Collectively, respiratory diseases are the leading cause of death worldwide. As a consequence respiratory research has increased by 500% and animal models have supported approximately 20% of this research. More than 900,000 mice have been used in the last decade alone to help understand human respiratory health, however, the physiological and anatomic differences between the human and mouse lungs have made it difficult to translate research findings into human therapies. Inhalation research focuses on the effect of inhaled chemicals, particles, bacteria, viruses, drug delivery systems and pollution in both healthy lung and diseased lungs. The most widely used human specific models of the human lung are cell-based models that focus on lung epithelial cells, the cells that make up the surface of the lungs. While these models are useful to model the response of epithelium to inhaled substances, the whole lung response is extremely important and encompasses complex cell-to-cell interactions and signalling, immune cells and extracellular matrix modifications that currently only mouse models can fully replicate, hence their over-use. We will address this gap in model platforms by creating a multi-cell type, immune competent model of human lung alveoli that is animal-free, capable of stretching and applicable to many areas of respiratory research. To achieve this we will use stem cell derived type II alveoli epithelial cells with macrophages, fibroblasts and endothelial cells in a synthetic hydrogel developed by our industrial partner Manchester BIOGEL. We will demonstrate application of the model to understand how atmospheric particulate matter, the most significant environmental pollutant in the world, affects lung health. The objectives below will provide an opportunity for a "next-generation" model of the human lung alveoli, capable of modelling epithelial response, fibrosis as well as that of the immune system and could provide important mechanistic, diagnostic and therapeutic information across many areas of respiratory research. 1): Combine hIPSC derived lung cell types into synthetic animal-free hydrogels. We will bring together our stem cell derived type II alveoli epithelial cells, with macrophages, fibroblasts and endothelial cells in a synthetic hydrogel to generate a 3D multi-cell-type, immune competent organoid model of the human lung alveoli. We will characterise structure of the organoid in different hydrogel formulations that mimic fibrosis both with and without stretch forces. We will then compare and validate individual cell behaviour to human precision cut lung slices cultured under equivalent conditions. 2) Understand the impact of particulate matter on lung alveoli tissue To further demonstrate application of the model we will expose the alveoli organoids to environmental particulate matter in different hydrogel and stretch characteristics identified in Obj 1. We will then characterise the response of each cell type and compare and validate the response using human precision cut lung slices as in Obj 1. 3) Educate and train new users in development and manipulation of hIPSC derived alveoli organoids. We will host a conference focussed on current animal free models of human health and disease to educate current and future research leaders in use and development of animal-free models of human health. We will also host a 1-week training course for researchers that would like to adopt and use our model for their own research purposes. Together these objective will create a "next generation" model of human alveoli tissue applicable that could reduce and replace animal models across many different respiratory diseases. Importantly, there will be training opportunities provided to other researchers that will allow them to adopt and modify the model to their own experimental needs, further enhancing the possibility that the platform will have a positive impact in reducing animal use in research.

Pancreatic cancer is a serious disease of the pancreas with very poor prognosis and low survival rate (<8% of patients survive the disease). Furthermore, as oppose to other cancers where we have seen significant improvement of survival due to novel treatment developments, there has been hardly any improvement over the last decades for pancreatic cancer. A key aspect that leads to the progression of the disease is the so-called tissue (tumour) microenvironment (TME), which is essentially a cocktail of cells and biomolecules which interact with the tumour, making it resistant to treatment and helping it metastasise. Typically, therapies for pancreatic cancer are tested on animal models or on tumour cells cultured in 2D static conditions. While the complexity associated with animal studies improves the disease insight, they are expensive, complex, difficult to reproduce and many times unrepresentative. 2D single cell cultures are easy to use, reproducible and cost-efficient, however, they are unable to reproduce topologically, mechanically, biologically and biochemically the complex TME. More recently, 3D spheroid type ('tissue-spheres') cultures from human and mouse pancreatic cancer represent the state-of-the- art as they can be cultured for longer than 2D systems, they are suitable for drug screening. These can be established from small biopsy specimens, so in principle can be used to identify some tumour characteristics of individual patients. However, the self-organising nature of spheroids and the lack of mechanical and biochemical integrity limits the tuneability of the environment, therefore reducing the versatility of these models. An in vitro system with robust control of the biophysical, biochemical and biomechanical environment is currently lacking and would benefit the patients and the research community substantially as there is clear evidence in the state of the art that the biomechanical and biophysical environment can affect the disease progression, metastasis and response to treatment. The aim of our project is to develop a high fidelity pancreatic cancer model, which will enable patient & disease specific treatment optimization via robust control of various biochemical, biomechanical and biophysical features of the TME. More specifically, tailoring in a controlled manner parameters of the tumour microenvironment like extracellular matrix composition, stiffness (at levels that realistically occur in PDAC in vivo), interstitial flow rates (mimicking high or low vascularisation or avascular tumours), vessel sizes or fibrotic levels (mimicking dense or less dense fibrotic reaction) will enable the conduction of long term fundamental studies unravelling the interaction of each of those parameters with different cells of the tumour microenvironment. Underpinning such interactions at multiple levels, i.e., genetic, metabolic, will enable a better understanding of the evolution of the disease as well as the role of different TME configurations on driving signaling pathways for migration and metastasis. Furthermore, such a robust, tuneable, representative model for pancreatic cancer will help improve the success rate of emerging therapies and constitute a platform for personalised medicine.

