Tips For Cell Cryopreservation

Cryopreservation is a well-established laboratory technique for storing cells and other biological material at temperature close to liquid nitrogen. It provides researchers with a backup in case growing cells are lost due to contamination and helps to minimize the occurrence of genetic drift by allowing early passage cells to be brought into use while the current cultures have been grown for an extended time.

Cryopreservation 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 is important to know best practices for cell freezing.

Check Cell Health Before Freezing

Cells should ideally be frozen at a low passage number, when cell properties have had little time to change as a result of extended passaging. Passaging cells or refreshing the growth media 1-2 days before freezing will ensure the cells are healthy and in an active phase of growth. The concentration at which cells are frozen may vary between cultures, but it is typically 1 x 10⁶–5 x 10⁶ cells/mL in freezing media. Freezing cells at too low or too high a density can impact viability and should be avoided.

Before freezing cells, it is important to perform a viability count using Trypan Blue or another live/dead stain, and to check for contamination via sterility evaluation and mycoplasma testing.

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 crystals that form at low temperatures.

Freeze Gradually

Freezing cells slowly is essential to prevent the formation of ice crystals and can be achieved using a freezing container with a freezing rate of 1°C/minute. This should be placed at -80°C for at least 4 hours but ideally overnight once the cell-containing cryovials have been added.

Label and Document

It is important to keep a record of the cells' identity and preservation details. Properly label and document the type of cells, passage number, date of cryopreservation, and any other relevant information to prevent mix-ups and ensure traceability.

Store in Liquid Nitrogen

Store cryopreserved cells in liquid nitrogen (-196 °C or lower) to ensure long-term preservation. To prevent damage to cell viability, cryopreserved cells should never be transferred from liquid nitrogen to the laboratory on wet ice. Dry ice or a liquid nitrogen container must always be used, especially when cells are to be transported over a considerable distance or time.

Monitor and Record Temperature

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

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

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