Why Your Frozen Cells Die and How to Save Them?

Cryopreservation uses cold temperatures to put cells or tissues in a state of suspended animation where few biological changes happen over a long period. The cold temperatures greatly slow enzymatic activity and other chemical reactions in the cells, allowing for storage periods of months or years.

To protect cell viability after thawing, it is important to prevent cryoinjury, or damage that can occur due to osmotic stress or ice formation within cells or tissues during freezing. It is also vital to prevent mishaps during cell thawing that can reduce post-thaw cell viability.

Cell Cryopreservation Techniques

The most common cryopreservation protocols involve using a cryoprotective agent and either slow-cooling or rapid-cooling techniques. Cryoprotective agents help reduce ice formation and damage to cells during freezing. Common cryoprotective agents include dimethyl sulfoxide (DMSO), glycerol, and trehalose, among others.

During slow cooling, lower the temperature by 1°C to 3°C per minute until reaching the proper temperature. Samples are then stored in liquid nitrogen vapor phase or in freezers at -80°C or below. Rapid cooling includes techniques for vitrification, or the formation of a glass-like state, in the frozen cells or tissues, and techniques involving direct immersion of sample vials in liquid nitrogen.

However, not all cell types and sample types can withstand each of these methods. Before cryopreserving your precious samples and cell lines, consider each step of the cell freezing, storage, and thawing processes, and research which techniques are compatible with your application.

Do The Following When Cryopreserving Your Cells:

• Use healthy cells: The healthier your cells, the more likely they are to survive the stress of cryopreservation and thawing. Damaged cells are more prone to membrane rupture and cell death during cryopreservation.
• Select proper cryogenic containers: Be sure to use cryovials or other appropriate containers designed for cryopreservation. Seal your containers tightly to prevent contamination.
• Use the right cryoprotectants: Use cryoprotectants such as Propylene Glycol, Glycerol, and Dimethyl Sulfoxide (DMSO) to protect cells from damage during freezing and thawing. Cryoprotectants increase the concentration of solutes and reduce the amount of ice that can form at low temperatures.
• Wear personal protective equipment and have proper training: When handling liquid nitrogen, always wear appropriate protective gear to avoid skin and eye exposure. Properly wearing thermally-insulated gloves, full-face visors, and splash-proof aprons protects you and the cell cultures. Ensure all personnel who are working with liquid nitrogen are properly trained.
• Work under a laminar flow hood: Your area should be clean and sterile. Using a laminar flow hood will help to keep your area free from contaminants.
• Handle one cell line at a time: While you may be required to work with more than one cell line, focus on one cell line at a time to reduce the chance of cross-contamination and minimize aerosol generation that could be potentially harmful.
• Freeze gradually: Slowly reduce the temperature to minimize the formation of ice crystals that can damage cells. Controlled-rate freezing devices are often used.
• Store in liquid nitrogen: Ensure long-term preservation. Store cryopreserved cells in liquid nitrogen (-196 °C or lower).

Cryopreservation is a delicate process. To maintain the long-term viability of your cells, be sure to implement best practices, proper procedures and specific protocols for your type of cell culture because each cell type may have distinct requirements for successful cryopreservation.

Learn more about cell cryopreservation step by step

View our complete line of cryogenic products