Monitoring neuron and astrocyte interactions with a 3D cell culture system.

Methods are described for the generation and analysis of 3D co-culture models in which astrocyte and neuronal behavior can be studied. Cells may be obtained from a variety of sources, then cultured within collagen hydrogels to explore cellular responses and interactions in response to substances under test or under conditions that mimic physiological or pathological environments. Cell populations are labelled then either mixed within gels or arranged as separate adjacent populations, with further options including directing the self-alignment of cells to form anisotropic 3D cultures. Immunofluorescence staining and confocal microscopy can be used to capture image data from 3D structures and detailed protocols are provided for obtaining reliable results. Finally, 3D image analysis of confocal microscopy data is discussed, providing guidance on how astrocyte and neuronal features can be quantified.