Stochastic to Deterministic: A Straightforward Approach to Create Serially Perfusable Multiscale Capillary Beds

Generation of <i>in vitro</i> tissue models with serially perfused hierarchical vasculature would allow greater control of fluid perfusion throughout the network and enable direct mechanistic investigation of vasculogenesis, angiogenesis, and vascular remodeling. In this work, we have developed a method to produce a closed, serially perfused, multiscale vessel network fully embedded within an acellular hydrogel, where flow through the capillary bed is required prior to fluid exit. We confirmed that the acellular and cellular gel-gel interface was functionally annealed without preventing or biasing cell migration and endothelial self-assembly. Multiscale connectivity of the vessel network was validated via high-resolution microscopy techniques to confirm anastomosis between self-assembled and patterned vessels. Lastly, using a simple acrylic cassette and fluorescently labeled microspheres, the multiscale network was demonstrated to be perfusable. Directed flow from inlet to outlet mandated flow through the capillary bed. This method for producing closed, multiscale vascular networks was developed with the intention of straightforward fabrication and engineering techniques so as to be a low barrier to entry for researchers who wish to investigate mechanistic questions in vascular biology. This ease of use offers a facile extension of these methods for incorporation into organoid culture, organ-on-a-chip (OOC) models, and bioprinted tissues.