Background/Purpose: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with multiple tissues contributing to its pathology, including the synovial membrane. The RA synovial membrane consists of various cell types, including fibroblast-like synoviocytes (FLS), macrophages (Mϕ), endothelial cells (EC), and immune cells. Interactions among these cells drive the inflammatory processes and local joint destruction, characteristics of RA. Traditional 2D cell culture is insufficient for modeling this complex cellular environment and its dynamic interactions. Thus, advanced 3D culture models that incorporate the diverse cell types are needed, as this would offer a more physiologically relevant platform for studying RA synovial pathophysiology and testing therapeutic interventions.

Methods: We assessed two different 3D culture systems, a Matrigel-based and a hanging drop system. For Matrigel 3D cultures, FLS alone or in combination with Mϕ, EC (HUVEC), and/or group 2 innate lymphoid cells (ILC2) were resuspended in Matrigel and plated into poly-HEMA-coated plates. For the hanging drop system, cells (as described above) were resuspended in FLS media, pipetted into poly-HEMA-precoated 96-well plates, which were then turned upside down and left untouched for one week. Media was changed every three days, with stimulation (TNFα 20 ng/ml) added after one week for both systems. 3D cultures were harvested after a total of three weeks and evaluated by H&E, immunofluorescence (IF) staining, and expression of mRNA by qPCR.

Results: We successfully developed a 3D Matrigel-based culture system that can incorporate FLS, Mϕ, EC, and ILC2. The 3D cultures mimicked physiological properties of the synovial membrane, forming a defined lining layer, as well as expressing specific FLS markers such as Podoplanin (PDPN) and CD55. IF staining revealed that the inclusion of Mϕ increased the basal expression of PDPN (0.006±0.010 vs 0.150±0.069, p=0.022, n=3), a marker for aggressive RA FLS. TNFα stimulation further increased FLS expression of both PDPN (0.150±0.069 vs 3.778±0.665, p< 0.0001, n=3) and CD55 (0.017±0.005 vs 0.592±0.619, p=0.020, n=3). Further, IL6 mRNA expression was significantly upregulated upon TNFα stimulation (p=0.001, n=3) and interestingly, 3D cultures with RA FLS showed increased IL6 mRNA expression compared to those with healthy FLS. To avoid non-physiological matrix components and growth factors present in Matrigel, a multicellular, long-term 3D model using the hanging drop system was developed. In this system, we found FLS to produce their own extracellular matrix, including the human synovial components collagen types I and IV. In addition, hanging drop cultures showed similar synovial characteristics, including CD55 and PDPN expression.

Conclusion: Here, we present two long-term 3D culture systems that incorporate synovial membrane-specific cells, including FLS, Mϕ, EC, and ILC2. Our model exhibits synovia-specific cellular architecture and RA-specific inflammatory responses, providing an excellent platform for testing novel therapeutic compounds over an extended period and studying specific signaling pathways and cell-cell interactions in a physiological manner independent of mouse models.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-long-term-synovial-tissue-3d-model-incorporating-fibroblasts-macrophages-endothelial-cells-and-other-immune-cells-such-as-innate-lymphoid-cells-enables-animal-model-independent-rheumatoid-arthrit/