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A key challenge of in vitro organogenesis is the development in timely manner tissue equivalents. Herein, we assessed the simultaneous effect of oxygen tension (0.5%, 2% and 20%), foetal bovine serum concentration (0.5% and 10%) and macromolecular crowding (75 μg/ml carrageenan) in human dermal fibroblast culture. Our data demonstrate that cells cultured at 2% oxygen tension, in the presence of carrageenan and at 0.5% serum concentration deposited within 3 days in culture more extracellular matrix than cells grown for 14 days, at 20% oxygen tension, 10% serum concentration and in the absence of carrageenan. These data suggest that optimal oxygen tension coupled with macromolecular crowding are important in vitro microenvironment modulators for accelerated development of tissue-like modules in vitro. Statement of Significance To enable clinical translation and commercialisation of in vitro organogenesis therapies, we cultured human dermal fibroblast at 2% oxygen tension, under macromolecular crowding conditions (75 μg/ml carrageenan) and at low foetal bovine serum concentration (0.5%). Within 3 days in culture, more extracellular matrix was deposited under these conditions than cells grown for 14 days, at 20% oxygen tension, 10% FBS concentration and in the absence of crowding agents. These data bring us closer to the development of more clinically relevant tissue-like modules.