(H-314) Optimizing Flow Conditioning to Enhance Endothelial Barrier Maturation of Vascularized Milliscale Tissues Anastomosed in Membrane-free Organ Chips

The circulatory system relies on blood vessels to transport and exchange nutrients, oxygen, and regulate temperature. Vascular endothelial cells are the primary building blocks that regulate these transport processes. 3D in vitro models enable the recapitulation of complex organ-level functions and have been exploited to accelerate the discovery of drugs and therapeutics. However, the ability to create bulk tissues with an anastomosed and perfusable internal vasculature is necessary to model clinically relevant aspects of vascular physiology such as barrier function and intravascular delivery of biologically active compounds. We leveraged stereolithography-based (SLA) mold fabrication with rapid prototyping capabilities combined with Polydimethylsiloxane (PDMS) soft lithography to engineer a milliscale model of bulk tissue vasculogenesis with continuous perfusable vasculature. We compared unidirectional pump-driven flow and pump-free oscillatory rocking as methods of flow conditioning to enhance endothelial barrier function.

HLF and HUVEC were incorporated in hydrogels composed of collagen type I and fibrin to create stromal vascular tissues. We engineered PDMS membrane-free organ chips with a central channel that enables patterning of vascularized bulk tissue, and adjacent media side channels. We seeded endothelial cells at high concentrations in the side channels to enable the formation of a complete endothelial cell monolayer that subsequently anastomosed with the internal bulk vasculature. The constructs were cultured under dynamic mechanical conditioning using unidirectional pump-driven flow and a gravity-driven oscillatory bidirectional rocking platform. For end-point analysis, the constructs were visualized using a light-scanning confocal microscope and perfused with FITC-dextran and nanoparticles to assess the barrier integrity of the formed vessels across different mechanical conditioning regimens. Fixed samples were stained with endothelial-specific lectins and phalloidin to label the actin cytoskeleton of all cells. We performed MATLAB-based morphometric analysis of 3D confocal image data to quantify the structural features of emergent vascular networks in the tissues.

Interconnected 3D vascular network assembly spanning the entire length of the bulk tissue was evident after 7 days of culture. Morphometric evidence of maturation included decreasing non-participating endothelial cell numbers, increasing vessel diameters, and increasing diffusion distances. Side channel seeding with a high density of endothelial cells after 2 hours of bulk tissue seeding results in immediate and complete coverage of the tissue interfaces. Numerous anastomoses with the perfusable internal vasculature form de novo by 9 days in culture. Fluid shear-induced maturation of vessels using mechanical conditioning improved vessel barrier integrity assessed by FITC-dextran perfusion testing. Perfusion in pumping conditioned devices demonstrated less leakage into the extravascular space compared to rocked and static devices. Perfused devices showed maximum intravascular fluorescence upon introducing FITC-dextran, while rocked devices had delayed vessel recruitment and gradual extracellular fluorescence increase due to vascular leakage. Pumped vasculature exhibited larger diameters, increased diffusion distances, and shorter vessel lengths. Flow-conditioned vasculature successfully delivered nanoparticles to bulk tissue, with aggregates observed near branchpoints and adherent particles near anastomosis points.
We have developed an adaptable 3D in vitro model of baseline perfusable vascularized tissue in the milliscale dimension and assessed mechanical flow conditionings to improve vascular morphometry and barrier integrity. The model can be used for studying vascular physiology, barrier function, and drug delivery in vascularized tissue. This advancement will contribute to developing treatments for various vascular-related pathologies and enhancing our understanding of complex vascular systems in the context of pathophysiology like cancer and inflammation.