Cryopreservation of primary neurons for tissue culture

We have developed new cryopreservation methods which allow storage of fetal rat central nervous system tissues for more than 1 week at 3-8 degrees C or for several months at -70 or -90 degrees C prior to tissue culture. For refrigeration, small brain regions (less than 2 mm thick) were placed intact into 35 mm petri dishes of 'hibernation medium' inside a humidified chamber. Optimal preservation was obtained with hibernation media of pH 6.8-7.4, containing 30-70 mM K+, 10-30 mM Na+, 5-50 mM PO4(2-), 20 mM lactic acid, 5 mM glucose, and less than 0.1 mM Ca2+. The media were made approximately isotonic by addition of sorbitol. For freezing, brain tissues were dissociated by gentle trituration (without enzymes) in the above medium supplemented with 5-10% dimethylsulfoxide. After refrigeration or freezing, neurons were very sensitive to damage from mechanical stress (e.g. centrifugation, harsh rinsing or trituration). Rapid changes in osmotic pressure or excessive polylysine on the tissue culture substratum also reduced neuronal survival after cryopreservation. Pretreatment of tissue culture substrata with media from lung cell cultures, or plating of neurons at higher density (2000 cells/mm2) improved neuronal survival after cryopreservation.