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Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold\u2010free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi\u2010omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate &lt; 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of &gt;1000\u00a0lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    = 0.97). Integration of multi\u2010omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGF\n                    <jats:italic>\u03b2<\/jats:italic>\n                    signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPAR\n                    <jats:italic>\u03b3<\/jats:italic>\n                    and CEBP activity resulting in improved adipogenesis.\n                  <\/jats:p>","DOI":"10.1002\/advs.202100106","type":"journal-article","created":{"date-parts":[[2021,6,24]],"date-time":"2021-06-24T11:59:53Z","timestamp":1624535993000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":89,"title":["3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis"],"prefix":"10.1002","volume":"8","author":[{"given":"Joanne 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