A self-assembly and cellular migration based fabrication of high-density 3D tubular constructs of barrier forming membranes.

Three-dimensional (3D) in vitro models, superior in simulating physiological conditions compared to 2D models, offer intricate cell-cell and cell-ECM interactions with diverse signaling cues like fluid shear stress and growth factor gradients. Yet, developing 3D tissue barrier models, specifically perfusable luminal structures with dense, multicellular constructs maintained for extended durations with oxygen and nutrients, remains a technical challenge. Here, we describe a molding-based approach for the fabrication of free-standing, perfusable, high cellular density tissue constructs using a self-assembly and migration process to form functional barriers. This technique utilizes a polytetrafluoroethylene (PTFE)-coated stainless-steel wire, held by stainless steel needles, as a template for a perfusable channel within an elongated PDMS well. Upon adding a bio-ink mix of cells and collagen, it self-assembles into a high cell density layer conformally around the wire. Removing the wire reveals a hollow construct, connectable to an inlet and outlet for perfusion. This scalable method allows creating varied dimensions and multicellular configurations. Notably, post-assembly, cells such as human umbilical vein endothelial cells (HUVECs) migrate to the surface and form functional barriers with adherens junctions. Permeability tests and fluorescence imaging confirm that these constructs closely mimic in vivo endothelial barrier permeability, exhibiting the lowest permeability among all in vitro models in the literature. Unlike traditional methods involving uneven post-seeding of endothelial cells leading to subpar barriers, our approach is a straightforward alternative for fabricating complex perfusable 3D tissue constructs and effective tissue barriers for use in various applications, including tissue engineering, drug screening, and disease modeling.