Spatial mechano-engineering using Engineered Living Microtissues

As 3D cell cultures more accurately resemble the complex microenvironment of native tissues than 2D cell cultures, many studies focus on advancing 3D cell culture techniques. However, scaffold-based techniques primarily prioritise cell-material interactions, often at the expense of cell-cell interactions. In contrast, scaffold-free systems, such as spheroid models, lack control over cellular behaviour through cell-material interactions. Cell-sized dextran microgels with tunable stiffness can be incorporated into spheroids to introduce controlled cell-material interactions. However, as native tissues frequently exhibit regions with varying stiffness, tissue engineering approaches are required to mimic this heterogeneity in vitro. This study aimed to develop a method to fabricate tissue models with region-specific mechanical properties by applying spheroid fusion of microgel-containing spheroids with differing stiffness. The proposed method consisted of two sequential seeding steps. Initially, spheroids containing stiff microgels were seeded into a non-adhesive, custom-designed well, resulting in a fused tissue with the desired geometric features. Subsequent seeding of spheroids containing soft microgels led to the formation of a fused tissue construct with distinct soft and stiff regions. The developed method was applied using pre-differentiated osteogenic (stiff) and chondrogenic (soft) spheroids to fabricate an in vitro model of the osteochondral interface with well-defined mechanical and geometrical properties.