Probing Multicellular Tissue Fusion of Cocultured Spheroids—A 3D‐Bioassembly Model

Abstract

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.

3D‐Bioassembly of VH‐Spheroids for Cartilage Regeneration: in Vitro Evaluation of Chondrogenesis, Fusion and Lateral Integration

“…To model the lateral integration of two native cartilaginous structures, two cartilage tissues (TS-TS) were bioassembled into a PCL cage. [5] Cartilage tissues, fabricated via standard centrifugation protocols, show similarity in ECM deposition compared to native cartilage tissue. However, cartilage tissue spheroids do have a higher cellular density compared to the low percentage of residing chondrocytes in native cartilage.…”

“…[4] Commonly used modular building blocks for bottom-up TE are cell-rich spherical tissues. [1,5,6] These cell-rich spherical tissues enable extensive cell-cell and cell-extracellular matrix (ECM) interactions, which support successful tissue regeneration. Besides, it is suggested that cellular aggregation resembles in vivo processes like organogenesis and enhance key cellular performances including cellular viability, cellular differentiation and ECM deposition in 3D spheroids.…”

“…[18] To overcome this size limitation, cell-rich cartilage spheroids can be applied as modular building block in bottom-up TE strategies. [1,5,6] These 3D spheroids necessitate meeting several ambitious design criteria, they should display reproducible, healthy and functional cartilage tissue formation within the building block, and support both fusion and integration with surrounding modular building blocks and native cartilage.…”

“…Using our previously developed VH platform as a bioactive, naturally derived ECM hydrogel, [19] we systematically evaluate the impact of the batch-to-batch variation of VH on the self-assembly capacity of cell-seeded VH hydrogels and chondrogenic differentiation of two clinically relevant cell types: hACs and hMSCs. Additionally, to investigate the applicability of the VH spheroids as building blocks for bottom-up tissue engineering strategies, their capacity of fusing with other VH spheroids and integrating with cartilaginous tissues is evaluated in vitro, using customized 3D-printed poly(ε-caprolactone) (PCL) cages, [5] see Scheme 1.…”

Biomanufacturing human tissues via organ building blocks

“…The differentiation or maturation state of these OBBs can also affect cell behavior and, hence, fusion. Lindberg et al found that fusion of mesenchymal stromal cell spheroids and articular chondrocyte spheroids, as well as matrix deposition, depends on the timing of contact relative to their maturation state (Lindberg et al, 2021). Hajdu et al found that their maturation state determines whether spheroids simply adhere or undergo envelopment and that fusion kinetics are related to ECM production (Hajdu et al, 2010).…”