The bone marrow is a site of health and disease. In health, it produces all of the blood cells that we rely on to carry oxygen and protect us from infection. However, the stem cells that produce the blood and that reside in the marrow, the haematopoietic stem cells (HSCs), age and can tip over into disease states, such as developing leukaemia. Factors such as smoking and treatment of cancers elsewhere in the body (toxic effects of chemotherapy/radiotherapy) can accelerate ageing, and therefore, drive the transition to disease. Further, it forms a home to other cancer cells, that leave their original tumour and move, or metastasise, to the bone marrow. Once in the marrow, they can become dormant, hiding from chemotherapies and activating sometime later to form devastating bone cancers. The cues that wake cancer cells from dormancy are largely unknown. If models of the bone marrow that contain human cells and that can mimic key facets of the niche in the lab, such as blood regeneration, cancer evolution and dormancy, can be developed it would be a big help in the search for better cancer therapies. We are developing the materials and technologies required to meet this challenge. In this programme of research, we will tackle three biomedical challenges: 1) HSC regeneration. Bone marrow transplantation (more correctly HSC transplantation) is a one-donor, one-recipient therapy that can be curative for blood diseases such as leukaemia. It is limited as HSCs cannot be looked after well out of the body. Approaches to properly look after these precious cells in the lab could allow this key therapy to become a one-donor, multiple recipient treatment. Further, the ability to look after the cells in the lab would open up the potential for genetically modifying the cells to allow us to cure the cells and put them back into the patient, losing the need for patient immunosuppression. 2) Cancer evolution. As we get older, our cells collect mutations in their DNA and these mutations can be drivers of cancer. Lifestyle choices such as smoking, and side effects of treatments of other diseases can also add mutations to the cells. As blood cancers develop, the bone marrow changes its architecture to protect these diseased HSCs. Our 3D environments will allow us to better understand this marrow remodelling process and how drugs can target cancers in this more protective environment. The models will also allow us to study the potential toxicity of gene-edited HSCs to make sure they don't produce unwanted side effects or are not cancerous in themselves. 3) Dormancy. What triggers dormancy and activation from dormancy are poorly understood. By placing our 3D environments in a miniaturised format where we can connect other models that include infection and immune response, we can start to understand the factors involved in the activation of cancer cells from dormancy. Our vision is driven by materials and engineering, as the bone marrow niche is rich in structural and signalling biological materials (proteins). Therefore, we will establish three engineering challenges: (1) Cells can be controlled by the stiffness and viscous nature of materials (viscoelasticity). We will therefore develop synthetic-biological hybrid materials that can be manufactured to have reproducible physical properties and that have biological functionality. (2) We will develop these materials to interact with growth factors and bioactive metabolites, both of which are powerful controllers of cell behaviours. These materials will be used to assemble the HSC microenvironments in lab-on-chip (miniaturised) format to allow high-content drug and toxicity screening. (3) We will develop real-time systems to detect changes in cell behaviour, such as the transition from health to cancer using Raman and Brillouin microscopies. The use of animals in research provides poor predictivity. We will offer better than animal model alternatives.

