Y37 - Gelatin-Based Hydrogels Support Functional Maturation of Hepatic Aggregates for In Vitro Liver Models

Introduction: : The liver plays a critical role in metabolism, detoxification, and homeostasis, yet current in vitro models fail to fully recapitulate its complex functions. Three-dimensional hepatic aggregates offer a promising alternative, but their maturation remains limited without a supportive extracellular matrix. Developing functional liver tissue in vitro is critical for advancing drug screening and regenerative medicine. Gelatin-based hydrogels enzymatically crosslinked with mTG, derived from denatured collagen, offer tunable biochemical and mechanical cues that mimic native liver microenvironments. In this study, we investigate how gelatin hydrogel properties influence the structural and functional maturation of iPSC-derived hepatic aggregates. We hypothesize that optimized gelatin formulations can enhance hepatic marker expression and metabolic function, supporting more physiologically relevant liver tissue models for drug screening and regenerative applications.

Materials and

Methods: : Human iPSCs were differentiated into hepatocyte-like cells (HLCs) over 21 days. Definitive endoderm was induced using the STEMdiff Definitive Endoderm Kit (STEMCELL Technologies). After foregut specification, for 15 days, Hepatocyte Culture Medium (HCM) with 20 ng/mL Oncostatin-M (days 16–21) was used to promote hepatic lineage commitment. Hepatic aggregates (HA) were generated between days 13–16 using Aggrewell 400 plates, seeded at 200 cells per microwell, and cultured in HCM + OSM until day 21. On day 21, HA were encapsulated in gelatin-based hydrogels enzymatically crosslinked with microbial transglutaminase (mTG) with varying stiffness achieved by using different gelatin concentrations (4%, 5%, and 6% w/v; designated as Gel4, Gel5, and Gel6, respectively). and cultured until day 26. Maturation was assessed using immunofluorescence (Albumin, HNF4A, AFP, A1AT), qRT-PCR (ALB, KRT19, APOB, SERPINA), and metabolic assays (albumin, urea secretion). 2D cultures were used as controls. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test.

Results, Conclusions, and Discussions:: Hepatic aggregates derived from iPSCs exhibited maturation when encapsulated in gelatin-based hydrogels of varying stiffness (Gel4, Gel5, Gel6) and cultured until day 26. Immunofluorescence revealed that HAs in Gel6 exhibited stronger expression of mature hepatic markers HNF4A and A1AT compared to Gel4 and Gel5, while fetal marker AFP was notably reduced. This pattern was supported by mean intensity quantification, confirming statistically significant differences (p < 0.01) in marker expression levels across conditions. Quantitative RT-PCR demonstrated that Gel6 aggregates showed significant upregulation of hepatocyte-specific genes such as ALB, APOB, and SERPINA1, and downregulation of immature or biliary markers like KRT19 and AFP, relative to both Gel4 and 2D controls. Albumin secretion was significantly increased in Gel6, as measured by Albumin ELISA, and supported by enhanced urea synthesis, indicating improved hepatic metabolic function. These findings suggest that the mechanical properties of the gelatin hydrogel microenvironment significantly influence hepatic maturation. Gel6, which presumably offers optimal matrix stiffness and porosity, provided a favorable niche for cell-cell and cell-matrix interactions, supporting both structural organization and functional gene expression. The 3D format also appeared to suppress fetal gene expression, promoting transition to a more adult-like hepatic phenotype.
In conclusion, this study demonstrates that gelatin hydrogels can serve as effective scaffolds for hepatic tissue maturation. By encapsulating hepatic aggregates at the hepatoblast stage and allowing extended culture to day 26, we observed enhanced hepatic marker expression, metabolic activity, and tissue organization. This platform provides a scalable, modular strategy for engineering hepatic tissue for applications in drug testing, disease modeling, and future regenerative therapies. Future work will aim to integrate endothelial co-culture to mimic liver structure and vascularization in vitro.

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