starvation slightly lowered basal respiration and glycolytic function, alongside upregulation of fasting response genes. Conclusions: Liver derived organoids showed bipotent characteristics and inter line variability, reaching partial hepatocyte like differentiation.

Culture conditions, including the choice of medium and matrix free system, influenced morphology, metabolism and gene expression, underscoring the need for detailed characterization of each established line prior to their use as models for studying liver biology and metabolic stress.