A Matrigel-Free 3D Chondrocytic Spheroid Model for Rheumatoid Arthritis-Associated Synoviocytes Invasion Studies

Yingjie Zhao,1,2,* Xuezhi Yang,3,* Feng Yao,1,2,* Ziwei Ouyang,2 Weirong Hu,2 Lin Li,4 Juan Cheng,5 Ke Wang,1,2 Jie Ding,2 Liang Zheng,1,2 Biao Qu,1,2 Cheng Sun,2 Shufang Li,1 Chen Jiang,2 Yanan Chen,2 Renpeng Zhou,1,2 Wei Hu1,2 1Department of Clinical Pharmacology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230601, People’s Republic of China; 2Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, Anhui, 230032, People’s Republic of China; 3Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui, 230032, People’s Republic of China; 4Department of Rheumatology and Immunology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230601, People’s Republic of China; 5Department of Laboratory Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230601, People’s Republic of China*These authors contributed equally to this workCorrespondence: Renpeng Zhou, Department of Clinical Pharmacology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230601, People’s Republic of China, Email [email protected] Wei Hu, Department of Clinical Pharmacology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230601, People’s Republic of China, Email [email protected]: The primary pathology of rheumatoid arthritis (RA) involves the invasion of the extracellular matrix (ECM) of articular cartilage by inflammation-activated fibroblast-like synoviocytes (FLS), a process targeted by most RA therapeutic drugs. However, the absence of an efficient in vitro model for evaluating FLS invasion hinders relevant drug screening and mechanism research. To address this, a novel three-dimensional (3D) chondrocytic spheroid model that mimics cartilage ECM has been developed, along with corresponding indices to quantify synoviocytes invasion.Methods: The matrigel-free 3D chondrocytic spheroid model was developed using an ultra-low attachment plate. The model was characterized using transcriptome sequencing, immunofluorescent staining. To explore the feasibility of this 3D chondrocytic spheroid model for evaluating the invasive capacity of synoviocytes, multi-interference strategies, including ADAMTS5 gene overexpression, inflammatory cytokine stimulation, and anti-inflammatory drug (Etanercept) treatment were involved. Additionally, specific indices—Invasion Depth Ratio (IDR), Invasion Counts (IC), Invasion Ratio (IR), and Invasion Area Ratio (IAR)—were designed to quantify synoviocytes invasion.Results: The 3D culture environment is more suitable for cartilage ECM synthesis by increasing cartilage anabolism-related gene (COL2A1) and reducing catabolism-related genes (ADAMTS5, MMP3, CCL2 and CDKN2A) expression. Moreover, the optimal conditions for developing the 3D chondrocytic spheroid model were identified. This model was sensitive to gene, inflammation and drug interference. Increased IDR, IC, IR and IAR was observed in ADAMTS5 overexpressed- and IL-1β-treated chondrocytic spheroid. Further, Etanercept could inhibit TNF-α induced synoviocytes invasion of chondrocytic spheroid.Conclusion: This matrigel-free 3D chondrocytic spheroid model offers an ideal platform for innovative drug screening and pathogenesis studies focused on synoviocytes invasion of cartilage.Keywords: RA, invasion, 3D, synoviocytes, chondrocytes, drug screening model