Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients

Part of the Methods in Molecular Biology book series (MIMB, volume 1687)


An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a “scratch” assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.

Key words

Duchenne muscle dystrophy Fibrosis Fibroblasts In vitro model Pirfenidone Toxicity Proliferation Collagen Migration Fibrotic nodules 


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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Muscle Cell Biology Lab, Neuromuscular Diseases and Neuroimmunology UnitFondazione IRCCS Istituto Neurologico “C. Besta”MilanItaly

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