While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D-bioassembly platform is introduced to primarily study fusion of cartilage-cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D-bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type-II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D-model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high-throughput 3D-bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis.

Lateral integration and fusion of cartilage tissue interfaces remain significantly challenging and limits success of cartilage tissue engineering (TE) strategies. In this study, self‐assembled vitreous humor (VH) spheroids are fabricated by seeding clinically relevant human articular chondrocytes (hACs) or human mesenchymal stromal cells (hMSCs) in four VH hydrogels with different glycosaminoglycans (GAG) and protein content. Quantitative and qualitative analysis of the VH spheroids revealed that hAC‐VH spheroids are dependent on the initial GAG content of VH hydrogels to achieve successful chondrogenesis. Remarkably, uniform GAG and collagen type II distribution is found in all hMSC‐VH spheroids, independent of the VH donor. HMSC‐VH spheroids are therefore further evaluated for downstream applications by tracking cellular migration, and evaluating neotissue formation at the tissue‐tissue interface of cartilage spheroids 3D‐bioassembled into an in vitro fusion model to asses fusion and integration. hMSC‐VH spheroids enhanced multidirectional cellular migration of both hACs and hMSCs toward the tissue‐tissue interface, and consequently supported dense GAG and collagen type II deposition at the integration region. Ultimately, hMSC‐seeded VH spheroids display successful chondrogenesis and endorse fusion and integration of cartilage tissue interfaces through upregulated cellular migration of clinically relevant cell sources – key elements for clinical translation of cell‐based TE strategies.

Self-assembled vitreous humor (VH) spheroids fulfill key requirements as reproducible building blocks for 3D-bioassembly of cartilage tissues. As described by Gabriella C. J. Lindberg and colleagues in article number 2200882, these VH hydrogels promote both cellular migration and neo-tissue formation in the active interface region of the assembloids, driving successful lateral fusion and integration between mature cartilage spheres while enabling fabrication of larger, clinically relevant, tissue constructs.