Laurens Spoelstra*1,2,3, Nuno Araújo-Gomes1, Mariia Zakharova2,Tim Welting4, Marcel Karperien1, Loes Segerink2, Séverine Le Gac31Developmental BioEngineering, TechMed Centre, University of Twente, Enschede, The Netherlands2BIOS Lab on a Chip group, MESA+ Institute, TechMed Centre, University of Twente, Enschede, The Netherlands3Applied Microfluidics for BioEngineering Research, MESA+ Institute, University of Twente, Enschede, The Netherlands4Laboratory of Experimental Orthopedics, Maastricht UMC, Maastricht, The Netherlands*Corresponding author: [email protected] affects millions of people globally and commonly involves synovial inflammation.Unfortunately, for many arthritic diseases, such as Rheumatoid Arthritis (RA) and Osteoarthritis (OA) inparticular, there is a limited number of disease-modifying treatments available. This is in part due to alack of physiologically relevant preclinical models to assess these inflammatory forms of arthritis.Recently, we have developed a Synovium-on-Chip (SoC) platform that modeled the synovial lining,consisting (mainly) of synovial fibroblasts and macrophages, and the synovial vasculature in the subintimawith endothelial cells (Figure 1A) [1,2]. However, this model had the limitation of possessing a ~20µmthick nonporous PDMS membrane acting as physical barrier separating the endothelial cells from thehuman synovial fibroblasts (hSFBs) and macrophages, limiting intercellular communication and thepossibility to mimic key pathogenic events such as monocyte extravasation [1].Here, we address these limitations and integrate a previously developed [3] porous 2µm thick PDMSmembrane (pore diameter of 5µm and a 30µm pitch). First, SoC devices were fabricated at a wafer scalewith up to 20 devices in a single process to enable higher throughput experiments (Figure 1B). Next, weco-cultured hSFBs from donors with THP-1-derived macrophages (top chamber) and Human UmbilicalVein Endothelial Cells (HUVECs, bottom chamber) for up to 10 days. All three cell types integrated wellin the devices (Figure 1C) and confluent cell layers were observed on both sides of the membrane (Figure1D-E). After verification of their integration and viability, the cells were challenged using 1 ng/mL TNF-α as a proof-of-concept for studying inflammatory arthritis (Figure 1F). After 4 days of stimulation (T10),RT-qPCR was performed on the hSFB and THP-1 cells (top chamber) for IL6, CCL2, MMP1, andTNFAIP6, which were all found to be upregulated by ~2-5 fold (Figure 1G).Surprisingly, microscopic analysis of the devices showed that HUVECs had started to delaminate fromthe walls of the channels while fibroblast-like cells were visible in the bottom channel (not shown). Thisprompted the hypothesis that the hSFBs could migrate through the porous membrane, which was testedin devices with nonporous membranes and in devices with only fibroblasts seeded in the top chamber.Interestingly, fibroblast migration was observed through the 5µm pores (Figure 2A), and 3D confocalmicroscopy revealed that the HUVECs formed a lumen-like structure (Figure 2B). Strikingly, hSFBs(labeled with green CellTracker) were found next to the HUVECs at the bottom of the chip and colocalized with the HUVECs in the lumen-like structure’s lining (Figure 2C). Future research will focuson how the interactions between HUVECs and hSFBs occur, leading to this lumen formation, assessingstability in long-term culture through microscopy analysis.In short, we have successfully developed a novel SoC model with an integrated porous membrane and infuture experiments, we will investigate different inflammatory stimuli, drug efficacy, monocyteextravasation, and the apparent self-organization of HUVECs and hSFBs.References1. Paggi, C. A., et al. Nat. Rev. Rheumatol. 18, 217-231 (2022).2. Paggi, C. A., et al. MicroTAS Conference (2021).3. Zakharova, M. et al. Adv. Mater. Technol. 6, 2100138 (2021
