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This study, therefore, is designed to define environmental requirements necessary to sustain the homeostasis of terminally differentiated hepatocytes. Our study reveals that the supplementation of dimethyl sulfoxide (DMSO) is indispensable in mitigating fate deterioration and promoting adaptation to the in vitro environment, resulting in the restoration of tight cell\u2013cell contact, cellular architecture, and polarity. The morphological recovery was overall accompanied by the restoration of hepatocyte marker gene expression, highlighting the interdependence between the cellular architecture and the maintenance of cell fate. However, beyond the recovery phase culture, DMSO supplementation is deemed detrimental due to the potent inhibitory effect on a multitude of hepatocyte functionalities while its withdrawal results in the loss of cell fate. In search of DMSO substitute, our screening of organic substances led to the identification of dimethyl sulfone (DMSO2), which supports the long\u2010term maintenance of proper morphology, marker gene expression, and hepatocytic functions. Moreover, hepatocytes maintained DMSO2 exhibited clinically relevant toxicity in response to prolonged exposure to xenobiotics as well as alcohol. These observations suggest that the stepwise culture configuration consisting of the consecutive supplementation of DMSO and DMSO2 confers the microenvironment essential for the fate and functional maintenance of terminally differentiated human hepatocytes.<\/jats:p>","DOI":"10.1096\/fj.202201292rr","type":"journal-article","created":{"date-parts":[[2023,1,6]],"date-time":"2023-01-06T10:35:09Z","timestamp":1673001309000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["<scp>Long\u2010term<\/scp>\n                    cell fate and functional maintenance of human hepatocyte through stepwise culture configuration"],"prefix":"10.1096","volume":"37","author":[{"given":"Go","family":"Sugahara","sequence":"first","affiliation":[{"name":"Department of Medicine, Division of Gastrointestinal and Liver Diseases University of Southern California, Keck School of Medicine  Los Angeles California USA"},{"name":"Research and 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