Preparation of glial extracellular matrix: a novel method to analyze glial–endothelial cell interaction

Abstract

Studies on the interactions of endothelial cells and glial cells are of increasing importance for the understanding of the formation of the blood–brain barrier (BBB) and for the reconstruction of BBB properties in cultured brain capillary endothelial cells in vitro. Many methods have been used to examine cell-cell interactions, including conditioned medium, co-culture, feeder layers, and many others. Here we describe how to prepare the extracellular matrix (ECM) secreted from cultured cells. Cells are known to produce and interact with their extracellular components in an organized matrix and to regulate the function of other cells through the ECM. The ECM plays a central role in the differentiation and function of the cells, and controls the proliferation and motility of these cells. The responses of cells to ECM molecules need to be clarified. As the ECM is situated between cerebral capillaries and astrocytes in the central nervous system, the ECM secreted by glial cells may also play an important role in the formation and maintenance of the BBB. In our previous studies, the ECM produced by glial cells elevated γ-glutamyl transpeptidase activity, which is an accepted marker enzyme for differentiated brain capillary endothelial cells, in cultured bovine brain capillary and aortic endothelial cells. Using the method described here, the cell-cell interaction via the ECM molecules can be examined.