Three-dimensional (3D) cell culture has tremendous advantages to closely mimic the in vivo architecture and microenvironment of healthy tissue and organs, as well as of solid tumors. Spheroids are currently the most attractive 3D model to produce uniform reproducible cell structures as well as a potential basis for engineering large tissues and complex organs. In this review we discuss, from an engineering perspective, processes to obtain uniform 3D cell spheroids, comparing dynamic and static cultures and considering aspects such as mass transfer and shear stress. In addition, computational and mathematical modeling of complex cell spheroid systems are discussed. The non-cell-adhesive hydrogel-based method and dynamic cell culture in bioreactors are focused in detail and the myriad of developed spheroid characterization techniques is presented. The main bottlenecks and weaknesses are discussed, especially regarding the analysis of morphological parameters, cell quantification and viability, gene expression profiles, metabolic behavior and high-content analysis. Finally, a vast set of applications of spheroids as tools for in vitro study model systems is examined, including drug screening, tissue formation, pathologies development, tissue engineering and biofabrication, 3D bioprinting and microfluidics, together with their use in high-throughput platforms.

Synthetic hydrogels often lack the load‐bearing capacity and mechanical properties of native biopolymers found in tissue, such as cartilage. In natural tissues, toughness is often imparted via the combination of fibrous noncovalent self‐assembly with key covalent bond formation. This controlled combination of supramolecular and covalent interactions remains difficult to engineer, yet can provide a clear strategy for advanced biomaterials. Here, a synthetic supramolecular/covalent strategy is investigated for creating a tough hydrogel that embodies the hierarchical fibrous architecture of the extracellular matrix (ECM). A benzene‐1,3,5‐tricarboxamide (BTA) hydrogelator is developed with synthetically addressable norbornene handles that self‐assembles to form a and viscoelastic hydrogel. Inspired by collagen's covalent cross‐linking of fibrils, the mechanical properties are reinforced by covalent intra‐ and interfiber cross‐links. At over 90% water, the hydrogels withstand up to 550% tensile strain, 90% compressive strain, and dissipated energy with recoverable hysteresis. The hydrogels are shear‐thinning, can be 3D bioprinted with good shape fidelity, and can be toughened via covalent cross‐linking. These materials enable the bioprinting of human mesenchymal stromal cell (hMSC) spheroids and subsequent differentiation into chondrogenic tissue. Collectively, these findings highlight the power of covalent reinforcement of supramolecular fibers, offering a strategy for the bottom‐up design of dynamic, yet tough, hydrogels and bioinks.

The integration of biomaterials with living cells and stimuli‐responsive materials can be employed to create bioinks capable of generating 3D in vitro models that better recapitulate native tissues. A cellularized, multilayered cylindrical model is introduced that combines such hybrid multifunctional materials to mimic the tunica adventitia arterial wall, and an extracellular matrix (ECM)‐based bioink for the tunica media artery layer. The stimuli‐responsive hybrid ink integrates inorganic (plasmonic nanoparticles) and organic (polymers) components, providing structural support and introducing diverse functionalities to the system. The cell‐laden bioink consists of human vascular smooth muscle cells within a hydrogel based on porcine artery‐derived decellularized ECM that fosters optimal cell growth and proliferation. An embedding bioprinting technique is employed for the fabrication of the multimaterial model consisting of functional concentric cylinders. The dimensions of the 3D model and the bioprinting parameters are fine‐tuned to ensure effective crosslinking of the multiple concentric layers resulting in the creation of self‐supporting constructs. The effectiveness of the hybrid bioink composition and bioprinting parameters in supporting cell viability and proliferation within the multilayered construct is demonstrated, expanding the possibilities of employing novel multi‐component materials for the fabrication of 3D vasculature models resembling the structure of native blood vessels.