Introduction

Tendons are a classic example of cell-sparse tissue, characterized by a scarcity of endogenous stem cells. This inherent biological constraint poses a significant challenge to achieving robust structural regeneration and complete functional recovery following tendon injury.
In regenerative medicine, patient-specific stem cell-derived organoids have demonstrated strong therapeutic potential for transplantation and organ repair, including the intestine and liver. Such customized organoid systems offer distinct advantages: they bypass donor supply limitations, mitigate immune rejection risks, and recapitulate native tissue architecture and function. As a result, the in vitro engineering of large-scale, transplantable stem cell-derived organoids is emerging as an auspicious direction for future development.
Despite these promising developments, the clinical translation of organoid technology confronts a fundamental obstacle: the "scale discrepancy". Conventional organoid models typically reach millimetre-scale dimensions, well below the centimetre-scale requirements for human tissue transplantation. This critical size limitation poses a challenge that requires overcoming to realize the full clinical potential of organoid-based therapies. Addressing this challenge through the development of functional, human tissue-scale organoids thus constitutes a central focus in advancing regenerative medicine applications.

(Image: Journal titled “Centimeter-Scale Self-Assembling Tendon Organoids Drive Tissue Regeneration)

Recent advances in regenerative medicine have yielded a critical breakthrough in addressing the fundamental challenge of scaling engineered tissues to clinically relevant dimensions. A research team led by Yin Zi and Chen Xiao from Zhejiang University conducted systematic investigations into the aforementioned "scale breakthrough" issue. The researchers published their findings in Advanced Science (Impact Factor: 14.1), under the title "Centimeter-Scale Self-Assembling Tendon Organoids Drive Tissue Regeneration."

The researchers proposed a synergistic strategy combining "optimized chemical signaling regulation with biomimetic 3D microcarriers." By recapitulating key factors in tendon development and utilizing 3D porous microsphere carriers, they successfully constructed centimeter-scale tendon organoids resembling natural tendons. This approach not only provides high-performance grafts for tendon repair but also explores new pathways for clinical translation of large-scale, transplantable organoids.

Research Background

In the pursuit of scaling up organoids, tendons have emerged as an ideal model for constructing large-scale stem cell-derived organoids due to their relatively simple cellular composition. Among the key components, tendon stem/progenitor cells (TSPCs) represent a core breakthrough—unique stem cell population within tendon tissue. TSPCs exhibit a distinctive combination of pluripotency, self-renewal capability, and inherent tenogenic differentiation potential, making them ideal "seed cells" for tendon development and regeneration.
However, a prerequisite for clinical application is achieving exponential expansion of TSPCs from minimal biopsy samples. Conventional serum-containing media exhibit significant batch-to-batch variability, complex composition, and limited regulatory precision, often leading to progressive functional deterioration of expanded cells. Thus, the development of customized, chemically defined culture media is urgently needed. Beyond biochemical signalling, maintaining stable cell function relies not only on chemical signalling but also on support from the complex physical microenvironment in vivo. Chemical signals alone are insufficient to meet the developmental requirements of centimeter-scale tendon-like organoids.
To tackle this challenge, CytoNiche's 3D TableTrix™ Microcarriers offer distinct advantages. These carriers not only possess high porosity and excellent biocompatibility but also contain arginine-glycine-aspartic acid (RGD) peptide sequences that directly promote collagen assembly. Additionally, they enhance stem cell adhesion, provide appropriate mechanical support and stiffness, and trigger positive differentiation signals in TSPCs by increasing the specific surface area. Importantly, their multi-chamber structure effectively addresses nutrient penetration challenges in traditional cultures, enabling smooth diffusion of nutrients into the tissue interior. This enhanced nutrient delivery provides critical support for the formation of centimetre-scale microtissues, effectively simulating the in vivo physiological environment and natural tissue structure.

(Image: 3D TableTrix™ Microcarriers)

Research Design

This study successfully developed transplantable tendon organoids exceeding 3 cm in length—conforming to the scale of human tissue—by employing a combination of optimized chemical signaling and biomimetic 3D microcarriers to simulate the tendon Extracellular Matrix (ECM).

