In vitro models of TGF-beta-induced fibrosis suitable for high-throughput screening of antifibrotic agents

Xu Q, Norman JT, Shrivastav S, Lucio-Cazana J, Kopp JB
Source: Am J Physiol Renal Physiol
Publication Date: (2007)
Issue: 293(2): F631-40
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Mesangial cells, Human (NHMC)
Species: human
Tissue Origin: kidney
Nucleofector® I/II/2b
Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective anti-fibrotic drugs. Here we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in 8-well chamber slides, transforming growth factor-beta1 (TGF-beta1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-beta1-induced matrix over-expression since they were mimicked by addition of collagen I to the cell culture medium. TGF-beta1-induced nodule formation was inhibited by vacuum ionized gas treatment of plate surface. This blockage was further enhanced by pre-coating plates with matrix proteins but prevented, at least in part, by poly-L-lysine (PLL). We have established two cell-based models of TGF-beta-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL coated 96-well plates, on which TGF-beta1-induced 2-dimensional matrix accumulation, 3-dimensional nodule formation and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the anti-fibrotic compound tranilast were shown to have inhibitory activities in both assays. Key words: collagen, smad3, tranilast, cell migration.