Given the lack of reliable, disease relevant in vivo models of fibroproliferative diseases, anti-fibrotic drug discovery relies heavily on phenotypic in vitro models. With the recent advancement of disease relevant 3D cell culture models taking favour over traditional 2D models, there is a clear need for the development of reliable inflammation-independent in vitro models of fibrosis and tissue remodelling [20, 21]. Here we have described a phenotypic in vitro collagen deposition assay enabling accurate quantification of rapidly deposited, insoluble, cross-linked collagen that can be combined with patients-derived cells in a medium throughput manner. The ‘scar-in-a-jar’ assay has been previously described [11, 13] and utilised to confirm efficacy of novel anti-fibrotic agents [2, 10, 14], however until now a high content, medium throughput screening assay has not been described. The ability to screen compounds using 10-point concentration response curves enables the generation of accurate potency and efficacy data for novel compounds inhibiting collagen deposition at multiple points during processing and maturation rather than focussing on transcriptional read-outs or soluble immature collagen monomers. Furthermore, this assay gives an early indication of potential cytotoxicity, by assessing nuclei size, morphology, and intensity. These parameters identify nuclei blebbing, shrinkage and fragmentation, all of which are indicators of apoptosis .
Here we have combined macromolecular crowding with the pro-fibrotic and pleiotropic cytokine TGF-β1 known to be elevated in IPF , to develop a robust, high content, phenotypic screening assay using patient-derived pulmonary fibroblasts. This assay has been used to screen over 150 novel compounds with an assay success rate of over 95%. The control compounds SB-525334 and CZ415 have proven to be reliable positive controls indicating the importance of the TGF-β1  and mTOR [2, 14] pathways in type I collagen fibrillogenesis. However, the biological heterogeneity between IPF patient cell lines was prevalent when evaluating the effects of PGE2 on collagen deposition. PGE2 exhibited a larger range in efficacy compared to other compounds with higher variability in potency estimation suggesting that PGE2 is not a robust assay control. This could be due to some IPF patients exhibiting a deficiency in PGE2 synthesis coupled with an inability to respond to exogenously added eicosanoid following downregulation of PGE2 receptors and signalling [18, 23,24,25]. On the one hand, this supports the use of patient-derived cells to more accurately reflect the disease biology, however this also highlights the challenges of using multiple patient primary lines to generate accurate potency data. While subtle differences may be observed at baseline between healthy control and IPF primary lung fibroblasts, these differences become undetectable upon TGF-β1 stimulation (data not shown). Recently we demonstrated that healthy control lung fibroblasts also exhibit a similar TGF-β1-induced collagen type I response .
While type I collagen is considered to be one of the most significantly upregulated ECM proteins, and a hallmark feature of fibroproliferative diseases , hits from the ‘scar-in-a-jar’ assay can be further validated against other markers of fibrosis. We demonstrate that in addition to quantifying type I collagen deposition, the ‘scar-in-a-jar’ assay also enables the visualisation and quantification of collagen type IV  and fibronectin deposition, as well as α-SMA expression. Indeed this assay could be utilised to quantify a range of ECM proteins, a number of which have previously been reported to upregulated by TGF-β1 and macromolecular crowding including collagens I, III, IV, V, VI, XII . Recently it has been demonstrated that culturing fibroblasts on stiff substrates such as tissue culture plastic induces cellular fibronectin production , perhaps causing the high basal fibronectin deposition. However while cellular fibronectin deposition was not elevated in the ‘scar-in-a-jar’ assay, there was a marked difference in fibronectin distribution and organisation; a characteristic considered to be an important pathological event in fibrosis , thus representing another potential parameter for high content analysis.
The ‘scar-in-a-jar’ assay offers several advantages over previous in vitro models of fibrosis. Unlike previous techniques, the ‘scar-in-a-jar’ assay enables the visualisation and quantification of rapid deposition and cross-linking of mature collagen fibrils, more closely resembling the disorganized fibres characteristic of IPF lesions [11, 13, 30]. In contrast, previous assays have focused on quantifying soluble collagens using the Sircol assay  or measuring soluble P1NP (procollagen type I N-terminal propeptide ), both of which reflect surrogate markers of ECM turnover . Other methods of quantifying ECM deposition include the histological Picro-Sirius red staining, however this lacks resolution and collagen specificity . Similarly, quantification of hydroxyproline, using reverse-phase HPLC, is non-specific and involves substantial sample processing and manipulation that is both low throughput and time-consuming .
The addition of macromolecular crowding agents to culture media has previously been demonstrated to mimic the dense extracellular microenvironment by imitating the features of the tissues from which the cells were isolated [35, 36]. Macromolecular crowding enhances ECM deposition as well as influencing the alignment, thickness, and architecture of ECM fibrils in vitro [11, 30, 35, 36]. Collagen chains which are synthesised in the endoplasmic reticulum, and undergo post translational modifications (PTMs) such as the hydroxylation of lysine and proline residues, followed by glycosylation of specific hydroxyl residues . Following PTMs, collagen chains (two proα1 and one proα2) form a triple helix to make pro-collagen which is released into the extracellular space . The N- and C- procollagen terminals are cleaved by ADAMTS and procollagen C-proteinase (bone morphogenic protein-1/BMP1)  and cross-linked to form mature collagen fibrils. Macromolecular crowding induces the phenomenon known as the excluded volume effect (EVE) to dramatically enhance enzymatic activity and accelerate ECM formation in vitro. In combination with TGF-β1, macromolecular crowding-induced EVE significantly elevates the deposition of mature, cross-linked ECM [11, 13, 30]. To increase the likelihood of identifying novel inhibitors of ECM synthesis and maturation with unknown mechanisms of action, a 3-h pre-incubation step is performed to allow compounds to reach equilibrium before TGF-β1 stimulation.
We have described the development of an IPF-relevant ‘scar-in-a-jar’ assay to screen novel anti-fibrotic compounds. Recently, we also utilised the ‘scar-in-a-jar’ assay to identify novel anti-fibrotic targets using CRISPR/Cas9 gene editing to explore the cellular phenotype in response to alterations in genotype [2, 38]. With the recent advances in whole genome CRISPR screening , the HCS ‘scar-in-a-jar’ assay represents an attractive approach to identify novel targets. Indeed, this assay is not limited to IPF research. The benefits of macromolecular crowding in tissue-specific cultures have been described for a number of other fibrotic and tissue remodelling assays  including using corneal fibroblasts , dermal fibroblasts  and bone marrow stroma-derived cells  affecting ECM deposition as well as modifying the cellular phenotype . Furthermore, this assay could be developed to explore the impact of ECM deposition in complex multi-cellular systems. For example, others have utilised macromolecular crowding-induced ECM to explore the effects of different tissue microenvironments on cell-ECM interactions including mesenchymal  and embryonic  stem cells. Recently, cancer-associated fibroblast (CAF)-derived ECM has been shown to be deposited in an organised, aligned manner facilitating cancer cell motility and enhancing tissue invasion [44,45,46]. In addition to its application for pulmonary fibrosis research as outlined in this study, the HCS ‘scar-in-a-jar’ assay could be utilised to study complex cell-cell and cell-ECM interactions in research areas including fibrosis (pulmonary, hepatic, renal, cardiac and dermal), tissue remodelling, wound repair and oncology.