Mesenchymal stem cell-derived spheroids and microtissues for biofabrication: advances in bioassembly and bioprinting strategies

Biofabrication integrates biological and engineering principles to generate functional tissues for regenerative medicine, drug screening, and disease modeling. Among the diverse cellular sources available, mesenchymal stem cells (MSCs) have emerged as a critical component due to their multipotency, immunomodulatory capacity, and scalability. This review focuses on MSC-derived spheroids and microtissues as building blocks for biofabrication, organizing recent advances in bioassembly and bioprinting strategies around the axis of undifferentiated versus pre-differentiated microtissues and how the timing of differentiation shapes construct functionality. Key technological innovations, including dynamic culture systems, scaffold-free assembly, and high-resolution bioprinting (e.g. droplet-based), have enabled enhanced control over microtissue maturation and structural fidelity. The role of the cellular microenvironment in determining MSC fate is examined, emphasizing how biochemical cues, mechanical properties, and oxygen gradients can be engineered during microtissue formation. Cellular heterogeneity is addressed as a path-dependent variable continuously reshaped by donor-specific factors (age, sex, health status) and process-imposed cues (isolation methods, expansion conditions, and mechanical stress) throughout thein vitrolife cycle, and concrete mitigation strategies are discussed, including prospective cell sorting and a proposed functional potency panel (extracellular matrix deposition, fusion competence, and secretome profiling), alongside recent advancements in automated and machine learning-based biofabrication platforms. Vascularization and immunological integration are examined as central translation bottlenecks, covering endothelial co-culture, prevascularized spheroids, sacrificial bioprinting, and MSC-driven modulation of the host immune response. By integrating the fabrication route, heterogeneity as a manufacturing process variable, and translational/regulatory bottlenecks into a single framework, this review highlights recent advances, discusses current limitations, and outlines future directions for clinical translation of MSC-based biofabrication.