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.