Establishment of a permanent rat brain‐derived glial cell line as a source of purified oligodendrocyte‐type 2 astrocyte lineage cell populations

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Abstract
A permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process-bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process-bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum-containing culture medium, most of the process-bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1 + or O4+ cells were rarely GFAP−, and GFAP+ cells were rarely LB1− or O4−. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process-bearing cells accumulated the neurotransmitter γ-aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the 0-2A (oligodendrocyte-type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of 0-2A lineage cells. The L3 cell line represents a unique source of large numbers of purified 0-2A-like lineage cells and may therefore represent a useful experimental system to analyze the functional and developmental properties of this glial population.