Optimisation and validation of 3D models of progressive human lung fibrosis

Why did we fund this fellowship?

This award aims to further develop an in vitro human 3D model of idiopathic pulmonary fibrosis by incorporating cytokines and multiple cell types, so that it can be deployed to replace the use of mouse models in some studies.

Idiopathic pulmonary fibrosis (IPF) is a degenerative, fatal lung disease in older adults where build-up of scar tissue, caused by excessive extracellular matrix deposition, prevents oxygen transfer into the blood leading to progressively impaired breathing. There is currently no cure for IPF, and treatments are only able to slow disease progression. In vivo and in vitro models are used to study IPF and research new therapeutic options. Mice are most commonly used to model IPF by intratracheally administering bleomycin under anaesthesia. This results in scarring of the lungs, however the damage, unlike in humans, is reversible, limiting the clinical translation of murine models. Current in vitro models use cells isolated from patient lungs, however, the cells do not recapitulate the remodelling of collagen typically seen in patients when they are grown in 2D cell culture conditions. This limits their use as an alternative to in vivo models.

With in-kind support from the Medicines Discovery Catapult, Dr Joseph Bell will further develop a human 3D model of IPF in which  fibroblastic cells resemble the stiffened scar tissue in IPF when treated with TGFβ. Joseph will use low oxygen culture conditions and add cytokines identified in IPF patient lung fluid, in addition to TGFβ, before incorporating further cell types to make the model more representative of the human condition. He will then use RNA sequencing to validate the model against existing patient data. Joseph will develop skills in 3D co-culture techniques, mass spectrometry and spatial gene expression analysis.

Idiopathic pulmonary fibrosis (IPF) is a disease affecting older adults which involves the build up of stiff scar tissue between cells that form the air sacs of the lung and the underlying blood vessels which transport oxygenated blood around the body. This scar tissue prevents the transfer of oxygen into the blood (and CO2 from the blood), and destroys the air sacs of the lung, leading to progressively impaired breathing and eventual death. IPF has no cure, and existing treatments only slow progression of the disease. In IPF, the cells responsible for producing scar tissue, fibroblasts, are found in small aggregates called fibroblastic foci which are thought to be the active areas of disease. Studies have also previously identified a molecule, transforming growth factor beta (TGFβ) which drives fibroblasts to produce scar tissue. Animals, principally mice, have been extensively used to study IPF. Mice treated with a drug called bleomycin develop a build-up of scar tissue in their lungs like that seen in IPF. Importantly, however, this scar tissue build-up is reversible in mice, in contrast to human disease, where it is permanent and progressively worsens. Replacing these mice with a different model which better reflects IPF in humans is the goal of this project.

One alternative to using animals to study disease mechanisms is growing human cells in flasks and subjecting them to various treatments to mimic how they behave within diseased tissue. However, cells in flasks grow in a single, 2D layer, in contrast to the 3D environment in the human body. Researchers in Southampton have developed a 3D fibroblast culture model which allows fibroblasts isolated from IPF lung tissue to grow more naturally. These 3D aggregates of fibroblasts resemble fibroblastic foci in the stiffened scar tissue they produce when treated with TGFβ. However, while TGFβ likely makes an important contribution to scar tissue formation, my previous research has identified that it is not solely responsible. For example, lack of oxygen in the densely packed fibroblastic foci also contributes to scar tissue formation, as does the presence of other mediators. In addition, my research has identified other cell types in fibroblastic foci, suggesting that the current model which just uses fibroblasts is too simplistic to accurately reflect the actual disease.

The aim of this project is to develop the 3D fibrosis model to better reflect a fibroblastic focus found in IPF lung tissue. I will treat 3D cell cultures with chemicals which mimic the effects of lack of oxygen on the cells, and will add other molecules identified in IPF lung fluid to the 3D cultures to better represent the environment of fibroblast foci in IPF. I will compare the 3D culture model to fibroblastic foci from people with IPF using a technique called RNA sequencing. This quantifies the genetic messages that control how cells behave, allowing changes in cell behaviour to be identified based on the change in these gene signatures. We already have RNA sequencing data from fibroblastic foci, so we can use that to compare how well the gene signatures from the different 3D culture conditions match those in actual disease. We will also use a separate technology which allows fast quantification of these gene signatures to validate these data, in collaboration with the Medicines Discovery Catapult (MDC).

After we have developed this model, we will seek to improve it further by adding other cells into it, reflecting the multiple cell types present in fibroblastic foci. This multicellular model will also be analysed by RNA sequencing and compared to fibroblastic foci. We will also collaborate with MDC to identify how the cells' genetic and metabolic signatures change spatially across the 3D model and fibroblast foci. If successful, this 3D cell culture model will give an alternative to animal models for the study of lung fibrosis and provide a novel testbed for evaluation of new treatments.

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Fellowship

Status

Inactive

Principal investigator

Dr Joseph Bell

Institution

University of Southampton

Grant reference number

NC/V002384/1

Award date

Apr 2021 - Mar 2023

Grant amount

£114,352