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Due to their importance, the use of hepatocytes has gained prominence in scientific research, mainly in the prediction of hepatotoxic drugs. As primary hepatocyte culture presents limitations, human induced pluripotent stem cells (hiPSC) have been used as a promising alternative. However, the differences between 2D and 3D culture during the differentiation of iPSCs into hepatocytes is limitedly explored. In this context, this study compared the differentiation of hepatocytes\u2010like (iHep) from hiPSC in two\u2010dimensional (iHep\u20102D), three\u2010dimensional (iHep\u20103D) and mixed (iHep 2D\u20103D) cultures to evaluate their impact on the acquisition of hepatocyte\u2010like characteristics. After 2D differentiation, transcriptomic analysis revealed that iHep\u20102D has a gene profile very similar to that of primary human hepatocytes, demonstrating that this could be a valuable protocol to use as a starting point for iHep 2D\u20103D differentiation. The cells were also evaluated for the presence of hepatocyte\u2010associated markers by immunofluorescence and our results showed that, although all three cultures generated iHep cells that were able to express specific hepatocyte markers, the iHep 2D\u20103D differentiation generated cells displaying a more pronounced hepatocyte\u2010like phenotype, based on Albumin and CYP3A4 expression. Therefore, in this study we suggest that combining 2D and 3D differentiation may promote a more pronounced hepatocyte\u2010like phenotype in iHeps derived from human iPSCs, which could serve as a valuable model to investigate liver disease.<\/jats:p>","DOI":"10.1002\/bit.70384","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T14:11:17Z","timestamp":1789567877000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Differences of 3D and Mixed 2D and 3D Culture in the Generation of Human iPSC\u2010Derived Hepatocytes"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4827-5436","authenticated-orcid":false,"given":"Cibele Ferreira","family":"Pimentel","sequence":"first","affiliation":[{"name":"Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro Rio de Janeiro Brazil"},{"name":"National Institute of Science and Technology\u2010 INCT 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