Figure 5. In vitro modelling of fibrillar collagen production and cross-linking in IPF.

We utilised a long-term 3D in vitro model of lung fibrosis using primary human lung fibroblasts from patients with IPF treated with the pro-fibrotic cytokine TGF-β₁ cultured for up to 6 weeks. (A–C) Characterisation of (A) total collagen normalised to total protein, (B) immature divalent (DHLNL) and (C) mature trivalent (PYD and DPD) hydroxyallysine-derived collagen cross-links at the culture times indicated following addition of TGF-β₁. Bars are mean +range (n = 2 IPF donors). (D) Relative gene expression analysis of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 using the ∆∆Ct method (n = 3 IPF donors, two experiments per donor). (E, F) Masson’s trichrome stain of histological sections from (E) the in vitro model at 6 weeks and (F) IPF lung tissue including a fibroblastic focus. Blue staining identifies fibrillar collagen. Scale bars are 100 μm (main image) and 500 μm (inset, showing location of enlarged image).