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Background & Aims: The use of differentiated hepatocytes from hiPSCs is increasingly favored for BAL,but,inferior function limited it application.In this study,we aimed to establish a 3D condition to mimic the environment that of hepatogenesis in vivo for large-scale culture and higher throughput BAL.Methods: We utilize a synthetic hydrogel to create the HLCs-based spheroids in the 3D condition and evaluate the maturity of spheroids function.Results: We differentiated hiPSCs to DE cells with EB formation in monolayer condition in the first stage,and then dissociated hiPSCs-DE into single cells to implante into hydrogel via self-aggregating to form spheroid.The HLCs-based spheroids showed expression of hepatocyte markers.The ALB secretion,Urea production and glycogen synthesis by HLCs in 3D conditions are significantly higher than the cells in 2D conditions,or HepG2.In addition,metabolic activities of the CYP450 enzymes were also improved while cells differented in 3D condition.Conclusions: This 3D hydrogel conditions can promote human induced pluripotent stem cells differentiated hepatocytes,and it may be have the potential application of for differentiated hepatocytes from hiPSCs as a souree for BAL.