Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains

Abstract

Simple methods for obtention of primary cultures of isolated astrocytes and microglia from adult simian brain have been developed. Characterization of these two glial cell populations were performed by morphological observations and by immunocytochemistry. The astroglial cultures were obtained by an indirect method. After L-leucine methyl-ester treatment and trypsinizations, more than 99% of cells expressed glial fibrillary acidic protein (GFAP), whereas no macrophages or microglia could be detected. Likely, the 1% remaining cells were immature astrocytes or cells that lost their GFAP expression. Cultured simian astrocytes expressed vimentin, laminin, and fibronectin. We also found a constitutively low expression of major histocompatibility complex (MHC) class II by cultured astrocytes which was significantly enhanced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha) treatments. Microglial cultures were obtained by a direct method of isolation using Percoll gradient separations and compared to simian monocyte-derived macrophages or alveolar macrophages. Microglial cells differed from macrophages by their proliferation upon granulocyte-macrophage colony stimulating factor (GM-CSF) treatment and by their typical morphology when observed by scanning electron microscopy. As macrophages, they expressed in vitro CD68, CD64, CD14, CD11b, MHC class II, and fibronectin. However, contrary to macrophages, simian cultured microglia expressed laminin. This observation suggests that microglia represent a new potential source of this extracellular matrix protein in the brain.