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.