Synovial Organoids and Cell Culture Models: An Ideal Platform for Exploring Traditional Chinese Medicine in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, inflammation, and pannus formation. Although conventional therapies are effective, they are often associated with significant side effects. Traditional Chinese medicine (TCM), known for its high safety profile and multitarget therapeutic effects, has shown unique potential in the treatment of RA. In vitro models, including synovial organoids and cell culture systems, serve as crucial platforms for replicating the structure and function of human synovial tissue. Synovial organoids closely mimic the pathological microenvironment of RA, providing an ideal tool for drug screening and efficacy evaluation. In contrast, cell culture models, which offer greater experimental flexibility, allow precise control of conditions to facilitate an in-depth investigation of the roles and mechanisms of individual or interacting cell types in RA pathogenesis. This review first covers the fundamental structure and pathophysiology of the synovium, followed by a detailed discussion of the development and construction techniques of synovial organoids. Building on this foundation, it explores the therapeutic potential of TCM using synovial organoids and cell culture models, highlighting how TCM exerts therapeutic effects by inhibiting synovial hyperplasia and modulating immune responses through its multitarget and multipathway mechanisms. Finally, the review envisions the broad application prospects of integrating synovial organoids and cell culture models with TCM in RA treatment, underscoring the importance of interdisciplinary collaboration and innovation. This study aims to provide a novel perspective on the integration of TCM with modern biotechnological approaches and promote innovative advances in RA therapy.

1. Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation, synovial hyperplasia, pannus formation, and progressive destruction of articular cartilage and bone tissue. , The synovium, a critical component of the joint structure, not only provides lubrication and protection but also plays a central role in the pathogenesis of RA. , Key pathological features of RA include aberrant proliferation of synovial cells, excessive secretion of pro-inflammatory cytokines, and the formation of invasive pannus tissue. , Current treatment strategies for RA, such as nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, and glucocorticoids, are effective in controlling symptoms but are often associated with significant side effects, particularly with long-term use, which can adversely impact patients' quality of life and treatment compliance. − Although newly developed biological agents and targeted small-molecule drugs have shown impressive therapeutic outcomes, their high cost poses a considerable financial burden on patients and healthcare systems alike. , Therefore, there is an urgent need to develop safe, effective, and affordable therapeutic alternatives for RA.

Traditional Chinese medicine (TCM), known for its favorable safety profile and low incidence of adverse effects, is gaining increasing global attention as a complementary or alternative therapeutic approach. In many Asian countries, the use of herbal medicine for RA has a history spanning thousands of years. This time-honored medical practice, supported by extensive clinical experience and an increasing body of scientific evidence, has demonstrated significant therapeutic potential. , One of the distinguishing features of TCM is its multitarget, multipathway mode of action, allowing it to exert therapeutic effects through diverse biological mechanisms. , Specifically, many Chinese herbal formulations possess strong antirheumatic properties, including anti-inflammatory, analgesic, and immunomodulatory effects, as well as the ability to inhibit abnormal synovial cell proliferation and pathological angiogenesis. , Numerous herbs and their active constituents have shown promising efficacy in suppressing synovial hyperplasia, providing novel insights and potential breakthroughs in RA therapy.

Organoids are three-dimensional cellular structures formed through intrinsic genetic reprogramming mechanisms, primarily initiated by tightly regulated soluble biochemical niche signals derived from either suspension cultures or three-dimensional extracellular matrix (ECM)-like systems that closely mimic in vivo environments. These signals are essential for promoting cellular self-organization and the development of tissue-specific organ-like structures. − In recent years, organoids have attracted considerable attention in biomedical research due to their ability to replicate the complex architecture and function of human organs in vitro. Their high fidelity in modeling organ development, disease progression, and drug response makes them powerful platforms for investigating organogenesis, elucidating disease mechanisms, and conducting drug screening and evaluation studies. ,

Among these, synovial organoids have emerged as critical models for recapitulating the structural and functional features of human synovial tissue in vitro. In recent years, they have shown substantial progress in the fields of disease modeling, pharmacological research, and tissue engineering. − The establishment of physiologically functional synovial organoids not only enhances our understanding of RA pathogenesis but also offers invaluable experimental systems for drug development, therapeutic evaluation, and tissue repair. TCM has demonstrated notable therapeutic effects in such models. For example, Danggui Buxue Decoction (DBD) has been shown to target SFRP4 and modulate the Wnt signaling pathway, thereby effectively suppressing inflammation in RA fibroblast-like synoviocytes (RA-FLS) and slowing disease progression. Similarly, Baihu Guizhi Decoction (BHGZD) inhibits synovial cell hyperproliferation and inflammation by suppressing Toll-like receptor 4-mediated NLRP3 inflammasome activation, offering promising support for symptom relief in RA patients. In an effort to further elucidate the mechanisms of TCM, researchers have developed an innovative in vitro coculture model in which peripheral blood mononuclear cells (PBMCs) from RA patients are used to stimulate FLS, mimicking the pathological immune environment of RA. Using this model, the TCM formula Huangqin Qingre Chubi Capsule (HQC) was found to exert significant anti-inflammatory effects, likely through inhibition of the hsa_circ_0091685/EIF4A3/IL-17 regulatory axis, thereby contributing to disease amelioration. Moreover, recent studies have identified sesamol as a novel p53 stabilizer with therapeutic potential in RA. Notably, sesamol not only alleviates RA symptoms but also inhibits the growth of synovial organoids, providing a new direction for TCM applications in RA management. Collectively, these findings highlight the promising results and future potential of TCM in single-cell, coculture, and synovial organoid-based models. They underscore the growing importance of TCM in RA treatment and suggest a substantial opportunity for its further development.

