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Three-dimensional (3D) cell culture is a technique to artificially create an environment close to the living conditions in vivo, which can permit cells to grow or interact with their surroundings in all three dimensions. Actually, most cells in vivo exist in complex 3D structures, which include different cell types within extracellular matrix. Compared to 2D cell cultures, 3D cell culture can provide more meaningful scientific conclusions and accurate insight into pathogenesis. Therefore, 3D cell cultures have been widely used in the study of disease mechanisms, drug discovery, drug testing, and tissue engineering.

At present, two categories techniques, scaffold-based methods and scaffold-free approaches have been developed for 3D cell culture. Scaffold 3D cell culture allows cells to grow in all directions on a scaffold, such as hydrogels or inert matrices. Scaffolds, which mimic the in vivo extracellular matrix (ECM), can provide attachment sites and microenvironment for cells. 3D cell culture can also be grown without the scaffold. Various methods, such as low adhesion plates, hanging drop plates, micropatterned surfaces, magnetic 3D bioprinting, and rotating bioreactors, can be used for scaffold-free 3D cell culture.