Large‐Scale Production of Expandable Hepatoblast Organoids and Polarised Hepatocyte Organoids From hESCs Under 3D Static and Dynamic Suspension Conditions

ABSTRACT To date, generating viable and functional hepatocytes in large scale remains challenge. By employing 3D suspension condition with the support of low concentration Matrigel, a novel culture system was developed to generate expandable hepatoblast organoids (HB‐orgs) and mature polarised hepatocyte organoids (P‐hep‐orgs) from human embryonic stem cells (hESCs) in both dishes and bioreactors. scRNA‐seq and functional assays were used to characterise HB‐orgs and P‐hep‐orgs. hESC‐derived HB‐orgs could proliferate at least for 15 passages, leading to 10 12 in total cells in 4 weeks. P‐hep‐orgs differentiated from HB‐orgs displayed characteristics of mature hepatocytes with polarisation. Moreover, single‐cell RNA sequencing exhibited that over 40% of cells in P‐hep‐orgs were highly fidelity with human primary hepatocytes. Eventually, large‐scale production of P‐hep‐orgs could be generated from massively expanded HB‐orgs within 1 week with similar number in bioreactors, which were achieved by the enhancements in energy metabolism contribute to the expansion of HB‐orgs and maturation of P‐hep‐orgs in bioreactors. By providing a cost‐efficient and robust platform, our study represents a significant step toward manufacturing large‐scale functioning hESC‐derived hepatocytes for cell‐based therapeutics, disease modelling, pharmacology and toxicology studies.