Abstract
Creation of biomimetic in vitro systems recapitulating tissue physiology requires developing technologies to recreate the tissue microenvironment including appropriate blood vessels and perfusion. Blood vessels and endothelial cells (ECs) exhibit a variety of specialised functions, reflected in a remarkable phenotypical heterogeneity. Here, we first describe the creation of tools to study this EC heterogeneity at the single cell level and characterise EC behaviour in different populations of arterial, venous and microvascular ECs. We implement high content imaging to decipher heterogeneity in terms of proliferation, junctional status and Notch signalling pathway. The information extracted are key to a better understanding of the molecular mechanisms involved in EC and blood vessel specialisation. To recapitulate this functional heterogeneity and obtain mature populations of vascular cells for future tissue engineering purposes, we then describe the differentiation of stem cell-derived vascular cells. By implementing and refining existing protocols, we report the creation of mature populations of ECs, pericytes and fibroblasts and study their functional ability to create mature blood vessels in vitro. Following the characterisation and derivation of vascular cells, we focus on the elaboration of microfluidic devices for the generation of functional blood vessels.Current Lab-on-chip (LOC) technology allows creating very complex in vitro systems allowing passive tissue perfusion and generation of capillary-like structures. However, these systems cannot recreate the physiologic microenvironment including continuous perfusion and physiologic sheer and normal stresses to blood vessel walls, key determinants of vascular homeostasis and ultimately adequate tissue functions. We present a new scalable 3D printingbased workflow to manufacture Lab-on-chip devices at microfluidic scales enabling creation of complex and continuously perfusable LOC devices. Our workflow from design to manufacture is significantly less expensive and more flexible than current technologies. We employ 3D printing to accelerate the prototyping and manufacture of vasculature-on-chip systems and refine an organotypic system to create capillaries with organ-specific endothelial cells and stromal cells. We also design a custom perfusion system to introduce a physiologic continuous perfusion in our device. This allows the validation of our tailored design for the perfusion of self-assembled microvascular networks and the study the maturation of blood vessels over time in a flow-dependent manner. We envision that this technology will empower fast prototyping, manufacture and validation of novel in vitro systems of increasing complexity including interconnected multi-tissue systems.
| Date of Award | 1 Jan 2023 |
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| Original language | English |
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| Supervisor | Trevor Coward (Supervisor), Agamemnon Grigoriadis (Supervisor) & Lorenzo Veschini (Supervisor) |