Mallanna, S. K.; Karanth, S. S.; Marturano, J. E.; Kudva, A. K.; Lehmann, M.; Morse, J. K.; Jamiel, M.; Norman, T.; Wilson, C. G.; Munarin, F.; Broderick, D.; Van Buskirk, M.; Uddin, E.; Ret, M.; Steele, C. A.; Cheema, M. K.; Black, J.; Vanderploeg, E. J.; Chen, C. S.; Bhatia, S.; Rezania, A.; Lowery, T. J.; Cazanave, S. C.; Chhabra, A.

Liver disease affects millions annually in the United States, with orthotopic transplantation as the only curative option for many patients. However, the scarcity of donor organs highlights a need for alternative cell-based therapies. Hepatocyte-based approaches are promising due to the cells\' inherent synthetic, metabolic, and detoxifying functions, but they face critical barriers, including the lack of a scalable source of functional hepatocytes and poor engraftment. In this study, we developed a scalable process for expanding primary human hepatocytes (PHHs) while preserving their identity and function. By leveraging heterocellular aggregation with stromal cells, we generated cryopreserved \"seed\" constructs that maintained viability and function post-thaw. Seeds demonstrated enhanced metabolic and detoxification functions and robust engraftment across multiple anatomic sites outside of the liver. Our approach addresses key limitations of hepatocyte-based therapies, offering a stable, scalable, and clinically viable platform for liver cell therapy applications.