Minimal requirements for long-term serum-free culturing of electrically active dissociated neurons in a monolayer

A method is presented for culturing dissociated fetal rat cerebral cortex and other brain areas. These cultures meet the following requirements: (1) they are maintained in a serum-free medium, (2) the cultured cells form a near monolayer allowing optimal access and visibility, (3) neurons survive up to at least six weeks, while (4) showing a high incidence of spontaneous action potential discharges. The dissociated neurons are grown on coverslips in a serum-free medium conditioned by astrocytes. Development of the cultures as monolayers was dependent on basic polymers, which prevent neuronal aggregation. Long-term survival and health (as indicated by abundant spontaneous action potential discharges) depended on insulin, transferrin, lipoic acid, vitamins A and E, albumin, and conditioning of the medium by astrocytes. Series of more than 300 nearly identical cultures can be routinely produced from one litter of rats, for use in morphological, biochemical and electrophysiological assays without adaptation