Human primary hepatocytes are considered as the golden standard for in vitro model systems regarding drug development, toxicity assessment and metabolism studies. However, their rapid loss in cell viability in conventional 2D cultures limit the usage for these cells. Human pluripotent stem (hPS) cell-derived hepatocytes have a great potential to become a future in vitro model for hepatocyte applications if they possess a relevant usage window and functionality, which has indeed been challenging to accomplish. However, our newly developed hepatocyte maintenance medium enables culturing of cryopreserved human primary hepatocytes as well as hPS cell-derived hepatocytes for 4 respectively 2 weeks with maintained viability and stable activities of several key cytochrome P450 enzymes (CYPs). Multiple analyses on cryopreserved hPS cell-derived hepatocytes, including RT-qPCR, immunostainings, functional assays such as albumin secretion and CYP activity assays demonstrate mature features and high functionality. Importantly, the hPS cell-derived hepatocytes show expression of essential genes of the drug metabolizing machinery, such as CYPs, phase II enzymes and transporters. An extended in vitro culture time for hepatocytes enables chronic toxicity testing and we show that our hPS cell-derived hepatocytes could be exposed to known hepatotoxins for up to 14 days. Cells respond expectedly to these toxic compounds demonstrating their utility for chronic toxicity studies. The hPS cell-derived hepatocytes also respond to insulin and have the ability to take up and store low-density lipoproteins as well as fatty acids. This novel maintenance medium presented here, maintains the viability of cryopreserved human primary hepatocytes for an outstanding time of culture and is in sharp contrast to existing hepatocyte maintenance media available on the market today. Also, this medium allows long term culture of cryopreserved hPS cell-derived hepatocytes, from multiple lines, with preserved functionality. This increased usage window of functional hepatocytes in 2D cultures will empower new areas of applications and research in the field of hepatocytes.