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.

Keywords: 3D-bioassembly; cartilage tissues; cocultured spheroids; high throughput; microtissues; spheroid fusion; tissue fusion.

Figure 1

A) Illustration of a stem cell dividing and undergoing condensation prior to tissue specific differentiation and aggregation into tissue structures, and subsequent fusion into larger, matured configurations. B) The tissue fusion step is key to tackling the biological complexity of engineering larger structures, and subsequent tissue anisotropy and organogenesis. It requires reliable 3D‐models proficient to systematically screen these fusion events with precise control over microenvironmental factors and in high‐throughput. Schematic of the developed 3D‐model used for probing multicellular tissue fusion, a steppingstone towards organ‐like growth as well as cartilage integration. An established, high‐throughput centrifugation method is used to fabricate hundreds of cartilage tissue spheroids.^{[ 25 ]} After one week of preculture, two mature tissue spheroids are placed adjacently within a custom‐made, 3D‐printed thermoplastic cage. The two tissue units can be made from single‐ or multicellular sources and combined in any desired patterns, enabling the study of monocultures as well as both indirect and direct coculture mechanisms. The flexibility of this modular 3D‐bioassembly approach enables the fusion of mature hAC and hMSC spheroids into larger tissues following 3 weeks of culture, coordinated through interactions between distinct cells sources and neighboring tissue interfaces.

Figure 2

Illustration of 3D‐printed fusion cages holding two spheroids in place.

Figure 3

A) The size of single hAC, hMSC, and mixed spheroids were analyzed after 1 week of preculture in chondrogenic differentiation media, prior to bioassembly within the fusion boxes. B,C) Illustration of two spheroids placed adjacently within a fusion box (B, top) with zoomed in images of an experimental example of the mixed spheroid tissue–tissue interfaces as a function of time (B,C), i.e., area of change. D) Quantification of the area of change as a function time and culture conditions.

Figure 4

Histological sections through the center of samples fused following 4 weeks of culture in chondrogenic differentiation media. One cell type at a time has been labeled with a fluorescent tracker and is represented by two distinct colors (hAC: red; hMAC: green). The florescent images have been overlaid onto brightfield images. HACs (red) were not detected in the fusion zones of either of the A) hAC/hAC, B) hMSC/hAC or C) mixed conditions. D–F) hMSCs (green) instead displayed a high migratory capacity and were found present in most regions of the newly secreted matrix.

Figure 5

Macroscale biochemical quantification of A) DNA, B) GAG, and C) GAG/DNA content of each of the four fusion conditions as a function of time (1, 3, and 4 weeks of culture). D) Visualization of safranin‐O/hematoxylin/fast green, DAPI (blue)/collagen type II (green)/collagen type I (red), DAPI (blue)/collagen type X (red), and DAPI (blue)/connexin 43 (red) stained cryosections of spheroids after 4 weeks of accumulative culture. E–H) Quantification of florescent intensity in the overall construct and fusion region, respectively. Error bars represent the mean ± SD of six samples. * indicates significant difference (p < 0.05).

Figure 6

Gene expression of common markers of fibroblastic and hyaline cartilage formation in fused spheroids after 4 weeks of accumulative culture. A housekeeping gene was used to normalize values (GADHP). The quantified gene expression is presented as relative values to hAC spheroids. * indicates significant difference (p < 0.05).

Figure 7

A) The developed 3D‐bioassembly model was applied with a wide range of tissue units, introducing both GelAGE (ii, iii), GelNOR (iv), and HepSH (ii, iii) as biomaterials to help gain control over the delivery of bioactive factors as well as mechanical properties during the study of tissue fusion. The proof‐of‐concept studies demonstrated that the developed 3D‐model is compatible with both biomaterial free spheroids (i) and biomaterial based spheroids (ii, iiii), which can be applied with a variety of cells: hACS (i–iii), hMSCs (iv), and hUVECs (iv). The developed 3D‐model herein sets the stage for the next tissue engineering era, advancing from tissues to organoid cultures, by providing a robust and flexible platform to study the link between cellular differentiation, tissue fusion, organ growth as well as host tissue integration. The developed 3D‐bioassembly model was further printed into a larger model, offering the ability to achieve greater throughput as well as fusion in both lateral and horizontal directions—simultaneously. B) As a proof‐of‐concept, the models were designed to hold up to 12 tissue modules. Several combinations of 7 days precultured tissue modules made from human nasal chondrocytes (HNC, red fluorescent tag) and human MSCs (green fluorescent tag) were bioassembled into biphasic, graduated, and assorted structures and cultured for 14 days (B). The constructs were imaged using fluorescent microscopy, highlighting the modular capability of the platform, scale bars = 100 µm (B). C) Two fluorescently tagged hMSC tissue modules and two nontagged hNC spheroids were furthermore precultured for 7 days followed by 14 days of bioassembly culture, scale bars = 100 µm. The constructs were imaged using brightfield and fluorescent microscopy, demonstrating tissue fusion and migration in both X and Y and directions (C).