A Versatile Microfluidic Extrusion‐Based Hydrogel Platform for Self‐Organization and Long‐Term Maintenance of Engineered 3D Lymphatic Endothelium

ABSTRACT 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.