A versatile microfluidic extrusion-based hydrogel platform for self-organization and long-term maintenance of engineered 3D lymphatic endothelium

Lymphatic endothelium is essential for interstitial fluid drainage, immune surveillance, and macromolecular transport. Existing in vitro models that accurately recapitulate its three-dimensional structure, barrier properties, and long-term stability are still limited. In this study, we propose a robust, adaptable, and scalable platform designed to generate engineered three-dimensional lymphatic endothelium using primary human dermal lymphatic endothelial cells (HDLECs). This system combines microfluidic extrusion-based fabrication allowing precise control of the tubular geometry with a fully defined extracellular matrix-derived hydrogel. The capability to adjust dimensions enables the creation of constructs that encompass physiologically relevant size ranges. A comprehensive matrix screen revealed a distinct four-component formulation - gelatin, Matrigel, hyaluronic acid, and fibrinogen - that promotes HDLEC self-assembly into stable, lumen-lining monolayers within a week. These engineered structures retain viability and structural integrity for a minimum of 30 days under static culture conditions. Functional permeability assays demonstrated selective tracer uptake characteristics of lymphatic transport. Comparative studies with blood vascular endothelial cells indicate that the proposed platform supports prolonged maintenance of lineage-specific expression profiles, junctional organization, and permeability properties under identical fabrication parameters. Altogether, this methodology offers a reproducible and controlled system for examining three-dimensional endothelial architecture, transport mechanisms, and extracellular matrix remodeling across diverse endothelial phenotypes, thereby addressing a pivotal gap in the modeling of lymphatic vasculature.