(F-238) Human serum perfused engineered microvessel on a chip: A test bed to assess microvascular integrity to uremic toxins.

Introduction::
Introduction: Maintaining the functional barrier integrity of vascular endothelium is key in the prevention or delay of vascular diseases. Accumulation of uremic solutes in circulation due to renal failure induces a breakdown of the endothelial barrier function, causes an increase in the endothelial injury, reduces endothelial repair, and results in endothelial dysfunction in patients with chronic kidney disease1. To-date, limited effort has been directed toward the study of the effects of the uremic environment on endothelial barrier dysfunction in a 3D physiological setting. Here, we demonstrate for the first time the possibility to maintain a healthy functional endothelium, an engineered 3D microvessel completely enveloped in a collagen scaffold, perfused with 100% human serum to investigate the effects of uremic toxin on microvascular integrity.


















Materials and Methods::
Material and Methods: HUVECs (1.8x10⁶cells/ml) were seeded in the cylindrical microchannels enveloped in a collagen type I scaffold to form monolayers resulting in engineered microvessels. The microvessels were optically checked for vessel coverage after 24 h and were reseeded if necessary to ensure uniform vessel coverage. 48 h since first seeding, we next changed the perfusion media from cell culture media to 100% human serum for 24 hours. To mimic the uremic environment, we added pathophysiological level (25µg/ml) of indoxyl sulfate (IS) to serum from healthy donors. We stained microvessles for nuclei, F-actin, and VE-cadherin to better assess the microvessel integrity and barrier function.








Results, Conclusions, and Discussions::
Results and Discussion: The engineered microvessel on chip platform consisted of a 4 mm long cylindrical channel with a diameter of 160µm completely enclosed in a collagen type I scaffold (Fig. 1A). Microvessels perfused with serum and cell culture media were characterized by distinct, pericellular VE-cadherin staining, indicating the formation of mature adherens junctions, while microvessels perfused with serum containing IS were characterized by diffuse VE-cadherin indicating immature or dysfunctional adherens junction integrity (Fig. 1B). To test the integrity of our engineered microvessel in a uremic environment we measured the kinetics of transport of 70 kDa dextran across the microvessel wall using confocal microscopy (Fig. 1C). Microvessels perfused with serum spiked with 25 µg/ml of IS exhibited significantly increased diffusive permeability to dextran (Pd = 1.33 × 10-4 cm/s) when compared to microvessels perfused with healthy serum (Pd = 7.61 × 10-6 cm/s) or cell culture media (Pd = 1.85 × 10-5 cm/s, Fig. 1D).






Conclusions: The engineered microvessel on chip platform presented here with the ability to maintain a functional endothelium when perfused with pure healthy serum can be utilized as a diagnostic screening tool for several uremic solutes and directing personalized treatments for patients with chronic kidney disease.











Acknowledgements (Optional): :

References (Optional): : Ref: Jourde-Chiche N, Dou L, Cerini C, Dignat-George F, Brunet P. Vascular incompetence in dialysis patients--protein-bound uremic toxins and endothelial dysfunction. Semin Dial. 2011,24(3):327-37.