This article aims to explore the potential of synovial organoids and cell culture models as innovative platforms for applying TCM in the treatment of RA. It provides a comprehensive review of the developmental trajectory and construction methodologies of organoids and discusses in depth the therapeutic applications and advantages of TCM in these systems. Through this investigation, we seek to offer new insights and perspectives on the integration of TCM with modern biotechnological approaches for advancing RA therapy.

2. Basic Structure and Pathophysiology of the Synovium

The human synovium, a membranous tissue lining the inner surface of articulating joints, plays a crucial role in maintaining joint stability and facilitating diverse physiological functions. The synovial membrane (SM) is composed of two distinct layers: the intimal layer and the subintimal layer. , The intimal layer, forming the innermost portion of the SM, typically consists of one or two layers of macrophage-like synoviocytes (MLS, also referred to as type A synovial cells) and FLS (type B synovial cells). These cells are primarily responsible for secreting synovial fluid (SF), which lubricates joint cartilage, supports chondrocyte function, and supplies essential nutrients. Notably, type B synoviocytes synthesize hyaluronic acid, a critical glycosaminoglycan that significantly contributes to the viscosity and lubricating properties of SF. , Beneath the intimal layer lies the subintimal region of the SM, consisting of two to three layers of synovial cells supported by a loose connective tissue matrix abundant in fibroblasts.

Synovial dysfunction is closely associated with the initiation and progression of joint disorders with synovitis being one of the most prominent manifestations. Synovitis is characterized by synovial hyperplasia, excessive cell proliferation, and massive infiltration of immune cells. These pathological alterations are accompanied by elevated levels of pro-inflammatory mediatorssuch as cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2)secreted by resident synovial cells. In the context of inflammatory arthritis, particularly RA, the synovium is regarded as the primary site of pathology. Persistent synovitis plays a central role in driving secondary damage to adjacent cartilage and bone structures. , During RA flareups, the synovium undergoes two major pathological changes. First, activation of FLS and MLS leads to a marked expansion of the intimal layer. This expansion facilitates adhesion and proliferation of inflammatory mediators and matrix-degrading enzymes, including selectins, integrins, and members of the immunoglobulin superfamily. Second, the infiltration of activated immune cells into the SM further amplifies the local inflammatory milieu, contributing to persistent inflammation and disease progression (Figure ).

1.

Basic structure and pathophysiology of the synovium.

3. Evolution of Synovial Organoid Models

3.1. From Monolayer Culture to Coculture and 3D Organoid Development

The evolution of synovial research models has undergone a significant transitionfrom basic to advanced systems and from two-dimensional (2D) to three-dimensional (3D) configurations. While each model offers distinct advantages, no single approach can fully capture the complex pathological landscape of synovial diseases (Figure ). Monolayer cultures and coculture systems remain widely used due to their operational simplicity and cost-effectiveness. Monolayer cultures, in particular, are well-suited for large-scale experiments and circumvent the technical challenges associated with coculturing heterogeneous cell populations. However, their limited ability to replicate tissue complexity and dynamic intercellular interactions often restrict investigations to isolated components rather than the system as a whole. To overcome these limitations, 3D culture systems and tissue explant models have been developed, providing more physiologically relevant platforms for studying synovial diseases such as RA. These models enable the simulation of intercellular communication and stromal interactions more accurately than 2D systems, making them valuable tools for drug screening and mechanistic research. As a result, they have significantly improved the translational efficiency of preclinical studies. ,

2.

Comparison of synovial organoids to other preclinical models.

With growing insight, researchers have recognized that 2D models, such as cultures of synovial fibroblasts or commonly used cell lines such as RAW 264.7 and K4IM, are insufficient to fully elucidate disease mechanisms. These models often fail to incorporate critical components such as synovial macrophages and ECM elements. , Consequently, newer strategies have emerged to simulate 3D tissue structures. These include the direct use of tissue explants and the construction of cell-scaffold composites, both of which represent promising research directions. Notably, the synovial joint capsule (SJC), which serves as a conduit between intra- and extra-articular environments and contains functionally important synovial tissue, has become a valuable model for investigating glycoproteinase hydrolysis mechanisms. However, anatomical variability among SJC samples presents challenges to standardization and reproducibility in experimental studies. , Interestingly, coculturing synovial and capsular tissues with articular cartilage has been shown to enhance glycosidase activity, offering insights into the complex interplay within the joint microenvironment. A major breakthrough was achieved by Stefani and colleagues, who developed a highly biomimetic, tissue-engineered synovial model by embedding bovine synovial fibroblasts and macrophage-like cells in Matrigel. In this system, Matrigel functions as a regulatory ECM scaffold that guides cellular behavior and mimics inflammatory responses. This model provides a powerful experimental platform for investigating the role of synovial cells in joint homeostasis and inflammation.

