ÉLise DELANNOY's thesis

"Models of blood vessels on a chip for studying the functions of the endothelial barrier".

Oral defence on 13 December 2021 at 2 p.m.
IEMN Amphitheatre - Central Laboratory - Villeneuve d'Ascq

Jury :

Julie GAVARD, CNRS Research Director, University of Nantes, Rapporteur
Séverine LE GAC, Associate professor, Twente University (Netherlands), Rapporteur
Vincent THOMY, University Professor, University of Lille, Examiner
Maria Carla PARRINI, INSERM Research Engineer, Institut Curie, Examiner
Anne Marie GUE, CNRS Research Director, LAAS, Examiner
Fabrice SONCIN, INSERM Research Director, Thesis Supervisor
Dominique COLLARD, CNRS Research Director, Thesis Co-Director
Anthony TREIZEBRE, Senior Lecturer, University of Lille, Supervisor

Summary:

The blood vascular system is a network of ducts that transports blood throughout the body. By transporting blood, blood vessels play an important role in supplying oxygen and nutrients to tissues and removing metabolic waste products. Blood vessels form a more or less permeable barrier that allows these exchanges and also regulates the immune response. The microenvironment also plays an important role in vessel functionality. The composition and physical properties of the extracellular matrix and perivascular cells strongly influence the physiology of endothelial cells and the vascular barrier. In the context of inflammation, vascular hyperpermeability is a public health problem and a well-known side-effect of certain treatments, particularly anti-cancer treatments.
To study these vascular phenomena, we are proposing new models combining microfabrication, tissue engineering and microfluidic technologies to build organs-on-a-chip. Organs-on-a-chip are a technological breakthrough that will enable us to answer complex biological questions. They aim to bridge the gap between 2D in vitro tests that are too unrepresentative of in vivo and expensive, time-consuming and species-specific animal models. We have designed vessel-on-a-chip devices in which an initial channel has been created in a collagen-based hydrogel using microfluidic approaches that rely either on laminar flow properties, flow focusing, or on the viscoelastic properties of solutions in microchannels, viscous digitation. The latter technique in particular has been developed and has enabled the formation of an initial channel that constitutes the future lumen of the vessel, which has been seeded with primary human endothelial cells. These cells formed a confluent and cohesive monolayer, reproducing the inner surface of a blood vessel.
This technique was carried out once or twice in succession, thus making it possible to vary the diameter of the final vessel depending on the channel used, and also to create a double cell layer. The integrity of the endothelial barrier was studied by assessing the quality of the distribution of vascular endothelial-cadherin at adherens junctions and zona-occludens-1 for tight junctions. In addition, the permeability of the endothelium was studied using a videomicroscopy approach to quantify the real-time diffusion of dextran-fluorescent through the vessel endothelium. The ability of endothelial cells to be activated by pro-inflammatory cytokines and their capacity to induce adhesion of immune cells and cancer cells to the inner surface of the vessel lumen were also assessed.
The biomimetic approach of this model was enriched by forming two distinct and concentric cell layers made up of endothelial cells and perivascular fibroblasts in order to approximate even more closely the structure and composition of a natural blood vessel and to observe the influence of interactions between the two cell types on vessel permeability. The presence of perivascular fibroblasts significantly strengthened the endothelial barrier, particularly in response to thrombin. Finally, these vessels-on-a-chip were designed in a standardised multi-well plate format with the aim of being used for robotic, high-throughput drug screening. Here we propose devices suitable for screening molecules that effect vascular activation and permeability, particularly in the context of anti-cancer treatments.

Abstract:

The blood vascular system is a network of conduits that transports blood throughout the body. Through their blood transport functions, blood vessels play important roles in the supply of oxygen and nutrients to tissues and the evacuation of metabolic waste. Thus, blood vessels form a more or less permeable barrier that allows these exchanges and also regulates the immune response. The microenvironment also plays an important role in vessel functionality. The composition and physical properties of the extracellular matrix and the perivascular cells strongly influence the physiology of the endothelial cells and the vascular barrier. In the context of inflammation, vascular hyperpermeability is a public health problem and a well known side effect of certain treatments, notably anti-cancer.
To study these vascular phenomena, we propose new models combining microfabrication, tissue engineering and microfluidic technologies to build organs on a chip. Organs-on-a-chip are a technological milestone to answer complex biological questions. They aim to bridge the gap between 2D in vitro tests that are not representative enough of in vivo and expensive, time-consuming and species-specific animal models. We have designed vessel-on-chip devices in which an initial channel has been created in a collagen-based hydrogel using microfluidic approaches that rely either on laminar flow properties, flow focusing, or on the viscoelastic properties of solutions in microchannels, viscous digestion. The latter technique was particularly developed and allowed to form an initial channel that constitutes the future lumen of the vessel that was seeded with primary human endothelial cells. These cells formed a confluent and cohesive monolayer, so as to reproduce the inner surface of a blood vessel.

This technique was performed once or twice in a row and thus allowed to vary, depending on the channel used, the diameter of the final vessel and also to create a double cell layer. The integrity of the endothelial barrier was studied by evaluating the quality of the distribution of vascular endothelial-cadherin at adherens junctions and zona-occludens-1 for tight junctions. On the other hand, the permeability of the endothelium was studied by implementing a videomicroscopy approach to quantify the real-time diffusion of dextran-fluorescent through the vessel endothelium. The ability of endothelial cells to be activated by pro-inflammatory cytokines and their ability to induce adhesion of immune cells and cancer cells to the inner face of the vessel lumen were also evaluated.
The biomimetic approach of this model was enriched by forming two distinct and concentric cell layers consisting of endothelial cells and perivascular fibroblasts in order to more closely approximate the structure and composition of a natural blood vessel and to observe the influence of interactions between the two cell types on vessel permeability. Thus, the presence of perivascular fibroblasts significantly strengthened the endothelial barrier, especially in response to thrombin. Finally, these vessels-on-a-chip were designed in a standardized multi-well plate format with the aim of being used for robotic and high-throughput drug screening.We propose here devices adapted to the screening of effector molecules of vascular activation and permeability, notably in the context of anti-cancer treatments.