Abstract
BackgroundExcessive extracellular matrix (ECM) deposition is a hallmark feature in fibrosis and tissue remodelling diseases. Typically, mesenchymal cells will produce collagens under standard 2D cell culture conditions, however these do not assemble into fibrils. Existing assays for measuring ECM production are often low throughput and not disease relevant. Here we describe a robust, high content, pseudo-3D phenotypic assay to quantify mature fibrillar collagen deposition which is both physiologically relevant and amenable to high throughput compound screening. Using pulmonary fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF), we developed the ‘scar-in-a-jar’ assay into a medium-throughput phenotypic assay to robustly quantify collagen type I deposition and other extracellular matrix (ECM) proteins over 72 h.ResultsThis assay utilises macromolecular crowding to induce an excluded volume effect and enhance enzyme activity, which in combination with TGF-β1 stimulation significantly accelerates ECM production. Collagen type I is upregulated approximately 5-fold with a negligible effect on cell number. We demonstrate the robustness of the assay achieving a Z prime of approximately 0.5, and % coefficient of variance (CV) of < 5 for the assay controls SB-525334 (ALK5 inhibitor) and CZ415 (mTOR inhibitor). This assay has been used to confirm the potency of a number of potential anti-fibrotic agents. Active compounds from the ‘scar-in-a-jar’ assay can be further validated for other markers of ECM deposition and fibroblast activation such as collagen type IV and α-smooth muscle actin exhibiting a 4-fold and 3-fold assay window respectively.ConclusionIn conclusion, we have developed ‘scar -in-a-jar is’ into a robust disease-relevant medium-throughput in vitro assay to accurately quantify ECM deposition. This assay may enable iterative compound profiling for IPF and other fibroproliferative and tissue remodelling diseases.
Highlights
Excessive extracellular matrix (ECM) deposition is a hallmark feature in fibrosis and tissue remodelling diseases
Tissue remodelling characterised by dysregulated extracellular matrix (ECM) deposition is a hallmark feature of numerous human pathologies including cancer and fibroproliferative diseases such as pulmonary fibrosis, chronic kidney disease (CKD) and non-alcoholic steatohepatitis (NASH) [1, 2]
We describe the optimisation and implementation of the ‘scar-in-a-jar’ assay using primary idiopathic pulmonary fibrosis (IPF) patient lung fibroblasts into a robust, medium-throughput, high content screening (HCS) assay for the identification and annotation of novel anti-fibrotic agents
Summary
Excessive extracellular matrix (ECM) deposition is a hallmark feature in fibrosis and tissue remodelling diseases. Using pulmonary fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF), we developed the ‘scar-in-a-jar’ assay into a medium-throughput phenotypic assay to robustly quantify collagen type I deposition and other extracellular matrix (ECM) proteins over 72 h. IPF is characterised radiologically by the presence of honeycomb lung on high-resolution computed tomography (HRCT) and histologically by the appearance of fibroblastic foci [1] containing myofibroblasts in a collagen-dense ECM. These structural changes in pulmonary tissue architecture result in extensive remodelling of the lung parenchyma, leading to loss of lung function and causing death due to respiratory failure [3]. With a prevalence rate of 50 in 100,000 in the UK [5] and an average life expectancy estimated to be less than 3 years [6], there is an urgent need to develop new medicines with improved efficacy and tolerability profiles for patients with pulmonary fibrosis [5, 6]
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