Cultivating cells “in a dish,” or in vitro, can be realized in two different ways: on one hand, on a surface where the cells can grow, proliferate, and, if necessary, be detached again, and on the other hand, as a suspension culture, floating in a nutrient medium, without direct attachment to a surface. The latter are referred to as 3D cell cultures when cell associations are present in several layers or as cell clusters, which allow direct interaction between the same or different cell types. Compared to two-dimensional cell cultures, which usually only have one cell type, these 3D cultures can represent individual or several aspects of the physiological conditions in living beings (in vivo) more accurately. Nevertheless, even in two-dimensional cultures, which by definition exist as single cell layers (monolayers), the presence of different cell types is possible. However, theoretically, these cells do not interact functionally with each other in multiple layers beyond normal cell contacts and do not form different compartments. For example, nerve cells and glial cells or endothelial cells and satellite cells can be cultivated as functional associations in a monolayer. However, for these cultures, as soon as a formation of three-dimensional structures of the cells with each other occurs, the term “3D” should rather be used. On the other hand, it is possible to cultivate a single cell type in three-dimensional cultures. This is often used, for example, to maintain precursor or stem cells in the undifferentiated stage, or to ensure subsequent differentiation. Examples are stem cells that are propagated as so-called spheres or spheroids, or the increasingly used tumor spheroids (see Sect. 22.2.5). An example of a 3D culture for initiating differentiation are the so-called Fig. 22.1, aggregates of pluripotent stem cells that preferably differentiate into cells of the three germ layers. Such rather simple spheres are contrasted by the so-called organoids, complex self-organizing 3D structures, to which we will dedicate a large part of this chapter.