In conclusion, the progression from monolayer culture to coculture systems and 3D organoid models has enabled increasingly realistic simulations of the synovial microenvironment. These advancements have greatly enhanced our capacity to investigate disease mechanisms and develop novel therapeutic strategies.

3.2. Construction of 3D Synovial Organoids

3.2.1. Starting Cell Types

In the development of synovial organoids, stem cells play a central role as the foundational cell source owing to their exceptional self-renewal capacity and multipotent differentiation potential. Among these, adult stem cells (ASCs)especially when cultured as spheroidshave emerged as promising candidates for organoid generation, as they can faithfully recapitulate tissue morphogenesis and perform at least one function characteristic of the target tissue or organ. Within the context of musculoskeletal regenerative medicine, synovial-derived mesenchymal stem cells (MSCs) have attracted growing interest due to their distinctive biological characteristics. These cells reside within a specialized synovial niche, where local fibroblasts contribute to ECM production and joint homeostasis by secreting essential biomolecules. To enhance the precision of patient-specific therapeutic modeling, researchers primarily utilize two sources of fibroblasts in synovial organoid construction: fibroblasts directly derived from synovial MSCs and those generated via induced pluripotent stem cell (iPSC) technology. Each cell source presents unique advantages and application prospects.

Synovial-derived MSCs (SMSCs), a relatively recent addition to the MSC family, have garnered attention for their robust proliferative capacity and pronounced chondrogenic potential. When seeded at high densities, SMSCs rapidly expand, making them suitable for clinical-scale applications. Moreover, under 3D culture conditions, SMSCs demonstrate superior biocompatibility and enhanced chondrogenic differentiation, establishing a solid foundation for synovial organoid development. , Concurrently, the differentiation behavior of fibroblastscritical for maintaining tissue architecture and functionhas become a key focus in organoid research. , Recent studies have highlighted the pivotal role of Notch signaling, mediated by vascular endothelial cells, in regulating the differentiation of CD90⁺ fibroblast subpopulations, offering new mechanistic insights into fibroblast behavior within synovial organoid systems. iPSCs, another essential fibroblast source, not only provide a renewable and scalable cell population but also originate from patients with diverse clinical backgrounds. This makes them particularly suitable for disease modeling and the development of personalized therapies. , iPSC-derived constructs offer significant potential in high-throughput drug screening and in vitro disease modeling, thus accelerating progress in precision medicine and therapeutic innovation. In one notable study, researchers constructed a 3D synovial organoid model by coculturing iPSCs with MSC-derived fibroblasts and macrophages while introducing polyethylene particles to simulate mechanical and pathological stimuli. This system successfully recapitulated native tissue architecture and physiological responses under a variety of clinical conditions, providing a robust platform for diagnostic evaluation and the development of novel therapeutic strategies (Figure A).

3.

Current understanding and research progress on synovial organoids. (A) Inducing differentiation of pluripotent stem cells and mesenchymal stem cells into the synovium. (B) Using poor culture medium and heterogeneous culture medium components in organoid culture. (C) Various methods and scans have been used to construct synovial organoids.

3.2.2. Culture Environment and Signaling Factors

In the construction of synovial organoids, the physical and biochemical characteristics of the culture environment are of critical importance (Figure B). In recent years, significant progress in tissue engineeringparticularly in the development of 3D microsystems, organoid platforms, and microfluidic organ-on-a-chip technologieshas enabled increasingly sophisticated exploration of the complex structure and function of synovial tissue. − Utilizing 3D synovial organoid culture systems, researchers have been able to investigate cellular mechanisms involved in synovial tissue formation and inflammatory remodeling, highlighting the pivotal role of FLS in driving synovial inflammation under physiologically relevant conditions. , Innovative models of synovial angiogenesis based on a spheroid culture have also been developed. These models employ growth factor stimulation to recapitulate the complex process of angiogenesis and integrate monocyte-derived macrophages to simulate in vivo intercellular interactions, thereby deepening our understanding of immune and vascular contributions to synovial inflammation. − Since 2006, researchers such as Kiener's group have introduced 3D synovial micromass organoid models that allow for the spontaneous formation of synovial-like structures. These models have been successfully employed to investigate the regulatory roles of FLS in inflammatory cascades. , To further dissect the interplay between synovial and cartilage tissues, hydrogel-based 3D coculture models have been proposed. These systems aim to mimic intertissue communication and provide insight into joint pathophysiology by replicating the dynamic crosstalk between synovial and chondral organoids. Technological advances have also led to the emergence of microphysiological systems and organ-on-a-chip platforms, offering integrated, dynamic, and high-throughput environments for modeling complex aspects of arthritic disease. These platforms provide novel tools for elucidating disease mechanisms and facilitating therapeutic development. A notable example includes the work of Ertl et al., who established a coculture model involving human FLS (using the synovial sarcoma cell line SW982), murine osteoclast precursors (RAW264.7), and bone marrow-derived MSCs (BMSCs). This system was employed to investigate how FLS migration and invasion contribute to bone erosion in RA, offering new insights into the mechanisms underlying joint destruction. In synovial organoid cultures, the use of solid ECM materials is fundamental for supporting 3D morphogenesis and cellular organization. ECM components such as gelatin and alginate, which closely mimic native ECM properties, have demonstrated excellent biocompatibility and structural versatility for building 3D bioscaffolds. , A recent study effectively leveraged these materials in combination with 3D printing technology to construct an in vitro model that recapitulates the pathological features of RA-associated pannus formation, underscoring the immense potential of 3D biomanufacturing in biomedical applications.

