Work

Cryopreservation of primary neurons for tissue culture

Year: 1986

Type: article

Abstract: 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.

Source: Brain Research

DOI: 10.1016/0006-8993(86)91222-9

Authors: Jerrolynn C. Kawamoto, John N. Barrett

Institution: University of Miami

Language: English

Topic: Neuroscience and Neuropharmacology Research

Subfield: Cellular and Molecular Neuroscience

Field: Neuroscience

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,360

Funders: National Parkinson Foundation, National Institute of Neurological Disorders and Stroke