Isolation and Cultivation of Porcine Astrocytes

A procedure for the isolation and cultivation of astrocytes from swine is described. More specifically, the donor animals are adolescent minipigs about 3 months in age and 10 kg in weight. About 20 g of cerebral tissue can be isolated from the piglet, yielding enough astrocytes of homogeneous genetics for experimentation after only one passage in culture. The astrocyte isolation procedure includes mechanical and enzymatic digestion of the brain tissue followed by separation of the brain fragments, based on size and density. Astrocytes are further purified from any residual nonastrocytes by differential attachment during the first passage. The resulting culture is purely astrocytes (>98%) based upon their appearance in phase-contrast microscopy and their uniform expression of glial fibrillary acidic protein. More importantly for our purposes, the astrocytes greatly enhanced the functionality of our in vitro blood-brain barrier model when cocultured with porcine brain capillary endothelial cells.