摘要Multicellular microtissues of primary human hepatocytes (PHHs) co-cultured with other supporting cell types are a promis-ing model for drug screening and toxicological studies. However, these liver microtissues (LMs) rapidly lose their functions during ex vivo culture. Here, in order to mimic the cellular and structural hepatic microenvironment, we co-cultured PHHs with human mesenchymal stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs) in the presence of cell-sized microparticles (MPs) derived from liver extracellular matrix (LEMPs). The microwell culture platform enabled biofabrication of size-controlled multicellular microtissues (PHH:HUVEC:MSC = 3:2:1) with efficient LEMP incorpora-tion (about 70％ at a 2:1 ratio of cells:MP). The biofabricated liver microtissues (BLMs) were cultured ex vivo for 14 days and compared to the cell-only LM in terms of gene and protein expression, functional activity, cytochrome P450 (CYP450) enzyme inducibility, and drug sensitivity. The results supported superior hepatic-related gene expression, functional activ-ity, and polarity for PHH in BLM compared to LM. CYP450 enzyme inducibility and dose-responsive sensitivity to toxic drugs were significantly higher in the BLM group. In conclusion, microtissue engineering by incorporation of tissue-specific microparticles within a multicellular microtissue can offer some advantages for drug discovery studies and cell transplanta-tion applications. In the near future, this approach could generate a scalable platform of several functional biofabricated microtissues representing different organs.