Automated and scalable expansion of human liver organoids for translational applications

BACKGROUND: Human tissue-derived organoids hold strong potential for personalized medicine and cell therapy, but this requires large cell quantities. Conventional organoid culture systems remain labor-intensive, are difficult to scale, and lack process control. Here, we present a novel strategy using an automated bioreactor platform that enables large-scale expansion of human liver organoids. METHODS: Human liver organoids were expanded for 14 days in a single bioreactor suspension culture bag and compared with spinner flasks and static dome cultures. Cell yield, viability, fold expansion, morphology, and phenotypic markers (LGR5, E-cadherin, Vimentin, Ki67) were assessed. The system’s uninterrupted workflow enabled seamless transition to differentiation: using integrated perfusion, we performed a direct medium switch from expansion to hepatic differentiation without harvesting or disrupting the culture. Commitment to the hepatic lineage was evaluated by expression of ALB, CYP3A4, MRP2, and HNF4A. RESULTS: By day 14, the bioreactor generated an average of 5.63 × 108 (± 1.1 × 108) viable cells, while spinner flasks reached 1.22 × 108 (± 4.26 × 107) cells, while static cultures yielded only 4.02 × 105 (± 2.81 × 105), making the bioreactor’s output ~ 1400 times greater than static cultures (p = 0.022) and nearly five times higher than spinner flasks. This substantial gain in absolute cell yield is a promising indicator for downstream translation. Organoids preserved phenotypic integrity and proliferative capacity as shown by sustained expression of LGR5, E-cadherin, and Ki67. Bioreactor-cultured organoids exhibited robust growth and intact cyst-like morphology with a large size, due to the absence of mechanical fragmentation and related cellular stress. As a proof-of-principle, bioreactor-grown organoids differentiated efficiently toward the hepatic lineage, as evidenced by a downregulated gene expression of LGR5 and Ki67, with elevated gene expression of ALB, CYP3A4, MRP2, and HNF4A, along with an upregulated secretion of Albumin. CONCLUSION: The system establishes a closed, monitored, and scalable upstream workflow for liver organoid expansion. This work represents a significant step toward organoid production for future cell therapy and regenerative medicine applications, while maintaining phenotypic stability and differentiation capacity.