The complexity of neuronal cell structures and functions requires specific methods of culture to determine how alteration in or among cells gives rise to brain dysfunction and disease. In this context, the primary culture of neuronal cells plays an important role in the study of this topic, especially related to neuronal cells survival and differentiation, nutritional requirements, but also neuronal development and spine formation. For all these investigations and applications, it is very important that primary neurons are cultured under conditions that resemble the in vivo environment as closely as possible. In this line, glia-neuron sandwich co-cultures are an extremely useful tool in vitro to evaluate cell-to-cell interaction relaying on the release of soluble factors and could be a suitable method in the study of the contribution of glia-secreted molecules to neuronal development and spine formation. To this end, this chapter describes the procedures to set up a sandwich co-culture system from primary rat glial cells and hippocampal neurons, and highlights advantages and disadvantages of this approach and its possible application in the investigation of individual glial factor impact on neuronal properties.