Introduction: Liver transplantation remains the gold standard for treating end-stage liver diseases; however, the limited availability of donor organs and the risk of graft failure pose significant clinical obstacles. Liver organoids—three-dimensional, self-organizing structures derived from stem cells or primary liver cells—have emerged as promising in vitro models for elucidating hepatic disease mechanisms and for advancing regenerative strategies in transplant medicine.

Methods: A three-dimensional (3D) culture system was established using liver-derived cell lines (HepG2, PLC/PRF/5, and HepaRG) as well as primary hepatocytes isolated from porcine and rat liver tissue, following protocols provided by STEMCELL Technologies. Both Matrigel-embedded and matrix-free culture approaches were employed for the generation of spheroids and organoids. Morphological and structural analyses of the 3D cultures were performed using bright-field, confocal, and light sheet fluorescence microscopy, on both live and sectioned samples.

Results: Organoid formation was successfully achieved using both Matrigel-based and matrix-free methods. Bright-field microscopy enabled rapid assessment of morphology and preliminary validation of staining. Confocal microscopy provided high-resolution imaging of 50 µm-thick sections, while light sheet fluorescence microscopy enabled three-dimensional reconstruction of optically cleared organoids, facilitating comprehensive visualization of internal architecture and staining distribution.

Conclusion: The described protocols support robust generation and imaging of liver organoids across a range of culture conditions and sizes. These methods represent a foundational step toward establishing a cell-based platform for modeling hepatic pathophysiology and developing translational applications in liver disease research.