Method for preparing cultures of central neurons: Cytochemical and immunochemical studies (neuroblasts/astrocytes/oligodendrocytes/glial fibrillary acidic protein/insulin)

We report a simplified method for culturing fetal central nervous system cells predominantly inducing neurons that grow, differentiate, and live in vitro for as long as 10 weeks. These central nervous system cells form a confluent cell culture in which about 80% of the cells are fully differen- tiated neurons producing interconnecting axons and dendrite processes and live upon a sparse underlying population of fi- brillary and protoplasmic astrocytes, oligodendrocytes, and fi- broblasts. Morphological and cytochemical characteristics of these cell types, based on immunofluorescent cell specific markers and silver staining of neurons, are presented. Large numbers of neurons can be cultured from dissociated fetal brain of several species of rodents by the technique de- scribed in this paper. The growth of central neurons is relatively unencumbered by the concurrent growth of fibroblasts or glial cells, and the fine structure of the neurons can be readily studied by light and electron microscopy. Neurons are easily identified by their cytological structure and their specific cytochemical properties and may be distinguished from the main classes of other cells growing together with them by the use of cell-spe- cific immunofluorescent markers. The method for readily culturing large numbers of neurons from the fetal central nervous system is based on many essential modifications of other methods used for isolation, growth, and maintenance of central neurons in culture for long periods of time (1-8). There are three major modifications: (i) use of a fetus chosen at a critical phase of early development-at the beginning of organogenesis-while there are many still dividing neuroblasts that possess ability to survive under tissue culture conditions and to differentiate into mature neurons; (ii) use of arabinosylcytosine, a specific inhibitor of DNA synthesis, to kill all still-dividing cells at a critical time in the cultures differ- entiation; and (iii) use of unprecedented high concentrations of insulin and glucose in the medium.