Development and characterization of non-malignant human hepatocyte lines with retained hepatic functional identity for in vitro applications

Abstract Advances in liver metabolism research are constrained by the lack of stable non-malignant hepatocyte models. Primary human hepatocytes (PHHs), although the gold standard, are limited availability, donor variability, and rapid functional decline, restricting long-term and high-throughput use. In contrast, cancer-derived lines, such as HepG2 and Huh7, display altered gene expression and impaired metabolic competence. To overcome these limitations, we generated two immortalized non‑cancer hepatocyte lines, IL‑HEP1 and IL‑HEP2, using SV40 large T antigen and human telomerase reverse transcriptase to extend proliferative lifespan. Comprehensive gene‑expression and functional analyses confirmed retention of key hepatocyte-associated traits following immortalization. Principal component analysis and hierarchical clustering revealed distinct yet physiologically relevant transcriptomic signatures. The IL‑HEP models also maintained controlled proliferation, contact inhibition, and stable epithelial morphology, avoiding the deregulated growth and tumor‑like metabolic reprogramming characteristic of HepG2. Functionally, IL‑HEP1/2 cells secreted albumin more robustly than THLE2, exhibited CYP2C9 activity exceeding HepG2 and CYP3A4 activity at approximately 45–50% of HepG2 levels, and displayed balanced mitochondrial respiration and moderate glycolytic capacity relative to HepG2 and THLE2. Together, IL‑HEP1 and IL‑HEP2 provide scalable, stable, and functionally competent hepatic models, offering a physiologically relevant alternative to hepatocarcinoma‑derived lines and enabling more predictive in vitro studies in pharmacology, toxicology, and regenerative medicine. It should be noted that these comparisons are made relative to HepG2 and THLE-2; metabolically optimized systems such as differentiated HepaRG cells, Upcyte hepatocytes, and primary human hepatocytes represent a substantially higher standard of xenobiotic metabolic competence than the IL-HEP lines currently match.