Sinton:Protocols/Freezing cells

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Safety Safety goggles Nitrile gloves Lab coat Biosafety cabinet Materials Cell culture Cell media Cryogenic tubes Sterile pipette tip Sample holder -80°C Freezer DI Water Glycerol Ethanol 1% Sodium hypochlorite solution Description Freezing cells is a way of preserving a culture for extended periods, often for several years. This procedure reduces the risk of contaminations and genetic changes. The cell culture must be healthy, and growing in log-phase for proper preservation. This procedure can be completed within the CMS common labs. Protocol Preparation The day before freezing cells, prepare a new culture in fresh sterile media. The cell line must be in log-phase for effective cryoperservation. In addition, prepare a 30% glycerol solution in DI water. Glycerol serves as a cryoprotectant, preventing cell membrane rupture. Autoclave the solution. Sample freezing In a biosafety cabinet, combine 500 μL of cultured cell media with 500 μL of your sterile 30% glycerol solution. Follow proper biosafety cabinet procedures, and wear proper PPE. Repeat for however many cryogenic cultures are to be prepared, and transfer to a sample holder. Place the sample holder in the -80°C Freezer. Be sure to minimize the time the freezer is open. Sample recovery To recover frozen cells, remove a cryogenic sample from the -80°C Freezer and move to the biosafety cabinet. Use a sterile pipette tip to scrape off a small icy piece from the frozen sample, and transfer to fresh media. Immediately return the frozen sample to the freezer. Do not let it thaw. Culture the inoculated media in an incubator.