Cell Cryopreservation: Common Protocols, Thawing Tips, and FAQs

Imagine this scenario: You’ve spent months carefully cultivating a unique cell line or editing a specific gene. The data looks promising, and it’s time to bank these precious samples for future verification. You freeze them down, confident they are safe. But six months later, when you need to revive them for a critical experiment, the viability count plummets. The cells are sluggish, or worse—gone.

Every researcher knows that sinking feeling. It highlights a critical truth in the lab: freezing is only half the battle; the real victory lies in the recovery.

Cryopreservation —the process of freezing live cells, 3D cultures, or tissues at ultra-low temperatures—is the backbone of modern bioscience. Whether you are preserving patient samples for longitudinal studies, banking cell lines for therapeutic product development, or simply preparing samples to share with collaborators, this technique is essential for pausing biological time.

However, successful cryopreservation is not just about throwing tubes into liquid nitrogen. Achieving excellent post-thawing cell viability requires meticulous planning, precise technique, and the right tools. The protocols you choose today significantly impact the biological integrity of your samples tomorrow.

In this guide, we will share some basic knowledge about cell cryopreservation, common cell cryopreservation protocols, practical thawing techniques, and answer some of the most frequently asked questions by researchers.

Cryopreservation uses cold temperatures to put cells or tissues in a state of suspended animation where few biological changes happen over a long period. This is achieved through the substantial reduction of enzymatic activity and other chemical reactions within the cells, enabling storage durations that can span months or even years without significant degradation.

A key aspect of effective cryopreservation is the mitigation of cryoinjury, which refers to the cellular damage that may arise from osmotic stress or the formation of ice crystals during the freezing process. Such injuries can compromise cell membranes, organelles, and overall viability upon thawing. Equally important is the careful management of the thawing phase to avoid additional stressors that could further diminish post-thaw cell health, ensuring that preserved samples retain their functional integrity for subsequent research or applications.

Before diving into specific protocols, it is crucial to understand what actually happens to a cell when the temperature drops. The biggest enemy of cell survival is not the cold itself, but the formation of ice crystals.

When water inside a cell freezes, it expands and forms sharp crystals that can puncture the cell membrane and damage organelles. To prevent this mechanical damage, we use Cryoprotective Agents (CPAs). Think of CPAs as a molecular “antifreeze.” They lower the freezing point of the medium and increase viscosity, minimizing ice crystallization and reducing osmotic stress.

While there are many variations, most laboratory protocols fall into two main categories:

This is the most widely used method for general cell lines (like HeLa, HEK293) and many primary cells.

• The CPA Mix: The standard “golden ratio” usually involves a freezing medium containing 10% DMSO (Dimethyl Sulfoxide) or Glycerol mixed with Fetal Bovine Serum (FBS) or culture media. DMSO is excellent at penetrating the cell membrane to displace water, though it can be toxic at room temperature.

• The Cooling Rate: The goal here is a controlled temperature drop, typically -1°C per minute. This slow pace allows water to leave the cell before it freezes, reducing the risk of intracellular ice formation.

• The Method: Researchers often use isopropanol-filled freezing containers (often called “Mr. Frosty” style containers) or automated controlled-rate freezers to achieve this pace. Once the samples reach approximately -80°C, they can be transferred to the vapor phase of a liquid nitrogen tank for long-term storage.

For more sensitive samples that cannot withstand ice formation at all—such as oocytes, embryos, or certain stem cells—slow freezing may not be effective. In these cases, vitrification is the answer.

• The Concept: Vitrification uses a much higher concentration of CPAs and an ultra-fast cooling speed to bypass the ice stage entirely. instead, the liquid turns directly into a glass-like (vitreous) solid state.

• The Method: This involves “flash freezing,” often by immersing the sample vials directly into liquid nitrogen. Because the temperature drop is instant, ice crystals simply don’t have time to form.

It is vital to remember that not all cell types are compatible with every method. Slow freezing may cause excessive osmotic pressure shocks to some cells, while the high concentration of dimethyl sulfoxide (DMSO) used in vitrification may be toxic to some cells.

Before you start banking your cells, review the specific requirements for your sample type. Whether you are freezing robust cancer cell lines or delicate 3D organoids, selecting the right protocol—and the right container to withstand these extreme conditions—is the first step toward successful recovery.

Successful cryopreservation and thawing of cell samples demand meticulous attention to detail to ensure viability and functionality post-process.

Initiating the process with low-quality cells, those harvested at an inappropriate growth stage, or potentially contaminated samples can significantly compromise outcomes. To mitigate this, cells should be collected when they are in robust health, ideally during the late logarithmic phase of growth, and rigorously tested for any contaminants prior to freezing.

