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Animal tests and membrane vesicle assay are currently being conducted to assess the risk of cholestasis in the preclinical stage. However, these methods have drawbacks, such as species differences with humans and difficulties in evaluating the effects of drug metabolism and other transporters, implying the need for a cholestasis risk assessment system using human hepatocytes. However, human hepatocytes hardly form functional, extended bile canaliculi, a requirement for cholestasis risk assessment. We previously established a culture protocol for functional, extended bile canaliculi formation in human iPSC-derived hepatocytes. In this study, we modified this culture protocol to support the formation of functional, extended bile canaliculi in human cryopreserved hepatocytes (cryoheps). The production of bile acids, which induces bile canaliculi extension, increased time-dependently during bile canaliculi formation using this protocol, suggesting that increased bile acid production may be involved in the extended bile canaliculi formation. We have also shown that our culture protocol can be applied to cryoheps from multiple donors and that bile canaliculi can be formed stably among different culture batches. Furthermore, this protocol enables long-term maintenance of bile canaliculi and scaling down to culture in 96-well plates. We expect our culture protocol to be a breakthrough for in vitro cholestasis risk assessment.<\/jats:p>","DOI":"10.1007\/s00204-024-03757-8","type":"journal-article","created":{"date-parts":[[2024,5,16]],"date-time":"2024-05-16T10:02:14Z","timestamp":1715853734000},"page":"2605-2617","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Formation of functional, extended bile canaliculi, and increased bile acid production in sandwich-cultured human cryopreserved hepatocytes using commercially available culture medium"],"prefix":"10.1007","volume":"98","author":[{"given":"Shinichiro","family":"Horiuchi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukie","family":"Kuroda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryota","family":"Oyafuso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Komizu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuya","family":"Maeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6502-508X","authenticated-orcid":false,"given":"Seiichi","family":"Ishida","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,5,16]]},"reference":[{"issue":"3","key":"3757_CR1","doi-asserted-by":"publisher","first-page":"447","DOI":"10.1124\/dmd.108.023465","volume":"37","author":"K Abe","year":"2008","unstructured":"Abe K, Bridges AS, Brouwer KL (2008) Use of sandwich-cultured human hepatocytes to predict biliary clearance of angiotensin II receptor blockers and HMG-CoA reductase inhibitors. 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