The 25 kDa glycoprotein lipocalin-2 (LCN2) is widely expressed and has diverse functions, ranging from physiological to pathophysiological processes. In the liver, LCN2 is primarily associated with inflammatory processes and is considered a potential biomarker in metabolic disorders. However, a significant challenge is the absence of a suitable human in vitro model for studying LCN2 and its associated signaling pathways. Therefore, we have successfully generated patient-derived liver organoids of both male and female origin, providing a novel in vitro model for LCN2 research. Our data show that the self-renewing organoids mimic essential architectural features of hepatocytes, as demonstrated by electron microscopy and F-actin staining. Consistent with the expression profile observed in liver tissue, the isolated 3D organoids exhibit minimal endogenous LCN2 levels. Next, the LCN2 expression was studied at the protein and mRNA levels under inflammatory conditions by treating the organoids with various cytokines and lipopolysaccharides (LPS). Our results show that LCN2 expression is significantly upregulated by IL-1β and TNF-α in an NF-κB-dependent manner, but remains unchanged with IL-6 or LPS. In conclusion, we have established human patient-derived liver organoids as a valuable model for investigating LCN2 signaling mechanisms. This study lays the foundation for future research on the role of LCN2 in liver pathologies, aiding in disease progression understanding and facilitating patient-specific treatment predictions.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its consequences represent a growing global health burden that urgently requires physiologically relevant in vitro models beyond conventional 2D culture systems. In this study, we report the successful establishment of 45 patient-derived liver organoid lines. These organoids were generated from healthy, steatotic and cirrhotic tissues collected from 207 liver surgeries at RWTH University Hospital Aachen, with an initiation success rate of 82%. The organoids were propagated for at least six passages using an optimized protocol. Multiplex immunofluorescence analysis revealed highly proliferative structures with approximately 40% Ki-67-positive cells expressing hepatocyte (Albumin and HNF4α) and cholangiocyte (CK19) markers. Intermittent LGR5 staining suggested the presence of liver progenitor cell features. Quantitative PCR results confirmed variable HNF4α expression, indicating inter-patient heterogeneity in differentiation status. Time-lapse imaging combined with mathematical modeling uncovered a biphasic growth dynamic with an initial linear expansion in the first 15 h, followed by exponential growth (doubling time ≈ 20.6 h) between 30 and 72 h. Overall, our workflow produced genetically and phenotypically stable liver organoids that recapitulate essential features of various hepatic conditions. This provides a solid foundation for disease modeling, potential drug testing, and quantitative systems biology.