The Dos and Don’ts of Cell Cyropreservation

A list of what and what not to do when cryofreezing cells for future use.

Cryopreservation helps to retain the original structure of cells and maintain them for future use. It is a delicate process that requires specific cryogenic products, a lot of patience, and the ability to follow a specific process. To ensure the integrity of your cell lines and minimize the risk of contamination, it’s important to know best practices for cell cryopreservation. We’ve developed a list of dos and don’ts to help you get started quickly, but be sure to review all best practices, proper procedures and specific protocols for your type of cell culture before you begin.

The dos of cryopreserving cells

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.

Maintain separate media bottles: Use separate bottles of media for each cell line to maintain consistency, track and control the quality of the media, and prevent cross-contamination.

Thaw slowly: Use a water bath to gradually thaw cells.

Freeze gradually: Slowly reduce the temperature to minimize the formation of ice crystals that can damage cells. Controlled-rate freezing devices are often used.

Let cells recover: Once your cells are frozen, remove the cryoprotectant to prevent damage. Let them recover so they can adjust to their new environment. Refer to your protocols for the specific time period.

Label and document: Prevent mix-ups and ensure traceability. Properly label and document the type of cells, passage number, date of cryopreservation, and any other relevant information.

Store in liquid nitrogen: Ensure long-term preservation. Store cryopreserved cells in liquid nitrogen (-196 °C or lower).

Monitor and record temperatures:  Monitor and keep accurate records of the temperatures during the freezing and storage process. Temperature fluctuations can negatively impact cell viability.

Examine for contamination: Check cell cultures and media daily for bacterial or fungal contamination, even when using commercially purchased media.

Mycoplasma testing: Regularly test cell lines for mycoplasma contamination, as mycoplasma can compromise cell culture integrity.

The don’ts of cryopreserving cells

Don’t do the following when cryopreserving cells:

Use contaminated materials: Ensure that all equipment, containers, and cryoprotectants are sterile and free from contamination.

Handle cells from unauthenticated sources: Use caution when working with cells from unauthenticated sources. It’s best to quarantine them until quality control checks are complete to avoid introducing unknown or contaminated cell lines into your experiments.

Use outdated media: Cell culture media should not be used past its shelf life.

Continuously use antibiotics: Prolonged use of antibiotics in cell culture can lead to the development of antibiotic-resistant strains of microorganisms. It’s also important not to use antibiotics to mask potential contamination issues.

Overpack cryopreservation containers:  Make sure you’re not overcrowding cryopreservation containers. Leave some headspace to allow for expansion during freezing and to prevent your vials from breaking.

Freeze cells directly: Never freeze cells directly in liquid nitrogen without proper cryoprotectants. This can cause ice crystal formation and cell damage.

Culture continuously: Cultured cell lines should be periodically returned to frozen stock. Continuous passaging without returning to frozen stock can lead to genetic changes and reduced cell line quality.

Overcrowd cells as they grow: Cell should not become fully confluent. Sub-culture cells when they reach 70 to 80 percent confluency to prevent overcrowding and reduce the risk of differentiation or other issues.

Skip labeling: You may be in a rush but don’t neglect proper labeling. It’s important to keep a record of the cells’ identity and preservation details. Remaining organized and in control of your process will result in an organized lab and successful experiments.

Thaw quickly: Rapid thawing can lead to cell stress and reduced viability. Gradual thawing is recommended, usually in a water bath at 37 °C.

Re-freeze thawed cells: Once cells have been thawed, do not re-freeze them because re-freezing can reduce cell viability.

Forget safety precautions: Don’t ignore safety when working with cryogenic materials. Don’t neglect safety precautions when handling liquid nitrogen, such as proper storage, handling, and wearing proper personal protective equipment.

Accumulate waste: Ensure that waste, especially within microbiological safety cabinets and incubators, is regularly disposed of to maintain a clean and safe working environment.

Final thoughts about cell cryopreservation

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 and freezing

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Sources

PubMed®, Principles of Cryopreservation, Accessed, October 26, 2023.

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