Researchers inoculated the isolated and purified TSPCs onto CytoNiche's 3D TableTrix™ microcarriers for culture, allowing the cells to self-assemble in vitro into organoids resembling natural tendon structures. Subsequently, TSPCs were separated from the 3D microcarriers to evaluate cell viability and proliferation capacity. Comparative analysis was conducted between 2D culture and 3D microcarrier groups regarding TSPCs' differentiation potential toward osteogenic, adipogenic, and chondrogenic lineages. Organoid morphology and function were evaluated by flow cytometry, microscopy, and various staining methods. Concurrently, scRNA-seq and RNA analysis were employed to systematically characterize the gene expression profiles and lineage-differentiation features of the tendon organoids. Finally, qualified tendon organoids were transplanted into animal tendon injury sites to assess their in vivo survival and their ability to promote tissue regeneration.

Research Content

Design and Fabrication of Tendon Organoids Under Customized Culture Conditions

Using 3D microcarriers and optimized biochemical culture conditions (Figure 1), the researchers successfully generated centimeter-scale macroscopic tendon-like tissues. Scanning electron microscopy observations indicate that the gelatin-based 3D microcarriers have an interconnected microporous structure with pore sizes of approximately 10–30 μm and an overall diameter close to 200 μm. This well-defined porous structure provides an optimal spatial environment for cell accommodation, offering substantial surface area for cellular attachment and sufficient void volume to support proliferation. The microcarrier design effectively facilitates essential cell-cell communication while enabling sufficient cell-extracellular matrix interactions during culture. Quantitative assessments confirmed strong cellular adhesiveness and maintained cell viability, demonstrating excellent biocompatibility and practical support for tendon stem/progenitor cell proliferation and tissue maturation.

Figure 1: Schematic Diagram of 3D Organoid Engineering Based on TSPCs

(A) Comparison of cell proliferation rates between the organoid group and the control TSPCs group after 1, 3, 5, and 7 days of culture.

(B) SEM images showing the self-assembly and proliferation morphology of TSPCs in the organoid group at days 3, 5, and 7.Green and yellow indicate the microcarrier scaffold and the tenocytes/tendon cells, respectively. (Scale bars: 20 µm, 50 µm)

Cell viability and proliferation within the microcarrier system were evaluated using live/dead cell staining. Results showed that cell viability and proliferation within the microcarriers were favorable (Figure 2). Under tendon-specific conditions for organoid culture, without digesting the microcarriers, CCK-8 assay showed that the proliferation activity was significantly higher than that of the traditional TSPCs group. DAPI staining further revealed substantial expansion of cell populations over time, and the TSPCs on the microcarriers gradually fused and self-assembled into micro-tendon-like structures (Figure 3).

Figure 2: Assessment of TSPC Viability and Proliferation on 3D TableTrix™ Microcarriers

Figure 3: Comparative Proliferation and Self-Assembly of TSPCs on 3D TableTrix™ Microcarriers

The tendon-forming capacity of the engineered constructs was systematically evaluated through ultrastructural and morphological analyses. Based on the transmission electron microscopy (TEM) observation, the collagen fiber assembly was more pronounced in the tendon organoid group. In contrast, fewer and slender collagen fibrils were observed in the 2D-cultured TSPCs group (Figure 4D–F). F-actin immunofluorescence staining results indicated that TSPCs within organoids exhibited more compact morphology, reduced cell size and volume, improved nuclear-to-cytoplasmic ratio, and a phenotype more reminiscent of native tendon cells (Fig. 4A–B).

After 28 days of customized culture, the research team successfully obtained tendon-like microtissues over 3 cm in length (Figure 4C). These findings collectively establish that tendon organoids can achieve high cell viability, efficient cell expansion, and self-assemble into microtissues in vitro, fully demonstrating their outstanding potential for simulating the natural tendon formation process.

Figure 4: Morphological and Structural Characterization of Tendon Organoids Demonstrating Superior Maturation

(A, B) Representative confocal images of F-actin (Phalloidin) staining and quantitative analysis of nucleus-to-cytoplasm ratio and cell area for the organoid group (n = 4) and the control group (n = 4) after 4 days of culture. (Scale bar: 60 µm)

(C) Overall view of the tendon organoid, approximately 3 cm in length, after 28 days of culture.

