Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.

Systemic sclerosis (SSc) is a rare autoimmune disease with limited treatment options that is characterized by fibrosis in various organs. To screen the effectiveness of new therapies, there is an urgent need for reliable in vitro models. Key is that diseased cells' characteristics are maintained, which is challenging in currently used setups. In this study, an in vitro 3D culture system is described using the biocompatible polyisocyanide (PIC‐RGD) hydrogel and SSc patient‐derived fibroblasts from affected (lesional cells) and from healthy‐skin (healthy cells). In contrast to the standard collagen‐coated 2D cultures, the cells in the 3D PIC‐RGD gels maintain the native phenotype and functionality of the primary cells. The functionality of the model is studied in the presence of the fibrosis stimulator transforming growth factor β1 (TGFβ1) and the suppressor tumor necrosis factor (TNFα). In this study, it is observed that lesional cells have a stronger fibrotic character with increased contraction, proliferation, and expression of collagen, and myofibroblast markers α‐smooth muscle actin and fibroblast activation protein. The high tunability of the hydrogel, which can maintain the native functionality of fibroblasts in in vitro cultures, delivers a crucial step in developing these materials into an effective tool for personalized medicine approaches of SSc patients.