A high content, phenotypic ‘scar-in-a-jar’ assay for rapid quantification of collagen fibrillogenesis using disease-derived pulmonary fibroblasts

Abstract

Background: Excessive 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. Results: This assay utilises macromolecular crowding to induce an excluded volume effect and enhance enzyme activity, which in combination with TGF-β 1 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. Conclusion: In conclusion, we have developed 'scar-in-ajar 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.

Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis

“…Currently, many in vitro models only allow investigations of fibrogenesis in acute studies of 1-3 days [28,34] although some models extend to a few weeks [44]. To our knowledge, this is the first time that a 12 days fibroblast culture has been combined with the investigation of ECM synthesis in the supernatant.…”

“…Interestingly, the addition of a crowding agent induced complete cleavage of the type I collagen C-propeptide, whereas the fibroblasts in the non-crowded culture conditions continued to secrete intact procollagen. Recently, Good et al described a 72 h high content screening Scar-in-a-Jar assay utilizing fibroblasts from IPF patients, that allowed the quantification of disease-relevant ECM deposition in vitro [34].…”

The Need for Basic, Translational, and Clinical Research in the Field of Hypertrophic Scars

“…In addition, 3D cell cultures can be used to more closely mimic the organization of skin as a tissue structure. Such "scar in a jar" models are likely to be useful moving forward [126]. It has also been suggested that media components may be altered to create a pseudo "crowded" environment in culture.…”

Engineering Advanced In Vitro Models of Systemic Sclerosis for Drug Discovery and Development

“…[ 157 ] A similar study, combined macromolecular crowding with TGF‐β to develop a robust, high throughput, phenotypic screening assay using pulmonary fibroblasts derived from patients affected by idiopathic pulmonary fibrosis. [ 158 ] The Scar‐in‐a‐Jar offers a novel pathophysiologically relevant screening and evaluation tool for antifibrotic compounds interfering with different key steps in the collagen biosynthesis pathway.…”