A new approach to neural cell culture for long-term studies

We have developed a new method for culturing cells that maintains their hea lth and sterility for many months. Using conventional techniques, primary n euron cultures seldom survive more than 2 months. Increases in the osmotic strength of media due to evaporation are a large and underappreciated contr ibutor to the gradual decline in the health of these cultures. Because of t his and the ever-present likelihood of contamination by airborne pathogens, repeated or extended experiments on any given culture have until now been difficult, if not impossible. We surmounted survival problems by using cult ure dish lids that form a gas-tight seal, and incorporate a transparent hyd rophobic membrane (fluorinated ethylene-propylene) that is selectively perm eable to oxygen (O-2) and carbon dioxide (CO2), and relatively impermeable to water vapor. This prevents contamination and greatly reduces evaporation , allowing the use of a non-humidified incubator. We have employed this tec hnique to grow dissociated cortical cultures from rat embryos on multi-elec trode arrays. After more than a year in culture, the neurons still exhibit robust spontaneous electrical activity. The combination of sealed culture d ishes with extracellular multi-electrode recording and stimulation enables study of development, adaptation, and very long-term plasticity, across mon ths, in cultured neuronal networks. Membrane-sealed dishes will also be use ful for the culture of many other cell types susceptible to evaporation and contamination. (C) 2001 Elsevier Science B.V. All rights reserved.