Nature is the prime example of complex and sophisticated manufacturing. The human body is constructed by cells and support matrices where a variety of biomolecules perform complex functions in development, normal function and regeneration. This delicate balance is disturbed in disease or trauma and confounded by the body's declining regenerative capacity with increasing age. Organ transplantation has saved many lives and millions of pounds to the NHS, however every day 4 people in the UK die while on the waiting list. Those fortunate to receive organ transplants require immunosuppressant drugs, making them prone to infection and increased risk of cancer. There is a dire need for artificially engineered organs and tissue grafts, that engraft successfully on implantation without the need for immunosuppression. Furthermore, cardiovascular disease is the top cause of death globally. This is caused by problems with the heart or the circulatory system. Transformative solutions are required to meet the rising unmet clinical need for organ transplantation and cardiovascular diseases. The aim of this project is to develop an adventurous manufacturing workflow to recreate the structural and cellular complexity of blood vessels by employing novel manufacturing strategies. The project combines advanced materials, 3D printing and advanced imaging to provide transformative solutions to key healthcare challenges facing our aging society. This project will address the growing demand for functional tissue grafts and organs for transplantation and drug discovery. To date, a major hurdle in engineering artificial tissue has been the inability to reproduce the blood vessel micro- and macro-architecture. Our novel manufacturing research idea is to develop a complex and sophisticated fluid delivery system, with a 3D printer to recreate blood vessels in the laboratory. Our research will enable the rapid production of blood vessels from small (width of hair) to large (centimetres) sizes, and harness advanced biomaterials, designed to change from solution to gel by mixing in the fluid delivery system, to achieve this goal.

According to the Global Burden of Disease, neurological conditions are the leading cause of disability and the second-leading cause of death worldwide. The debilitating nature of these conditions can have a devastating effect on an individual's quality-of-life and their ability to undertake activities of daily living. This exerts a heavy strain on families, carers, society and healthcare systems, moreover, the medical costs, care costs and loss of productivity arising from disorders of the brain have been estimated to cost the UK economy over £100 billion per year. In order to design preventative and therapeutic strategies, we need to understand how neurological conditions arise and how they affect the human brain. However, the human brain is relatively inaccessible to study as a living organ, while post-mortem biopsies cannot be used to study the function of brain tissue. Meanwhile, differences in brain anatomy mean that animals are often unsuitable for studying human neurology. Over the last decade, a new approach to studying the human brain has emerged: the use of "brain organoids" generated from 3D clusters of stem cells. These organoids provide an alternative to animal studies and have been used to model human brain development and neurological conditions, such as microcephaly. A major limitation of brain organoids is the lack of control exerted over their formation and development, which leads to organoids that are geometrically and biologically symmetric. This is a problem because the human brain is a naturally asymmetric structure with different regions formed from an elongated cell structure, known as the neural tube. As a result, symmetric brain organoids cannot be used to study the asymmetric aspects of brain development or the asymmetric processes present in many neurological conditions. This limitation will be directly addressed in this Fellowship by developing a suite of technologies that can break the symmetry of brain organoids to produce models of the human brain that enable the study of complex neurological conditions. These technologies will be adapted from previous methods that I have developed for growing muscle and cartilage. Ultrasound patterning will be used to remotely assemble stem cells into elongated neural tubes, which will controllably develop different regions of the brain under the influence of chemical gradients slowly released from a biomaterial. Ultrasound will also be used to remotely pick up, move and fuse different brain organoids to assembly complex cerebral structures. These asymmetric organoids will be used to study asymmetric processes in common neurological conditions: the failure to form different regions of the brain in holoprosencephaly, the dysfunctional migration of neurons in many psychiatric disorders (e.g., schizophrenia, autism) and the spread of toxic proteins in Alzheimer's disease. For each of these processes, the symmetry-broken organoids will be used to assess the contribution of different environmental and genetic risk factors, providing new knowledge that will inform future preventative or therapeutic strategies. Moreover, these research outputs have a scope that extends far beyond neuroscience, with the capacity to address similar challenges in other organoids (e.g., pancreatic, endometrial). To benefit a wide range of users, the symmetry-breaking technologies will be refined into user-friendly toolkits, while high-throughput manufacturing methods will be developed for the symmetry-broken organoids. Academic collaboration, industry partnerships and product commercialisation will be used to disseminate these toolkits and organoids to academic groups, biotechnology industry and pharmaceutical industry. This will ensure far-reaching impact beyond the immediate goals of this Fellowship by providing researchers from different fields with the tools to grow their own complex organoids for the study of development, disease and drug response.