Shuichi Takayama, PhD (Georgia Institute of Technology)
Eva Dyer, PhD (Georgia Institute of Technology)
Melissa Kemp, PhD (Georgia Institute of Technology)
Nael McCarty, PhD (Emory University)
Krish Roy, PhD (Georgia Institute of Technology)
Microscale high-thoughput phenotypic assay to evaluate ECM remodeling in pulmonary fibrosis
In fibrotic disease, dysregulation of matrix remodeling generates excessive deposition of fibrous extracellular protein that can interfere with the architecture and function of tissue. The pathogenic role of aberrant fibrin remodeling is particularly interesting in idiopathic pulmonary fibrosis (IPF), which has proven largely unresponsive to conventional anti-fibrosis therapies. Due to the substantial variety factors that combinatorially influence extracellular matrix (ECM) turnover, there exists the need for a phenotypic assay to evaluate cumulative effects involving cell-mediated fibrinolysis and collagen deposition. The goal of this project was to develop a novel in vitro assay that mimics fibroblast-mediated remodeling of the provisional fibrin matrix, in order to establish a model system for fibrotic scar formation and evaluate potential therapeutic compounds.
This work introduces and evaluates new methods to analyze ECM turnover in a high-throughput, label-free format. An aqueous two-phase printing technique was established to enable generation of microscale fibroblast-laden fibrin gels, which resemble the provisional fibrin matrix in wound healing. In a first variant of the assay, addition of exogenous plasminogen enabled cell-mediated activation of plasmin for gradual degradation of the fibrin matrix. A second variation of the assay implemented higher fibroblast seeding densities with serum-supplemented media to facilitate remodeling of the fibrin matrix through concurrent fibrinolysis and collagen deposition. Live-cell imaging provided time-course brightfield micrographs that were analyzed through an automated image processing protocol for high throughput evaluation of different stages of remodeling. Application of this assay allowed convenient evaluation of healthy and diseased donors of pulmonary fibroblasts and assessment of anti-fibrotic compounds to determine their effects on different stages of remodeling.
This analysis of fibroblast-mediated remodeling of fibrin demonstrated characteristics of fibrotic ECM remodeling that are not assayed in other in vitro models of fibrosis. By investigating the cumulative effects of fibrinolysis and collagen deposition on fibroblastic remodeling of fibrin, this assay may provide a new resource for advancing understanding of fibrosis pathogenesis and for evaluating potential anti-fibrosis therapeutics.