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

We have developed a new method for culturing cells that maintains their health and sterility for many months. Using conventional techniques, primary neuron cultures seldom survive more than 2 months. Increases in the osmotic strength of media due to evaporation are a large and underappreciated contributor to the gradual decline in the health of these cultures. Because of this 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 culture dish lids that form a gas-tight seal, and incorporate a transparent hydrophobic membrane (fluorinated ethylene-propylene) that is selectively permeable to oxygen (O(2)) and carbon dioxide (CO(2)), 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 technique to grow dissociated cortical cultures from rat embryos on multi-electrode arrays. After more than a year in culture, the neurons still exhibit robust spontaneous electrical activity. The combination of sealed culture dishes with extracellular multi-electrode recording and stimulation enables study of development, adaptation, and very long-term plasticity, across months, in cultured neuronal networks. Membrane-sealed dishes will also be useful for the culture of many other cell types susceptible to evaporation and contamination.