A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents

Abstract

Ependymal cells form an epithelium lining the ventricular cavities of the vertebrate brain. Numerous methods to obtain primary culture ependymal cells have been developed. Most of them use foetal or neonatal rat brain and the few that utilize adult brain hardly achieve purity. Here, we describe a simple and novel method to obtain a pure non-adherent ependymal cell culture from explants of the striatal and septal walls of the lateral ventricles. The combination of a low incubation temperature followed by a gentle enzymatic digestion allows the detachment of most of the ependymal cells from the ventricular wall in a period of 6h. Along with ependymal cells, a low percentage (less than 6%) of non-ependymal cells also detaches. However, they do not survive under two restrictive culture conditions: (1) a simple medium (alpha-MEM with glucose) without any supplement; and (2) a low density of 1 cell/µl. This purification method strategy does not require cell labelling with antibodies and cell sorting, which makes it a simpler and cheaper procedure than other methods previously described. After a period of 48h, only ependymal cells survive such conditions, revealing the remarkable survival capacity of ependymal cells. Ependymal cells can be maintained in culture for up to 7-10days, with the best survival rates obtained in Neurobasal supplemented with B27 among the tested media. After 7days in culture, ependymal cells lose most of the cilia and therefore the mobility, while acquiring radial glial cell markers (GFAP, BLBP, GLAST). This interesting fact might indicate a reprogramming of the cell identity.