The formation of synovial organoids depends on specific signaling factors that initiate and guide the self-assembly of cellular developmental pathways at defined temporal stages. Although multiple construction protocols have been reported, a unified standard for optimal practices has yet to be established. One study employing a 3D in vitro organ culture system of FLS identified cadherin-11 as a critical mediator of FLS adhesion. Cadherin-11 was shown to interact with the actin cytoskeleton, promoting structural remodeling and playing a decisive role in the organization and stabilization of synovial-like structures in vitro. In efforts to optimize scaffold design for synovial organoid construction, researchers have investigated how architectural features of biomaterials influence the cellular mechanical microenvironment. Hydrogels, as cellular carriers, were found to more evenly distribute and transmit mechanical forces compared to porous scaffolds. − Moreover, coculturing FLS with human umbilical vein endothelial cells (HUVECs) and monocyte-derived macrophages within a 3D collagen-based scaffoldsupplemented with vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (bFGF)significantly promoted spheroid growth and increased the density of both FLS and ECs in the growth region, in contrast to unstimulated controls. To further enhance the structural and functional fidelity of engineered synovial tissue, researchers have adopted advanced volumetric extrusion bioprinting techniques. By utilizing gelatin methacryloyl (GelMA) as a bioink, they successfully fabricated bioartificial synovial tissue constructs that recapitulate key features of native tissue and are well-suited for in vitro experimentation (Figure C).

In conclusion, the construction of synovial organoids is a multidisciplinary and complex endeavor involving the selection of appropriate starting cell types, precise control of the culture environment, regulation of key signaling pathways, and incorporation of cutting-edge technologies. These advances provide powerful platforms for investigating synovial inflammation and offer a robust foundation for developing targeted therapeutic strategies for diseases such as RA.

4. Involvement of Synovial Organoids and Cell Culture Models in TCM Treatment for RA

4.1. Single-Cell Cultivation in TCM Therapy for RA

The introduction of single-cell cultivation technology has significantly advanced our ability to mimic the microenvironment of synovial cells in RA, allowing for precise investigation of the mechanisms by which individual compounds and complex formulations from TCM influence cell proliferation, differentiation, and function. This approach not only deepens our understanding of RA pathogenesis but also provides valuable experimental evidence for identifying molecular targets of TCM therapies.

Through the application of single-cell cultivation systems, the active ingredients and pharmacological mechanisms of various TCM agents in RA treatment have been progressively elucidated. For instance, Forsythiaside A has shown potent antimigratory and anti-inflammatory effects in an in vitro RA model using IL-1β-stimulated MH7A cells. Its activity is mediated through modulation of the JAK/STAT signaling pathway, identifying novel molecular targets for cell-level RA therapy. Similarly, Qufeng Zhitong Capsule (QFZTC) directly targets RA cells, significantly reducing cell viability and alleviating inflammatory responses, thereby underscoring the unique regulatory capacity of TCM in modulating RA cellular behavior. Hedyotis diffusa Willd (HDW) has been shown to mitigate RA progression by targeting the MMP9/miR-204–5p/MIAT axis, providing further evidence of the multipathway, multitarget potential of TCM in disease intervention. Siegesbeckiae Herba (SBH) offers a novel strategy for inflammatory control by promoting apoptosis in RA-FLS and reducing immune-mediated inflammation. Xinfeng Capsules (XFC), a well-characterized TCM formulation, suppress inflammation and oxidative stress by upregulating LINC00638 and activating the Nrf2/HO-1 pathway. XFC also modulates the expression of lncRNA MAPKAPK5-AS1 to induce apoptosis in RA-FLS, expanding the application spectrum of TCM in RA therapy. , Triptolide further demonstrates efficacy by significantly inhibiting RA-FLS proliferation and inflammation via the hsa-circ-0003353/microRNA-31–5p/CDK1 axis and downregulating ENST00000619282, introducing novel molecular avenues for RA intervention. , Complex formulations such as Huangqin Qingre Chubi Capsule (HQC) have been shown to delay RA progression through regulation of the CUL4B/Wnt pathway, illustrating the systemic therapeutic effects of TCM. Astragaloside, another TCM-derived compound, inhibits RA-FLS proliferation in rat models by modulating the lncRNA LOC100912373/miR-17–5p/PDK1 axis, providing strong support for its application in precision medicine approaches. Notably, combination therapies such as mangiferin and cinnamic acid, key components of Baihu-Guizhi Decoction (BHGZD), have been found to inhibit TLR4/NF-κB/NLRP3-mediated pyroptosis. Berberine also exhibits significant antiproliferative and antiadhesive effects on RA-FLS through modulation of the RAS/MAPK/FOXO/HIF-1 signaling pathways, providing yet another promising strategy for RA management. For a detailed summary of research findings regarding the role of single-cell cultivation technology in TCM-based RA treatment, refer to Table .

