Fabricating Microfluidic Co‐Cultures of Immortalized Cell Lines Uncovers Robust Design Principles for the Simultaneous Formation of Patterned, Vascularized, and Stem Cell‐Derived Adipose Tissue

Abstract In vitro co‐culture processes supporting simultaneous formation of vessel networks alongside differentiation toward mature parenchymal tissue have numerous clinical and agricultural applications but remain unrealized due to contrasting culture requirements. Of specific interest is lab‐grown vascularized adipose tissue to study diabetes, obesity, metabolic syndrome, and cardiovascular diseases, and to advance cultivated meat technologies. A microfluidic 3D hydrogel culture device capable of supporting live‐imaging of fluorescent reporter cell lines and generating counter‐current gradients of vasculogenic and adipogenic growth factors is reported. For the first time, experimental conditions capable of reproducibly forming diverse microvascular networks from telomerase immortalized endothelial and mesenchymal stem cells in both 2D and 3D hydrogel‐embedded cultures are reported. This novel microfluidic culture design demonstrates the generation of growth factor environments which support the 3D co‐formation of integrated robust microvascular networks and lipid‐producing adipocytes after 31‐days gradient culture. Microvascular networks substantially support parenchymal stromal cell differentiation to mature adipose tissue (67.4% lipid coverage), unachieved in avascular cultures (1.86% lipid coverage). It is attempted to validate the co‐culture model by applying inhibitors of vessel‐mediated lipogenesis (spermidine and VO‐OHpic), which are demonstrated to be ineffective in this novel human preclinical model.