(D–F) TEM (Transmission Electron Microscopy) images showing longitudinal and cross-sectional views of collagen fibers in the in vitro organoid group (n = 4) and the control group (n = 4). The organoid group exhibited a significantly larger range of collagen fiber diameters.

Systematic Evaluation of Centimeter-Scale TSPC Micro-Tissues

Systematic evaluation of the centimeter-scale micro-tissues formed by TSPC aggregation revealed that the constructed tendon organoids not only possess the ability to form complete micro-tendon structures but also highly express canonical tendon markers and rejuvenation-related markers (Figure 5).

Figure 5: Schematic and Time-Course Characterization of TSPC Self-Assembly and Tenogenic Differentiation on 3D Microcarriers

(A) Schematic diagram and gross appearance (or external view) of the tendon organoid.

(B, C) Fluorescence micrographs of Lamin B1 staining in TSPCs within the organoids at days 0, 3, 7, and 14, along with quantitative analysis of mean fluorescence intensity, positive rate, and cell count per microsphere (n = 3). (Scale bar: 40 µm)

(D, E) Fluorescence micrographs of Col3 staining in TSPCs within the organoids at days 0, 3, 7, and 14, along with corresponding quantitative analysis. (Scale bar: $40\ \mu\text{m}$)

(F, G) Fluorescence micrographs of EGR1 staining in TSPCs within the organoids at days 0, 3, 7, and 14, along with quantitative analysis (n = 3). (Scale bar: 40 µm)

(H, I) Fluorescence micrographs of MKX staining in TSPCs within the organoids at days 0, 3, 7, and 14, along with quantitative results for mean fluorescence intensity, positive rate, and cell count per microsphere. (Scale bar: 40 µm)

(J) Representative immunofluorescence confocal images of EGR1, EYA2, Lamin B, and Nestin in the in vitro uninduced tendon organoids at day 14.

(K, L) Light sheet microscopy images of the engineered organoids, stained for COL1 and COL3. (Scale bar: 500 µm)

Analysis of Pluripotency and Anti-Aging Characteristics of TSPC-Derived Tendon Organoids

To determine whether TSPCs retained stem cell properties during expansion, the research team conducted a systematic evaluation through multi-lineage differentiation potential assays and analysis of TSPC surface marker expression. The results showed that TSPCs in the organoids stably retained stem cell potential after successive passages and exhibited excellent multi-lineage differentiation capacity and regenerative characteristics.

According to the transcriptomic analysis, TSPCs in organoids maintained strong regulatory programs for differentiation and self-renewal at the molecular level. This result clearly demonstrates the organoid system's ability to efficiently simulate the precise formation and regeneration of complex tendon microtissue within a controlled in vitro environment (Figure 6).

Figure 6: Transcriptomic and Molecular Validation of Enhanced Tenogenic Lineage Commitment and Self-Renewal Potential in TSPC Organoids

(A) Principal Component Analysis (PCA) plot showing the distinct separation of the organoid group (n = 3) from the control group (n = 3) at Day 3.

(B) Heatmap of sample-to-sample distances constructed using rlog-transformed values.

(C) Volcano plot of differentially expressed genes (DEGs) between organoid culture and control-cultured TSPCs (Organoid vs. TSPCs; log₂ fold change > 1.5 or < -1.5; p < 0.05; n = 3).

(D) Heatmap of DEGs related to tenogenesis, osteogenesis, and inflammation, where color intensity from blue to red represents low to high gene expression levels.

(E) mRNA expression levels of TPPP3, EGR1, FOS, NES, ACAN, SOX9, MMP3, and ADAMTS5 in the organoid group compared to the control TSPCs (n = 3).

(F) Gene Ontology (GO) analysis showing enriched biological processes in the organoid group compared to the control group.

(H) Gene Set Enrichment Analysis (GSEA) of tendon-related genes. NES = Normalized Enrichment Score; FDR = False Discovery Rate.

