Microtissue size and hypoxia in HTS with 3D cultures

The three microenvironmental factors that characterize 3D cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. Although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. We make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in drug discovery. We further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. We offer guidance, depending on HTS objectives, for rational 3D culture platform choice for better emulation of in vivo conditions.