1. Synovial Organoid Involvement in TCM Treatment of RA.

evolution	reference	cell (model)	pathophysiological mechanism	index	axis	effects	TCM
monocell culture	[]	MH7A	inflammation	TNF-α, IL-6, IL-8	JAK/STAT	Forsythiaside A exhibits antimigratory and anti-inflammatory effects in an RA in vitro model (IL-1β-stimulated MH7A cells) by modulating the JAK/STAT pathway	forsythiaside A
	[]	RA-FLS	inflammation	IL-6, IL-1β, TNF-α, VEGF	MAPK	QFZTC inhibits cell viability and inflammatory response in RA	Qufeng Zhitong capsule (QFZTC)
	[]	RA-FLS	identification of specific mRNA- miRNA- lncRNA networks	MMP9, miR-204–5p, MIAT	MMP9/miR-204–5p/MIAT	HDW may affect the progression of RA by regulating the MMP9/miR-204–5p/MIAT axis	Hedyotis diffusa Willd (HDW)
	[]	RA-FLS	immune inflammation	Bax, caspase3, caspase8, caspase9, COX2, TNF, and IL-17	-	Siegesbeckiae Herba can promote apoptosis of RA-FLS and alleviate the immune inflammatory response of RA	Siegesbeckiae Herba (SBH)
	[]	RA-FLS	inflammation, oxidative stress	IL-6, IL-17, ROS, SOD2, RNS	LINC00638/Nrf2/HO-1	XFC inhibits inflammation and oxidative stress in RA by up-regulating LINC00638 and activating the Nrf2/HO-1 pathway	Xinfeng capsules (XFC)
	[]	HUVEC, RA-FLS	synovial hyperplasia, angiogenesis	VEGF, CD31	VEGFR/PI3K/AKT	EAF effectively inhibits synovial proliferation and angiogenesis in arthritic joints, a mechanism attributed to the modulation of the VEGFR/PI3K/AKT signaling pathway	ethanol extract of Anemone flaccida Fr. Schmidt (EAF)
	[]	RA-FLS	bone destruction	FGFR1, p-FGFR1, ERK1/2, p-ERK1/2, RANKL, MMP2, MMP3	FGFR1	JBQGF may play a therapeutic role in preventing and treating RA joint bone destruction by inhibiting the phosphorylation of FGFR1	Juanbi Qianggu Formula (JBQGF)
	[]	RA-FLS	migration, oxidative stress	ROS, MDA, SOD, MMP2, MMP9	Nrf2-Keap1	PCA reduces H2O2-induced migration and oxidative stress of RA-FLS through activation of the Nrf2-Keap1 signaling pathway	protocatechuic acid (PCA)
	[]	RA-FLS	proliferation, adhesion	MMP-1, MMP-3, RANKL, TNF-α	RAS/MAPK/FOXO/HIF-1	Berberine suppresses RA-FLS cell proliferation and adhesion via the RAS/MAPK/FOXO/HIF-1 signaling pathway, offering a potential treatment for rheumatoid arthritis	berberine
	[]	RA-FLS	inflammation	TNF-α, IL-1β, IL-6, IL-10	Wnt/β-catenin	DBD inhibits RA progression and may control the disease by suppressing inflammatory cytokine responses through Wnt signaling, targeting SFRP4, to delay RA development	Danggui Buxue Decoction (DBD)
	[]	RA-FLS	cell growth, inflammatory response	IL-1β, IL-6, IL-4, IL-10	hsa-circ-0003353/microRNA-31–5p/CDK1 axis	Triptolide inhibits fibroblast-like synoviocyte growth and inflammation by modulating the hsa-circ-0003353/microRNA-31–5p/CDK1 axis	triptolide
	[]	MH7A	inflammation, apoptosis	MMP-1, MMP-3, IL-1β, IL-2, IL-6, IL-17, IFN-γ, MCP-1, IL-10	PI3K/AKT, JAK/STAT	IBC promotes cell apoptosis through the PI3K/AKT and JAK/STAT signaling pathways, ultimately inhibiting the progression of synovitis	isobavachalcone (IBC)
	[]	RA synovial cells	inflammation and chemotaxis	TNF-α, IL-1β, CCL5, CXCL10, CXCR2, IL2	Toll, TNF	Dioscin significantly inhibits the proliferation and induces apoptosis of synovial cells in RA rats by reducing the secretion of TNF-α and IL-1β, suppressing the abnormal expression of CCL5, CXCL10, CXCR2, and IL-2	dioscin
	[]	MH7A	inflammation	Bax, Bcl-2, IL-6, IL-8, COX-2, iNOS, MMP-1, MMP-2, MMP-3, MMP-9	NF-κB, MAPK	Corilagin exerts antiproliferative and anti-inflammatory effects in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways	corilagin
	[]	MH7A, RAW264.7	inflammation, pyroptosis	TLR4, NLRP3, ASC, caspase-1, GSDMD-NT, IL-1β, IL-18	TLR4/NFκB/NLRP3	The novel drug combination of mangiferin and cinnamic acid in BHGZD alleviates rheumatoid arthritis by inhibiting TLR4/NF-κB/NLRP3 activation-induced pyroptosis	Baihu-Guizhi decoction (BHGZD)
	[]	HFLS-RA	inflammation and apoptosis	TNF-α, IL-6, MMP-1, MMP-3, COX-2, PGE2	NF-κB, MAPKs	FLA inhibits inflammatory mediator expression and synthesis in HFLS-RA by suppressing NF-κB and MAPK signaling pathways while inducing apoptosis via the mitochondrial pathway	Fuzi lipid-soluble alkaloids (FLA)
	[]	MH7A	inflammation	IL-1β, IL-6, IL-8, IL-17A		DA extracts exert anti-inflammatory and anti-arthritic effects on human rheumatoid arthritis fibroblast-like synoviocytes and distinct mouse arthritis	Deer velvet antler (DA)
	[]	RA-SF	monocyte adhesion	VCAM-1, CD11b	MEK1/2-ERK, p38, AP-1	Antcin K inhibits VCAM-1-dependent monocyte adhesion in human rheumatoid arthritis synovial fibroblasts	antcin K
	[]	RA-FLS	apoptosis, inflammation	IL-4, IL-17, Bax, Bcl-2	lncRNA MAPKAPK5-AS1	XFC promotes RA-FLS apoptosis and attenuates inflammation by regulating lncRNA MAPKAPK5-AS1	Xinfeng Capsules (XFC)