(G) Immunofluorescence staining of Nes and Lamin B in TSPCs isolated from the organoid group and the control group at Day 4 (Organoid-derived TSPCs vs. conventionally cultured TSPCs). (Scale bars: 30 µm, 20 µm)

High Tendon Maturity and Specific Lineage Differentiation of TSPC-derived Organoids

Systematic characterization of cellular states within tendon organoids confirms that this system not only efficiently induces directional differentiation toward tendon-specific lineages in vitro but also recapitulates the in vivo differentiation process from stem cells to mature tenocytes observed. This provides robust validation for its use as a reliable model in tendon organoid research (Figure 7).

Figure 7: Single-Cell Transcriptomic Analysis Reveals Enhanced Tenocyte Lineage Commitment and Reduced Heterogeneity in TSPC Organoids

(A) Schematic diagram of the three culture conditions for TSPCs: organoid, 2D-cultured TSPCs, and 3D MSs-cultured TSPCs.

(B) UMAP visualization of integrated data from the 2D, 3D, and organoid groups.

(C) Dot plot of marker genes for each cell cluster.

(D, E) Distribution plots of cell subpopulation proportions.

(F, G) Feature plots of marker genes.

(H, I) Cytotrace differentiation potential analysis plots.

(J) Overall tenocyte phenotype score.

(K) Dot plot of overall tendon marker gene expression.

Single-Cell Transcriptomic Analysis: Tendon Organoids as an Intermediate State

When evaluating the molecular similarity between tendon organoids and natural tendon tissues, researchers discovered that the organoids exhibit transitional molecular characteristics intermediate between in vitro-cultured TSPCs and natural tendon tissue. They possess higher maturity to simulate native tissue function while simultaneously retaining sufficient proliferative potential to support regeneration needs. This unique combination positions the developed centimeter-scale tendon organoid as a highly valuable model for advancing tendon regeneration research and facilitating successful translation of tissue engineering (Figure 8).

Figure 8: Comparative single-cell analysis reveals tendon organoids as an intermediate state between in vitro-cultured TSPCs and native tendon tissues.

(A) Schematic diagram of TSPCs cultured under three conditions: organoids, human neonatal tendon, and adult tendon.

(B) UMAP visualization of integrated tendon-related subpopulation data from 2D, 3D scaffold-only, organoid groups, human neonatal tendon, and adult tendon.

(C) Distribution of cell subpopulation proportions across samples.

(D) Dot plot of marker genes for each cluster.

(E) Cytotrace differentiation potential analysis plotted by cluster.

(F) Cytotrace differentiation potential analysis plotted by sample.

(G, H) GO enrichment analysis of Cluster 1 and Cluster 4.

(H) RidgePlot of marker genes.

(I) Feature plot of marker genes.

Tendon Organoids Enhance Self-Assembly via ECM Enrichment

To further elucidate the regulatory mechanisms underlying the maintenance of cellular function and suppression of phenotypic loss in organoids, researchers performed RNA sequencing. The results indicated that cellular activity and directional differentiation potential toward tendon lineages in tendon organoids primarily depend on ECM-enrichment signals. These signals function through downstream cytoskeletal regulatory pathways, thereby mediating the cell assembly and regulating differentiation potential (Figure 9).

Figure 9: Tendon organoids enhance self-assembly through ECM remodeling and cytoskeletal reorganization

(A) GO enrichment analysis of ECM and stem cell-related biological processes in the organoid group compared to the control group.

(B) GSEA analysis of ECM-related pathways.

(C) Heatmap of ECM-related DEGs, with colors ranging from blue to red indicating low to high gene expression levels (n = 3).

(E) mRNA expression levels of COL6A1, COL10A1, DES, EPHA4, ECM1, COL13A1, COL6A2, SDC1, THBS2, and CCN2 in the organoid group versus control TSPCs (n = 3).

(G) Cytoskeletal morphology of the organoid group, TSPCs group, and in vivo TSPCs.

(D–G) Representative confocal images showing immunofluorescence staining results for EGR1, Nes, and Lamin B under DMEM and Y27632 treatment conditions, along with quantitative analysis of positive rates.

