Development of a bioreactor and volumetric bioprinting protocol to enable perfused culture of biofabricated human epithelial mammary ducts and endothelial constructs

Abstract Tissue function depends on the 3D spatial organization of cells, extracellular matrix components, as well as dynamic nutrient gradients and mechanical forces. Advances in biofabrication technologies have enabled the creation of increasingly sophisticated tissue models, but achieving native-like tissue maturation post-fabrication remains a challenge. The development of bioreactors and microfluidic systems capable of introducing dynamic culture platforms and controlled mechanical and biochemical stimulation for biofabricated tissue analogues is therefore imperative to address this. In this technical note, we introduce a multi-step pipeline to fabricate, seed and perfuse geometrically complex hydrogel constructs with quality control protocols through the computational analysis of confocal multispectral 3D imaging data for each step of the process. Employing ultra-fast volumetric bioprinting, chips with tunable channel architectures were fabricated. Furthermore, an autoclavable and transparent perfusion bioreactor inspired by open-source designs was developed to enable controlled, long-term perfusion (up to 28 days) and real-time monitoring of cell behavior. As proof-of-concept, employing this pipeline, we fabricated a human mammary ductal model and an endothelialized vessel on-a-chip, demonstrating the compatibility of the platform with epithelial and endothelial cell lines, and investigated the effect of dynamic culture on tissue-specific cell organization. Dynamic perfusion underlined the influence of mechanical stimulation on cell organization and maturation. Various chip architectures, capable of recapitulating tissue-specific features (i.e. lobules) were printed, enabling the mono- and co-culture of human mammary epithelial and endothelial cells. Our pipeline, with the accompanying protocols and analysis scripts presented here, provide the potential to be applied for the dynamic culture of a wide range of tissues.