	[]	RA-FLS	apoptosis, inflammation	TNF-α, IL-1β, IL-6, IL-8, IL-4, IL-10	lncRNA ENST00000619282	Triptolide decreases ENST00000619282 to stimulate the apoptosis and relieves the inflammation of RA-FLS	triptolide
	[]	RA-FLS	inflammation	IL-1, IL-6, IL-8, MMP3, Fibronectin	circ_0015756/CUL4B/Wnt	RA pathogenesis is delayed by the traditional Chinese medicine compound Huangqin Qingre Chubi Capsule via the CUL4B/Wnt pathway	Huangqin Qingre Chubi Capsule (HQC)
	[]	RA-FLS	proliferation	PDK1, AKT	LOC100912373/miR-17–5p/PDK1	Astragaloside regulates lncRNA LOC100912373 and the miR-17–5p/PDK1 axis to inhibit the proliferation of fibroblast-like synoviocytes in rats with rheumatoid arthritis	astragaloside
	[]	HFLS-RA	inflammation, proliferation	EGFR, MMP9, IL2, MAPK14, KDR	Rap1, PI3K-Akt, Ras	ARF can partially weaken RA by regulating the expression of multiple targets in the inflammatory immune system	antirheumatic arthritis fraction (ARF) of G. yunnanensis
	[]	RA-FLS	autophagy	Atg1, Atg5, Atg14, LC3-II, PI3K, Akt, mTOR	PI3K/Akt/mTOR	JWJGC regulates autophagy by inhibiting the PI3K/Akt/mTOR pathway in RA	Jinwu Jiangu Capsule (JWJGC)
	[]	RA-FLS	pyroptosis	caspase-1/3/4/5, NLRP3, GSDMD, ASC, IL-1β, IL-18, LDH	NLRP3/CAPSES/GSDMD	JWJGC inhibits pyroptosis through the NLRP3/CAPSES/GDMD pathway and treats fibroblast like synovial cells in rheumatoid arthritis	Jinwu Jiangu Capsule (JWJGC)
	[]	FLS	inflammation	IL-1β, IL-6, IL-10, IL-4	JAK2/STAT3	NBTL ameliorates rheumatoid arthritis in rats through inhibiting the JAK2/STAT3 signaling pathway	New Bi Tong Ling (NBTL)
	[]	RA-FLS	inflammation	NLRP3, caspase-1 p20, IL-1β, TNF-α	NLRP3	The myrtenal and β-carboxymethyllene oxides selected from PEL can inhibit the activation of the NLRP3 inflammasome, thereby alleviating RA symptoms	Liquidabaris Fructus (LF) petroleum ether extract (PEL)
	[]	MH7A, RAW264.7 cells	immune inflammation, pyroptosis	caspase-1, IL-1β, LDH, NLRP3, ASC, TLR4, GSDMD	Toll-like signaling	BHGZD alleviates rheumatoid arthritis by inhibiting NLRP3 inflammasome activation mediated by Toll-like receptor 4	Baihu-Guizhi decoction (BHGZD)
	[]	RA-FLS	energy metabolism, apoptosis, autophagy	ROS, ATP, PKM2, GLUT1, HK2, Pklr, Aldob, Pgam2, Pfkm, Acss2, caspase 3, Bax, LC3-II/LC3-I	PI3K/AKT/mTOR	Shikonin induces programmed death of fibroblast synovial cells in rheumatoid arthritis by inhibiting energy pathways	shikonin
	[]	RA-FLS	inflammation, bone destruction	TNF-α, NO		Inhibition of rheumatoid arthritis using bark, leaf, and male flower extracts of Eucommia ulmoides	Eucommia ulmoides Oliv.
	[]	RA-SF	immune inflammation	IL-17A, TNF-α, IFN-γ, IL-6, TLR2, TLR4		The inhibitory effect of asarinin drug serum on RASFs may be achieved by inhibition of T helper cell (Th)1/Th17 cytokines through suppression of TLR2 and TLR4	asarinin
	[]	RA-FLS	inflammation, proliferation	IL-6, IL-1β, FOX3	miR-155/FOX3	Paeonol prevents TNF-α-induced FLS proliferation and cytokine release by reducing the expression of miR-155 and upregulating its target FOXO3	Paeonol (Pae)
	[]	MH7A	inflammation, proliferation	IL-1β, IL-6	MAPKs	XAN selectively regulates MAPK signaling and has subsequent antiproliferative and anti-inflammatory activities on MH7A cells	1,7-dihydroxy-3,4-dimethoxyxanthone (XAN)
	[]	MH7A	apoptosis, lipid accumulation, oxidative stress	ROS, CHOP		HO treatment induces lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells. CHOP plays a downstream antirheumatoid factor role in MH7A cells	hempseed oil (HO)
cell coculture	[]	Coculture of RA-PBMC and RA-FLS	inflammation	IL-17, IL-6, IL-23, TNF-α	hsa_circ_0091,685/EIF4A3/IL-17	HQC may exert anti-inflammatory effects by blocking the hsa_circ_0091685/EIF4A3/IL-17 axis in RA	Huangqin Qingre Chubi Capsule (HQC)
	[]	Coculture of rat peripheral blood lymphocytes and FLS	chemotaxis, inflammation	IL-6, TNF-α, IFN-γ, IL-4, CXCL10, CXCR3	CXCL10/CXCR3	Di-Long active ingredients exhibit good therapeutic effects on rheumatoid arthritis by inhibiting CXCL10/CXCR3 chemotaxis	Pheretima vulgaris
	[]	Coculture of T cells and synovial cells	inflammatory response, imbalance of Th17/Treg cells	IL-17, IL-1β, TNF-α, IL-10, Foxp3, RORγt	-	DTYMT significantly reduced the proliferation ability of RA fibroblast-like synovial cells under normal conditions, as well as the proliferation ability of T cells cocultured with RA fibroblast-like synovial cells after IL-6 activation	Duanteng-Yimu Tang (DTYMT)
3D organoids	[]	Synovial organoids	ubiquitin	p53, p21, PUMA, CCNB1, CCNA2, CDK1, CDK2, BIRC5, vimentin	p53	Sesamol serves as a p53 stabilizer to relieve rheumatoid arthritis progression and inhibits the growth of synovial organoids	sesamol

