Genome-wide association studies (GWAS) have now correlated hundreds of genetic variants with complex genetic diseases and drug efficacy. Functional characterization of these factors remains challenging, particularly because of the lack of human model systems. Molecular and nanotechnological advances, in particular the ability to generate patient-specific PSC lines, differentiate them into diverse cell types, and seed and combine them on microfluidic chips, have led to the establishment of organ-on-a-chip (OoC) platforms that recapitulate organ biology. OoC technology thus provides unique personalized platforms for studying the effects of host genetics and environmental factors on organ physiology. In this review we describe the technology and provide examples of how OoCs may be used for disease modeling and pharmacogenetic research.

Metabolic dysfunction associated with steatotic liver disease (MASLD) is the result of disturbed lipid metabolism. In MASLD, the accumulation of free fatty acids (FFAs) in hepatocytes causes lipotoxicity mediated by oxidative stress. Coffee compounds are known for their beneficial effects in MASLD; however, the mechanisms still need to be further explored. The aim of this study was to elucidate the protective mechanisms of coffee compounds against palmitate-induced lipotoxicity in primary hepatocytes. Methods: Primary hepatocytes were isolated from male Wistar rats and treated with palmitate (1 mmol/L) in combination with caffeine (CF: 1 mmol/L) or chlorogenic acid (CGA: 5 µmol/L). Mitochondrial ROS production, palmitate-induced necrosis, antioxidant response, ER stress markers and lipid droplet (LD) formation were assessed. Monoacylglycerols 2-SG (2-Stearolylglycerol), 2-OG (2-Oleoylglycerol) and SCD-1 (Stearoyl-CoA Desaturase 1) inhibitors were used to modulate LD formation. LD formation in steatotic Zucker rat hepatocytes was also investigated. Results: CF and CGA prevented palmitate-induced cell death and reduced ROS production. CF and CGA induced the antioxidant response, especially HO-1 expression, but had no significant effect on ER stress markers. CF and CGA increased LD formation in palmitate-treated cells. This effect was significantly reduced by 2-SG and SCD-1 inhibitors but enhanced by 2-OG. Lipid droplets were associated with lower palmitate toxicity and reduced ROS production. Conclusions: CF and CGA protect hepatocytes from lipotoxicity via modulation of the antioxidant response and enhance lipid droplet formation via an SCD-1-dependent mechanism. Oxidative stress-related toxicity in hepatocytes can be prevented by enhancing LD formation.

Background/Objectives: Nutraceuticals, including short-chain fatty acids (SCFAs) and antioxidants (AOXs), are nutrient-derived bioactive compounds considered as potential treatments for metabolic-associated steatotic liver disease (MASLD). However, in vitro studies of their effects are limited by inconsistent experimental conditions, including differences in cell lines, methods of steatosis induction, and culture media, and by reliance on qualitative rather than quantitative assessments. Here, we systematically evaluate the anti-steatotic potential of eight commonly used nutraceuticals—three SCFAs (butyrate, acetate, and propionate) and five AOXs (resveratrol, curcumin, berberine, chlorogenic acid, and vitamin E)—using a standardized in vitro approach. Methods: Following a systematic literature review to identify common experimental conditions, we developed an assay to validate steatosis induction and quantified the effects of the nutraceuticals. For our studies we used the HepG2 liver cancer cell line and the Fa2N-4 immortalized hepatocyte cell line. Steatosis was modeled by stimulating cells with free fatty acids and fructose for 48 h. Nutraceuticals were added either concurrently with steatotic stimulation, to assess preventive effects, or after 24 h to assess therapeutic effects. Anti-steatotic drugs (resmetirom, semaglutide, obeticholic acid, and a DGAT2 inhibitor) were included as positive controls. Intracellular triglyceride levels were measured to quantify steatosis. Results: A systematic review of 46 studies revealed large differences in culture conditions, steatosis induction, and nutraceutical assessment. In our experiments, most nutraceuticals did not reduce intracellular triglycerides, with the exception of vitamin E. Surprisingly, butyrate, berberine, and curcumin increased triglyceride accumulation. Resmetirom was the only drug that significantly decreased triglycerides, while obeticholic acid, semaglutide, and the DGAT2 inhibitor showed minimal or inconsistent effects. Fa2N-4 cells were generally more sensitive than HepG2 cells, showing larger absolute changes in triglyceride levels in response to both nutraceuticals and resmetirom. Conclusions: We established a standardized in vitro assay to evaluate the anti-steatotic potential of nutraceuticals. Using this system, we found that SCFAs and AOXs did not consistently reduce intracellular triglycerides, highlighting the need for quantitative assessments and careful validation when studying anti-steatotic interventions in vitro.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide. In vitro MASLD studies predominantly rely on hepatocellular carcinoma-derived cell lines such as HepG2 that are poorly differentiated and exhibit cancer-associated metabolic reprogramming that suppresses key adult hepatic functions. Primary human hepatocytes offer greater physiological relevance and capture inter-individual biological variability that reflects human population diversity, but their use is limited by high costs, availability, and rapid dedifferentiation in culture. Here, we characterized the immortalized primary human hepatocyte cell line Fa2N-4 as an alternative in vitro model for MASLD. We performed a comparative analysis of Fa2N-4 and HepG2 cells, assessing their genomic architecture, lipid-induced steatosis, and pharmacological responsiveness. The Fa2N-4 and HepG2 cell models exhibited pronounced differences, including distinct karyotypes, divergent MASLD-associated genetic risk variant profiles, and markedly different transcriptional responses to lipid loading and drug treatment. We further examined the hepatic response to resmetirom, a first-in-class US Food and Drug Administration-approved therapy for treating metabolic dysfunction-associated steatohepatitis. Resmetirom, a thyroid hormone receptor-β agonist, reduced intracellular triglyceride accumulation in Fa2N-4 cells but not HepG2 cells, and this was accompanied by transcriptional changes in mitochondrial glycolysis and oxidative phosphorylation pathways. These findings demonstrate that hepatocyte model selection critically influences experimental outcomes in MASLD research and highlight Fa2N-4 cells as a physiologically relevant platform for mechanistic and translational studies of MASLD therapeutics.

Human liver organoids (HLOs) and liver-on-a-chip (LoC) systems are emerging as physiologically relevant human models for studying liver function, disease, and drug metabolism, often in combination with human induced pluripotent stem cell (hiPSC)-derived tissues. However, hiPSC-derived models often display batch-to-batch variation and incomplete maturation, and the contribution of microfluidic flow to hepatic maturation remains insufficiently characterized. Here, we developed a cryopreservable and scalable workflow to generate hiPSC-derived hepatic organoids that can be directly matured to either static HLOs or LoC systems, enabling matched comparison of both platforms. Transcriptomic and functional characterization revealed progressive hepatic maturation during organoid differentiation, including increased expression of liver-specific metabolic pathways, enhanced albumin secretion, and increased CYP3A4 activity. Compared to mature HLOs, LoCs exposed to continuous microfluidic flow exhibited transcriptomic profiles suggesting further maturation, with increased enrichment of pathways related to lipid metabolism, xenobiotic metabolism, transport, and tissue organization. These findings demonstrate that microfluidic perfusion promotes hepatic metabolic specialization compared to static organoid culture while maintaining donor-specific characteristics. Together, this study establishes a robust hiPSC-derived LoC platform and highlights the potential of flow-based systems for improved modeling of human liver physiology, disease mechanisms, and drug responses.