TSPC Organoids Enhance Transplanted Stem Cell Retention and Promote Tendon Regeneration

To validate the practical efficacy of TSPC organoids in promoting tendon regeneration in vivo, researchers first transplanted them into the dorsal region of nude mice. The results suggest that the structural integrity and mechanical properties of regenerated tendons in the organoid transplantation group were significantly superior to those in the 2D-cultured TSPCs group and the 3D porous microcarriers-only group. This finding fully confirms that TSPCs within the organoid microenvironment can significantly enhance tendon regeneration capacity. Compared with traditional strategies involving direct TSPC implantation, tendon organoid transplantation is more effective at driving the tendon repair process (Figures 10, 11).

Figure 10. Analysis of the tendon regeneration capacity of TSPC-derived organoids at 2 weeks post-operation.

(A) Fluorescence imaging of tendon organoids implanted in the dorsal region of nude mice (n = 4).

(B) Schematic diagram of organoid implantation in the SD rat patellar tendon defect model.

(C) H&E staining, Masson's trichrome staining, and polarized light microscopy images of the repaired rat patellar tendon at 2 weeks post-operation. Scale bars: 100 µm (H&E and Masson), 200 µm (polarized light).

(D) Histological scores of the repaired tendons in rats (n = 5).

(E, F) TEM images of the cross-sectional (E) and longitudinal (F) views of the repaired collagen fibers; (E) average collagen fiber diameter, (F) frequency distribution of collagen fiber diameters (n = 5).

(G) DiI immunofluorescence staining and statistical analysis of the repaired tendons at 2 weeks post-operation.

Figure 11: Analysis of the tendon regeneration capacity of TSPC-derived organoids at 4 weeks post-operation.

(A) H&E staining, Masson's trichrome staining, and polarized light microscopy images of the repaired rat patellar tendon at 4 weeks post-operation. Scale bars: 100 μm (H&E and Masson), 200 μm (polarized light).

(B) Histological scores of the repaired tendons in rats (n = 5).

(C) Analysis of collagen content in the repaired tendon tissue (n = 5).

(D–F) TEM images of the cross-sectional and longitudinal sections of the repaired collagen fibers. (Scale bar: 200 nm)

(G) DiI immunofluorescence staining of the repaired and regenerated tendons at 4 weeks post-operation. (Scale bar: 50 μm)

(H) Mechanical properties (stiffness, tensile strength, and Young's modulus) of the regenerated tendons at 4 weeks post-operation (n = 8).

(I) Immunofluorescence staining for Col1 and TNMD in the repaired and regenerated tendons at 4 weeks post-operation. (Scale bar: 50 μm)

Research Conclusion

In summary, this study successfully constructed centimeter-scale tendon organoids for the first time, significantly promoting the proliferation and differentiation of TSPCs in vitro while enhancing their rejuvenation characteristics. It is particularly noteworthy that the 3D porous microcarriers, as the core support system, provide a biomimetic microenvironment for TSPCs through their high porosity, excellent biocompatibility, and surface-active peptide sequences. On one hand, they enhance cell adhesion and expansion efficiency by increasing the specific surface area; on the other hand, the multi-chamber structure ensures nutrient penetration, establishing the key physical foundation for ECM deposition and organoid self-assembly. It is precisely this synergistic model of "chemical signal regulation + 3D carrier support" that achieves a precise balance between TSPC proliferation and differentiation, efficiently driving massive cell expansion and micro-tissue self-assembly.

In vivo experiments results substantiate that these tendon organoids significantly improve post-transplantation retention and promote the regeneration of both structure and function in injured tendons. The observed efficacy stems from the organoids' capacity to replicate native tendon hierarchy while maintaining vital stem cell niches. The demonstrated success in functional restoration underscores the platform's significant potential to advance regenerative therapies for tendon disorders, offering a viable pathway toward clinical translation.

References

Fang, T., Zhang, H., Xie, Y., Li, X., Liu, X., Wang, Z., Xue, Y., Xia, X., Wang, Z., Lei, T., Lin, R., Shen, W., Wu, B., Chen, Y., Du, Y., Chen, X., & Yin, Z. (2025). Centimeter‐Scale Self‐Assembling tendon organoids drive tissue regeneration. Advanced Science, 12(43), e09453.

https://doi.org/10.1002/advs.202509453