Development and Characterisation of a Vessel-On-a-Chip Model of Inflammation and Inflammatory Haemostasis


Type
Thesis
Change log
Authors
Riddle, Rebecca 
Abstract

Excessive immune cell infiltration occurs during many chronic inflammatory diseases. To develop new therapies for these diseases, models of inflammation are required that recapitulate how immune cells are recruited and interact with each other and the surrounding environment.

While traditional mouse models are useful tools in drug discovery, they cannot provide accurate responses to candidate therapies due to physiological differences between mice and humans. Conversely, 2D in vitro cultures of human cells do not capture the complexity of in vivo microenvironments. Recent advances in bioengineering have led to development of ‘organ-on-a-chip’ models, where human cells are cultured on ‘chips’ in 3D environments in vitro, enabling cells to behave more physiologically. In this thesis, an organ-on-a-chip model of immune cell transmigration was developed and characterised.

The OrganoPlate (Mimetas) was used to develop an inflammation-on-a-chip model consisting of a vessel of human umbilical vein endothelial cells (HUVEC) against a 3D extracellular matrix (ECM). Stimulation with TNF-α induced HUVEC inflammatory cytokine expression and promoted neutrophil transmigration towards a chemoattractant gradient. Differences in neutrophil transmigration were observed depending on ECM composition. Neutrophils migrated in higher number, further, and did not require a chemoattractant gradient in vessels cultured against a mixed matrix of geltrex and collagen I, compared to collagen I alone. Response to pharmacological inhibitors was also influenced by ECM composition.

The potential use of the model in drug discovery was investigated during a secondment at the funding body, AstraZeneca. Several small molecule inhibitors were tested and siRNA-mediated knockdown of endothelial gene expression was optimised. Modification of the model for different disease indications was also explored, including incorporation of peripheral blood mononuclear cells instead of neutrophils, and induced pluripotent stem cell-derived endothelial cells instead of HUVEC.

Finally, platelets were incorporated into the model to explore their role in inflammation, and haemostasis of leaky vessels. In unstimulated vessels, platelets were protective, reducing leakage of small molecules. However, in inflamed vessels, platelets played a dual role, promoting permeability and enhancing neutrophil transmigration, whilst simultaneously preventing red blood cell (RBC) leakage at transmigration sites. Platelets were also protective during angiogenesis, preventing leakage of small molecules and RBCs from newly formed vessels.

Overall, this thesis developed and characterised a humanized in vitro vessel-on-a-chip model and demonstrated its use in studying cell-cell interactions during inflammation, alongside its application in testing of anti-inflammatory therapies. The commercial availability of the OrganoPlate will allow other researchers to easily adopt this model, increasing the feasibility of organ-on-a-chip models becoming mainstream tools in both basic science and drug discovery.

Description
Date
2023-03-28
Advisors
Harper, Matthew
Jennbacken, Karin
Hansson, Kenny
Keywords
3D cell culture, extracellular matrix, inflammation, inflammatory haemostasis, neutrophils, organ-on-a-chip, platelets
Qualification
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Sponsorship
AstraZeneca-Pharmacology PhD Studentship