Establishment of a stable three-dimensional fibrotic cell culture model for reproducible baseline and experimental applications.

Systemic sclerosis (SSc) is characterized by progressive fibrosis and complex alterations in extracellular matrix dynamics; however, experimental modeling of fibrotic processes remains limited by poor control of baseline signaling and spontaneous phenotypic drift in vitro. Such variability complicates the interpretation of perturbation-driven responses and reduces the reliability of antifibrotic and toxicological studies. In this study, we developed and characterized a three-dimensional (3D) fibrotic in vitro platform designed to maintain a stable and controlled baseline state across distinct microenvironmental conditions. Primary dermal fibroblasts derived from a bleomycin-induced scleroderma model and human idiopathic pulmonary fibrosis fibroblasts (CCL-191) were embedded in collagen-fibrinogen-based bioinks (Matrigel and Cellink Skin) and fabricated using extrusion-based 3D bioprinting. Across all conditions, the 3D constructs preserved structural integrity, sustained cell viability, and maintained consistent myofibroblast features, including α-SMA expression, fibrotic gene transcription (TGF-β, IL-6, IL-1, collagen I), and canonical signaling activity (pSmad2/3). Importantly, no evidence of spontaneous overactivation or loss of fibrotic signaling was observed, indicating tight regulation of baseline pathway activity independent of bioink composition. These findings demonstrate that fibrotic signaling can be stabilized within a controlled 3D microenvironment, minimizing baseline drift and enabling reproducible molecular and biochemical readouts. By establishing a non-fluctuating fibrotic baseline, this platform provides a robust framework for future perturbation-based studies and enhances the interpretability of antifibrotic and toxicological screening approaches.