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. This 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. In 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.

中文翻译：

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一种高含量、表型“罐子里的疤痕”测定，可使用疾病来源的肺成纤维细胞快速定量胶原纤维生成。


细胞外基质（ECM）过多沉积是纤维化和组织重塑疾病的标志特征。通常，间充质细胞在标准二维细胞培养条件下会产生胶原蛋白，但它们不会组装成原纤维。现有的用于测量 ECM 产生的测定通常通量低且与疾病无关。在这里，我们描述了一种稳健、高含量、伪 3D 表型测定，用于量化成熟纤维状胶原沉积，该测定既具有生理相关性，又适合高通量化合物筛选。使用源自特发性肺纤维化 (IPF) 患者的肺成纤维细胞，我们将“罐中疤痕”测定法开发为中等通量表型测定法，以稳健地定量 I 型胶原蛋白沉积和其他细胞外基质 (ECM) 蛋白72 小时。该测定利用大分子拥挤来诱导排除体积效应并增强酶活性，与 TGF-β1 刺激相结合可显着加速 ECM 的产生。 I 型胶原蛋白上调约 5 倍，对细胞数量的影响可以忽略不计。我们证明了该检测的稳健性，检测对照 SB-525334（ALK5 抑制剂）和 CZ415（mTOR 抑制剂）的 Z 素数约为 0.5，变异系数 (CV)% < 5。该测定已用于确认许多潜在抗纤维化药物的效力。来自“罐中疤痕”测定的活性化合物可以进一步验证 ECM 沉积和成纤维细胞活化的其他标志物，例如 IV 型胶原和 α-平滑肌肌动蛋白，分别显示 4 倍和 3 倍测定窗口。 总之，我们已将“罐子里的疤痕”开发成一种强大的疾病相关中等通量体外测定法，以准确量化 ECM 沉积。该测定可以实现 IPF 和其他纤维增殖和组织重塑疾病的迭代化合物分析。