Figure 4. A variety of experimental co-culture methods are essential to determining metabolic interactions among different cell types.

In 2D culture (top row), conditioned media from one cell type can be applied to a different cell type (A), cells can be co-cultured using a permeable insert so that they either share soluble factors through the media (left) or directly contact each other through the membrane (right, B), or cells can be directly co-cultured together in a dish (C). These same techniques can be used for 3D cell cultures (middle row), with cell spheroid media applied to an in vitro vessel-like network (D), cell spheroids co-cultured with a cell monolayer using a permeable membrane (E), or cell spheroids directly cultured on a vessel-like network (F). Finally, stimuli such as flow can be applied to co-cultures to examine metabolic interactions under more physiologically relevant conditions (bottom row). Flow can be applied to one set of cells using a cone and plate device, and conditioned media from the flow-adapted cells can be applied to another cell culture (G). Microfluidics can also be used to apply flow to cells in contact with another type of cells through a permeable membrane (H), or to 3D cell cultures like spheroids surrounding a perfused engineered vessel (I). Figure created with BioRender.