In summary, single-cell cultivation has become a pivotal tool in the exploration of TCM therapies for RA. It has not only facilitated the dissection of active compounds and their molecular mechanisms but also laid a solid foundation for the development of more precise and effective therapeutic strategies. However, despite its widespread application, owing to operational simplicity and cost-effectiveness, the single-cell model has intrinsic limitations. Specifically, it primarily reflects localized pathological processes, making it insufficient to fully capture the systemic physiological and pathological complexity of the RA.

4.2. Coculture Models in TCM Treatment of RA

Building upon single-cell models, the development of coculture systems enables the simulation of complex interactions among synovial cells, immune cells, fibroblasts, and other cell types, offering new opportunities to dissect the intricate immune networks involved in RA and the therapeutic mechanisms of TCM. The integration of both individual active ingredients and compound TCM formulations within coculture platforms has demonstrated broader therapeutic efficacy, thereby reinforcing the holistic treatment strategies characteristic of TCM for RA.

In exploring how coculture technology facilitates TCM research in RA therapy, a number of TCM formulations have revealed distinct mechanisms of action. For instance, Huangqin Qingre Chubi Capsule has demonstrated notable anti-inflammatory effects in coculture systems, such as models incorporating PBMCs from RA patients and RA-FLS. Specifically, this formulation acts by blocking the hsa_circ_0091685/EIF4A3/IL-17 regulatory axis, thereby modulating the inflammatory microenvironment of RA. These findings reveal a complex regulatory network and offer a novel mechanistic basis for the application of TCM in coculture-based models. Additionally, active compounds extracted from Di-Long (earthworm) have shown significant efficacy in suppressing inflammation by inhibiting CXCL10/CXCR3-mediated chemotaxis. This effect, validated in a coculture model of rat peripheral blood lymphocytes and RA-FLS, effectively disrupts the migration and accumulation of inflammatory cells, highlighting the crucial role of TCM in modulating intercellular communication and immune cell behavior in RA. Notably, the traditional formula of Duanteng-Yimu Tang significantly reduced the proliferation of RA-FLS under normal conditions in coculture models. Furthermore, it inhibited the proliferation of RA-FLS in the presence of T cells following IL-6 activation. These results demonstrate not only a direct suppressive effect on synovial fibroblasts but also the ability to modulate immune cell-synoviocyte interactions, offering promising insights into the immunomodulatory potential of TCM in RA treatment. For a detailed overview, refer to Table .

In conclusion, coculture models serve as a powerful experimental platform for elucidating the mechanisms by which TCM exerts its therapeutic effects in RA. By replicating the complex intercellular interactions within the synovial microenvironment, these models provide deeper insight into how TCM modulates pathological processes, laying a robust scientific foundation for the development of more precise and effective treatment strategies. However, despite their value in reflecting multicellular dynamics, coculture systems still face limitations, particularly in their inability to fully recapitulate the highly complex and dynamic in vivo environment. These challenges must be addressed to further advance their application in TCM-based RA research.

