A Versatile Microfluidic Extrusion-Based Hydrogel Platform for Self-Organization and Long-Term Maintenance of...

Lymphatic endothelium is essential for interstitial fluid drainage, immune surveillance, and macromolecular transport. However, in vitro models that capture three-dimensional (3D) architecture, lineage stability, and long-term barrier properties remain limited. Here, we present a microfluidic extrusion-based platform that generates size-controlled 3D lymphatic endothelium from primary human lymphatic endothelial cells. Coaxial extrusion of an alginate shell around a hydrogel core yields tubular constructs whose inner diameter can be tuned from ∼50 to ∼300 µm by adjusting flow rates. We identified a four-component hydrogel that supports self-assembly of lymphatic endothelial cells into lumen-forming monolayers within 1 week. These engineered tubes maintain viability, lymphatic marker expression, and selective macromolecular permeability for at least 30 days under static culture. Over time, the cells remodel the core into a stratified wall that includes a fibronectin-rich perivascular zone. RNA sequencing shows that 3D lymphatic tubes are enriched in markers for lymphatic morphogenesis, matrix organization, and maturation compared with two-dimensional monolayers. In parallel, the same matrix supports vascular endothelial cells that form long-lived 3D tubes with key lineage-specific transcriptional and barrier properties. Altogether, this platform provides a robust and tunable system for modeling lymphatic endothelium and dissecting structure-function relationships in engineered 3D microvessels.