A 5D dynamic bicellular in vitro model recapitulates the aetiopathogenic triad of systemic sclerosis: a preliminary proof-of-concept study

Abstract

The present work aimed at modelling in vitro the interplay between immune complexes (ICs) containing scleroderma-specific antibodies, vasculopathy and skin fibrosis to reproduce the multifaceted etiopathogenesis of systemic sclerosis (SSc). Endothelial cells (ECs) were grown in a bioreactor comprising apical and basal chambers separated by a porose membrane reproducing the endothelial barrier. ECs were seeded in the apical chamber in hydrogel. Skin fibroblasts were isolated from normal healthy subjects (NHS) and cultivated in acellularized 3D scaffold in a single-chamber bioreactor. The two bioreactors were interconnected via a fluidic system supplied by a peristaltic pump. ICs were purified by PEG precipitation from sera of SSc patients with anti-centromere or anti-DNA topoisomerase antibodies, systemic lupus erythematosus (SLE) or NHS. ECs were treated with SSc-ICs, SLE-ICs, NHS-ICs, TNFα or culture medium. Sections of ECs/hydrogel and fibroblasts/scaffold were evaluated by histological/immunohistochemical and immunocytochemical analysis. ICs and soluble mediators (IL-6, IL-8, CCL2/MCP-1) were measured in supernatants collected at different sites along the fluidic circuit. Upon stimulation with SSc-ICs, ECs in hydrogel developed unorganized endothelial tubular structures. The porous membrane prevented the passage of ICs but not of soluble mediators. Treating ECs with SSc-ICs induced the formation of fibrous septa, increased expression of collagen IV and αSMA, and reduced expression of CD34 in fibroblast scaffold. This innovative dynamic bicompartmental in vitro system incorporating 5 degrees of complexity (5D) allows reproducing the SSc environment, since stimulation of ECs with SSc-ICs is sufficient not only to disrupt vascular architecture but also to induce skin fibrosis.