Why Aren't My Cells Surviving Cell Thawing?: Cryopreservation Best Practices for Cell Freezing and Thawing

Cryopreservation—freezing live cells, 3D cultures, or tissues to preserve them—is an essential technique in many labs. Common uses include preserving patient samples for research, preparing samples for sharing with colleagues, preserving therapeutic products, or saving cell lines or 3D cultures for later use.

Successful cryopreservation with excellent post-thawing cell viability requires planning and care. The techniques and protocols used in sample cryopreservation and cell thawing significantly impact cell viability, and the best techniques vary based on the sample type. This article offers some best practices, practical tips for cryopreservation and thawing, and how to avoid common pitfalls.

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.

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.

Successful sample cryopreservation and thawing require research and careful planning. Here are some common pitfalls to watch out for, along with tips for avoiding them.

Using low-quality samples, cells at the wrong point in the growth cycle, or samples with possible contamination can impact cryopreservation success. Cells should typically be harvested for cryopreservation when they are healthy and in the late logarithmic phase of growth and have been tested for contamination.

Research the best cryoprotective agent and concentration based on your cell type or tissue type, the anticipated duration of storage, and the cryopreservation process you will use. For example, glycerol is commonly used for cryopreservation of yeasts, bacteria, and mammalian red blood cells and gametes, while DMSO is often a better choice for complex mammalian cells. Meanwhile, trehalose has low toxicity and can be helpful in the preservation of sensitive cell types, but it can be more expensive than other options.

Also, research the best concentration of cryoprotectant agent to use. Too-low concentrations can reduce post-thawing cell viability, while too-high concentrations can cause chromosomal instability in the cells.

Some labs take a do-it-yourself approach to slow cooling, using insulated boxes as freezing chambers, but this can be unreliable and may produce non-uniform results. Devices for controlling the rate of cell cooling, such as Corning^® Cool Cell^® containers, can help you cool cells closer to the ideal rate of 1°C to 3°C per minute, helping to prevent temperature shock.

Colder temperatures generally result in more extended periods of successful storage. Storage in the vapor phase of liquid nitrogen is often considered ideal due to the very low temperature and safety considerations. Still, storage directly in liquid nitrogen is also an option. You can store cell samples in freezers kept at -80°C or below, but this tends to result in a shorter shelf life for preserved samples.

Avoid incorrect cell thawing techniques to promote better post-thaw cell viability and health. Rapid thawing is typically recommended to minimize damage to cells and tissues. This can be accomplished by immediately transferring vials to be thawed to a 37°C water bath, or, for a lower risk of contamination, a water-free warming device.

Thawed cells should typically be washed in prewarmed cell culture medium to remove cryoprotectant, as cryoprotectant can be toxic to growing cells. Then immediately, but gently, transfer the thawed cells to prewarmed culture media for recovery.

These tips for cryopreservation and cell thawing apply to most cell types. Still, other considerations may come into play if you are cryopreserving tissues, products for clinical use, or sensitive cell types. For example, human induced pluripotent stem cells (iPSCs) are sensitive to temperature injury and may benefit from alternative cryoprotectants or specialized cell freezing and thawing techniques.

Corning's experienced scientists can help with your cryopreservation questions and needs. Learn more at Corning's Ask the Expert session, The Dos and Dont's of Cryopreservation.