3D spheroid or organoid culture is becoming mainstream method for studying physiologically relevant cell behaviors, and used in applications such as cell-based diagnostic, therapy, disease modeling and drug screening. Organoids/spheroids function best when maintaining size of <200μm diameter and in perfusion culture. To achieve this, we describe in this chapter various methods of constraining spheroid size during the formation and culture processes such that the spheroids can maintain high-level cell functions for characterization and applications. We describe methods based on substrate tethering, physical-constraints such as covering spheroids under porous membranes, inside macroporous sponges, or in microfluidic channels. The chemical and physical properties of the cell-contacting surfaces and devices are carefully engineered to enable the formation and maintenance of spheroid functions over time. Pitfalls of these methods and proposed solutions in the contexts of respective applications will also be discussed.
Keywords: Microfluidic; Organoid; Perfusion culture; Primary cell; Spheroid; Substrate stiffness; Surface modification.
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