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. 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 custom-optimized extracellular matrix-derived hydrogel. The capability to tune dimensions enables the creation of constructs that encompass physiologically relevant size ranges. Through a systematic matrix screen, we identified a unique four-component formulation--gelatin, Matrigel, hyaluronic acid, and fibrinogen--that supports the rapid self-assembly of HDLECs into stable, lumen-forming monolayers within one week. These engineered structures maintain 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 preserves maintenance of lineage-specific expression profiles, junctional organization, and permeability properties under identical fabrication parameters. Altogether, this approach offers a reproducible and controlled system for studying 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. HighlightsO_LIA microfluidic extrusion platform allows primary human lymphatic endothelial cells to self-organize into 3D hollow tubes. C_LIO_LIThe internal dimensions of the hybrid cell/hydrogel constructs can be precisely controlled by tuning the flow rates. C_LIO_LIA custom-defined four-component extracellular matrix provides essential biochemical cues for stable monolayer formation and for the in vitro organization of lymphatic endothelial vessels. C_LIO_LIMatrix composition significantly influences cell proliferation and migration, thereby impacting on the spatial arrangement of the tissue. C_LIO_LIThe three-dimensional lymphatic tissue configuration sustains cell viability, consistent expression of lineage-specific markers, and barrier function which lasts for at least 30 days. C_LIO_LIThe system maintains morphological and functional properties not only for lymphatic endothelial cells, but also for blood endothelial cells in long-term culture conditions. C_LI