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Microphysiological systems (MPS) have been suggested as promising new approaches to model liver physiology and predict chemical-induced cholestasis in humans. This study comprehensively compared both basal function and toxicant-induced effects in 2D cultures and three liver MPS (i.e., 2-lane OrganoPlate, 3-lane OrganoPlate and PhysioMimix LC12) that were seeded with either HepaRG cells, primary human hepatocytes (PHH), or human induced pluripotent stem cell (iPSC)-derived hepatocytes. PHH and iPSC-derived hepatocytes (iHeps) were tested up to 7\u00a0days while HepaRG were evaluated over 30\u00a0days. Albumin, urea, CYP3A4 activity, and bile acids were measured. HepaRG and PHH showed comparable function in 2D and PhysioMimix LC12, with albumin higher for HepaRG and urea higher for PHH. HepaRG maintained production of biomarkers for up to 30\u00a0days in both 2D and PhysioMimix LC12. In both OrganoPlate models, HepaRG produced higher levels of albumin and urea as compared to iHeps; still, HepaRG function in OrganoPlate was lower than that in 2D or PhysioMimix LC12. Bile acid synthesis (after 7\u00a0days) was much higher with PHH in the PhysioMimix LC12 as compared to 2D PHH or 2D HepaRG. Upon exposure to cholestatic agents (bosentan, 2-octynoic acid, \u03b1-naphthyl isocyanate), robust CYP3A4 induction was observed in HepaRG and PHH treated with bosentan and \u03b1-naphthylisocyanate. Only in PhysioMimix LC12, both HepaRG and PHH, all compounds elicited decreased bile acid release into cell culture medium, a biomarker for cholestasis. In summary, the hepatocyte functional markers (CYP3A4, albumin, urea) were comparable between PHH and HepaRG in 2D and PhysioMimix LC12 MPS. However, the effects of cholestatic agents on PHH and HepaRG, specifically, bile acid release were detected only in the PhysioMimix LC12 with PHH showing more consistent responses compared to HepaRG.<\/jats:p>","DOI":"10.1007\/s00204-025-04263-1","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T19:18:39Z","timestamp":1765221519000},"page":"1033-1047","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Exploring the potential of liver microphysiological systems of varied configurations to model cholestatic chemical effects"],"prefix":"10.1007","volume":"100","author":[{"given":"Katharina S.","family":"Nitsche","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Courtney","family":"Sakolish","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul L.","family":"Carmichael","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip","family":"Hewitt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piyush","family":"Bajaj","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen S.","family":"Ferguson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah M.","family":"Lloyd","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah S.","family":"Wilson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hans","family":"Bouwmeester","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9340-7384","authenticated-orcid":false,"given":"Ivan","family":"Rusyn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"issue":"1","key":"4263_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.cld.2016.08.001","volume":"21","author":"OA Almazroo","year":"2017","unstructured":"Almazroo OA, Miah MK, Venkataramanan R (2017) Drug metabolism in the liver. 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