This review discusses the characteristics of three-dimensional cell culture systems on and without carriers (scaffolds). Scaffolds are used to simulate the extracellular matrix, as well as to reproduce the natural physical and structural microenvironment of cells, similar to living tissue. The review examines the types of scaffolds (hard and gel-like, natural and artificial, degradable and non-degradable), their characteristics, advantages and disadvantages, features of cell distribution in them. The use of decellularized and devitalized organs and tissues as scaffolds is discussed. The review also considers matrix-free cultivation of cells in the composition of three-dimensional multicellular structures -spheroids. The structure and biology of spheroids is discussed. The features of spheroid formation under static (self-assembly) and dynamic (under the influence of external forces) cultivation conditions are considered. The role of spheroid size for cell survival is discussed.