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.

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.