Probing Multicellular Tissue Fusion of Cocultured Spheroids-A 3D-Bioassembly Model.

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.

Advances in Extrusion 3D Bioprinting: A Focus on Multicomponent Hydrogel-Based Bioinks.

3D bioprinting involves the combination of 3D printing technologies with cells, growth factors and biomaterials, and has been considered as one of the most advanced tools for tissue engineering and regenerative medicine (TERM). However, despite multiple breakthroughs, it is evident that numerous challenges need to be overcome before 3D bioprinting will eventually become a clinical solution for a variety of TERM applications. To produce a 3D structure that is biologically functional, cell-laden bioinks must be optimized to meet certain key characteristics including rheological properties, physico-mechanical properties, and biofunctionality; a difficult task for a single component bioink especially for extrusion based bioprinting. As such, more recent research has been centred on multicomponent bioinks consisting of a combination of two or more biomaterials to improve printability, shape fidelity and biofunctionality. In this article, multicomponent hydrogel-based bioink systems are systemically reviewed based on the inherent nature of the bioink (natural or synthetic hydrogels), including the most current examples demonstrating properties and advances in application of multicomponent bioinks, specifically for extrusion based 3D bioprinting. This review article will assist researchers in the field in identifying the most suitable bioink based on their requirements, as well as pinpointing current unmet challenges in the field.

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

BACKGROUND AND AIMS: Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. METHODS: Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. RESULTS: LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. CONCLUSIONS: PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.