A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

Abstract

The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five daysin vitro(DIV); cell growth was still substantial at 14 DIV. Neurons were identified byβ-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting ofβ-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.