Protocol to fabricate elastomer microwells for three-dimensional culture of primary adipocytes

Our understanding of how adipocytes influence metabolic signaling, immune function, and cancer progression remains limited as the culture of primary adipocytes is challenging. Here, we present a protocol to fabricate elastomer microwells for three-dimensional culture of collagen-embedded adipocytes. We describe steps to cure and functionalize elastomer microwells and to isolate and embed primary adipocytes. We then detail how to culture and analyze adipocyte-collagen gels. This protocol provides broad applications to improve our understanding of adipocyte biology in health and disease.