4.3. 3D Organoids in TCM Treatment of RA

The emergence of 3D organoid technology has significantly transformed in vitro modeling approaches for RA. By constructing 3D synovial organoids, researchers can more faithfully replicate the histopathological changes and drug response patterns observed in RA. This approach not only enhances translational research but also lays a foundation for the development of personalized treatment strategies. A recent study led by Lin and colleagues successfully established a synovial organoid model derived from RA-FLS to investigate the therapeutic potential of sesamol in RA. Their results revealed that sesamol exerted a dose-dependent inhibitory effect on synovial organoid growth. Intriguingly, organoids derived from different individuals exhibited varied sensitivities to sesamol, underscoring the feasibility of using organoid systems to guide personalized treatment decisions. To further elucidate the molecular mechanisms underlying sesamol’s action, the authors employed RNA sequencing (RNA-seq), which demonstrated that sesamol significantly upregulated the p53 signaling pathway and apoptosis-related genes but downregulated the key regulators of cell cycle progression and DNA replication. This transcriptional profile strongly supports sesamol's capacity to reverse pathological phenotypes in RA-FLS. Furthermore, the study revealed that sesamol enhances p53 pathway activity by inhibiting its ubiquitination and subsequent degradation, thereby functioning as a novel p53 stabilizer. The novelty of this research lies in its multidimensional validation of sesamol's therapeutic efficacy, utilizing not only traditional in vivo models such as collagen-induced arthritis mice and predictive modeling but also the RA-FLS-derived synovial organoid platform. This comprehensive approach offers valuable new insights and strategies for advancing TCM-based therapies for RA. Despite its advantages, the widespread application of 3D organoid technology remains constrained by several factors, including complex operational protocols, technical challenges, and relatively high costs. These limitations continue to pose barriers to their large-scale adoption and deeper exploration in basic and translational research (Table ).

In conclusion, the progression from single-cell cultures to coculture systems and ultimately to 3D organoids reflects the continuous advancement of biotechnological approaches in RA research. This evolution has opened new frontiers for exploring the therapeutic potential of TCM, particularly through the lens of synovial organoid models. With ongoing technological innovation and expanded research efforts, TCM strategies, especially those grounded in organoid-based platforms, are poised to play an increasingly critical role in the prevention, management, and treatment of RA.

5. Conclusions and Outlook

Synovial organoids and cell culture models represent promising platforms for studying the therapeutic potential of TCM in the treatment of RA. However, their development and application still face numerous notable challenges. First, while single-cell culture models offer operational simplicity, they fail to capture the full complexity and systemic nature of RA pathophysiology. Coculture models, though more representative of in vivo conditions, remain limited in their ability to fully simulate the dynamic and multifactorial environment of the human joint. Second, the current technology for constructing synovial organoids is still in its infancy. The efficient and stable generation of physiologically functional synovial organoids remains a significant technical hurdle. Additionally, the integration of TCM with synovial organoid research presents additional limitations that warrant careful attention: (1) Variability in Herbal Formulations: The multicomponent and multitarget nature of TCM prescriptions leads to considerable batch-to-batch variability in active compounds, making it difficult to achieve reproducible dose–response relationships. (2) Standardization Challenges: The lack of universally accepted quality control protocols for TCM extracts hinders the cross-laboratory validation of experimental findings across different laboratories and organoid platforms. (3) Technical Reproducibility: Differences in organoid generation methods (e.g., cell sources, scaffold materials, and cytokine cocktails) may result in inconsistent responses to TCM interventions. Overcoming these challenges will require coordinated interdisciplinary efforts. Key steps include the establishment of standardized TCM identification and optimization of organoid culture protocols among other solutions. Looking ahead, future research should focus on breaking through current technical barriers in organoid fabrication, developing more advanced and physiologically accurate in vitro models that better reflect the pathogenesis of RA, and further integrating TCM theories with modern biotechnological tools. Such advancements will significantly accelerate the translational application of organoid-based platforms in RA research, particularly in disease modeling, drug screening and evaluation, and design of personalized treatment regimens.

Glossary

Abbreviations

ASCs

Adult stem cells

BHGZD

Baihu Guizhi Decoction

DBD

Danggui Buxue Decoction

ECM

Extracellular matrix

FLS

Fibroblast-like synovial cells

HQC

Huangqin Qingre Chubi Capsule

iPSC

Induced pluripotent stem cell

NO

Nitric oxide

PBMCs

Peripheral blood mononuclear cells

PGE2

Prostaglandin E2

RA

Rheumatoid arthritis

SF

Synovial fluid

SM

Synovium

TCM

Traditional Chinese medicine

2D

two-dimensional

3D

three-dimensional

XFC

Xinfeng Capsules

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

All authors read and approved the final manuscript. F.W. and J.L. conceptualized and designed the study. F.W. and J.W. prepared the original draft. Y.S. and M.H. analyzed and interpreted the results. J.L. reviewed and edited the final draft.

This study was supported by the following projects: National Nature Fund Program (82274490, 82205090); National Traditional Chinese Medicine Inheritance and Innovation Project Fund (Development and Reform Commission[2022] No. 366); National High Level Key Discipline of Traditional Chinese Medicine, Traditional Chinese Medicine Arthralgia (number Chinese Medicine Ren Jiao Han [2023] No. 85).

No potential conflict of interest was reported by the author(s).

The authors declare no competing financial interest.