It is imperative to select the most suitable cryoprotective agent and its optimal concentration tailored to the specific cell or tissue type, the expected storage duration, and the chosen cryopreservation method. For instance, glycerol is frequently employed for yeasts, bacteria, mammalian red blood cells, and gametes, whereas dimethyl sulfoxide (DMSO) is often preferred for more complex mammalian cells. Trehalose, with its relatively low toxicity, may be advantageous for sensitive cell types, though it tends to be costlier. Concentrations that are too low can diminish cell viability after thawing, while excessively high levels may induce chromosomal instability.

Inadequate temperature management can lead to cellular damage from rapid ice formation or uneven cooling. While improvised methods, such as using insulated containers, might seem convenient, they often yield inconsistent results. Employing controlled-rate cooling devices can achieve the recommended gradual decrease of 1°C to 3°C per minute, thereby reducing the risk of thermal shock.

Lower temperatures generally support longer-term viability. Maintaining samples in the vapor phase of liquid nitrogen is widely regarded as optimal, offering extremely low temperatures while minimizing safety risks associated with direct immersion. Alternatively, direct storage in liquid nitrogen or in ultra-low temperature freezers at -80°C or colder is feasible, though the latter typically results in reduced shelf life.

Errors in thawing technique can undermine cell integrity, making rapid thawing essential to prevent prolonged exposure to damaging conditions. This is best achieved by promptly placing vials in a 37°C water bath or, to reduce contamination risks, utilizing a water-free warming system.

Immediately after thawing, cells should be washed in prewarmed culture medium to eliminate residual cryoprotectants, which can be harmful to proliferating cells, followed by a gentle transfer to fresh, prewarmed media to facilitate optimal revival and growth.

When cells are stored in liquid nitrogen at −196°C, all metabolic activity is essentially halted, which allows their biological characteristics to remain stable for extremely long periods. Under these conditions, the theoretical storage duration is unlimited, and many cell types can be recovered even after decades without significant loss of viability.

As a practical quality-control measure, newly frozen cell batches are often thawed once within a short period to confirm that the cells tolerate the cryopreservation procedure. For established cell lines, many laboratories also recommend thawing and re-freezing a vial approximately once a year to verify viability and ensure the long-term reliability of the cell bank.

This is a very common issue. The success of cryopreservation depends on several key factors, including cell collection, the choice and use of cryoprotective agents (CPAs), storage vessels, cooling rate, low-temperature storage, and the thawing process. Yet in practice, problems with cell viability often become noticeable only after thawing and seeding. To improve post-thaw survival, it is important to ensure that the cells are healthy and at an appropriate density before freezing, as healthier cells consistently show better recovery. Freezing about 2 × 10⁶ cells per cryovial(GenFollower’s cryo tubes are recommended). Excessively high densities may result in insufficient nutrients or CPA levels to maintain optimal cell condition. Cells should also avoid prolonged exposure to dissociation reagents or CPAs before freezing, and should not remain at room temperature for too long during collection.

Another critical factor is the cooling rate. When placing cryovials containing the resuspended cells and freezing medium into a −80°C freezing container, the temperature should decrease at approximately −1°C per minute, which requires using an appropriate freezing container along with the correct type and concentration of CPA. During the subsequent transfer to long-term storage at −196°C, it is essential to prevent the cells from experiencing any temperature rise. Finally, thawing must be performed rapidly, followed by proper removal of the CPA to minimize cytotoxicity and osmotic stress.

Cryoprotective agents (CPAs) are generally classified as intracellular or extracellular. Intracellular CPAs are small molecules capable of permeating the cell membrane, such as glycerol, ethylene glycol, propylene glycol, and commercial formulations like the Cell Banker series. Extracellular CPAs, by contrast, are larger molecules added to the freezing medium, including sucrose, dextrose, methylcellulose, and polyvinylpyrrolidone (PVP).

DMSO remains widely used across many cell types, including stem cells and dendritic cells for cell therapy, and it is commonly combined with FBS or HSA at a final concentration of 10%. However, several alternatives have been explored. PVP, for example, has been studied as a substitute for DMSO and fetal calf serum (FCS) in the cryopreservation of human adipose-derived stem cells, where 10% PVP combined with human serum produced recovery rates comparable to DMSO-based protocols (Source: Cryopreservation of Human Stem Cells for Clinical Application: A Review). Methylcellulose has also been evaluated either alone or in combination with reduced DMSO levels, with studies showing that 1% methylcellulose can achieve similar outcomes to protocols using as little as 2% DMSO in apoptosis assays.

Yes, this is completely normal. Even under well-optimized conditions, cryopreservation is inherently stressful for cells. Each freeze–thaw cycle exposes them to osmotic changes, ice-crystal formation, and CPA-related stress, so cells that are thawed, refrozen, and thawed again typically show much lower viability than cells that have undergone only a single thaw. Viability loss becomes more pronounced with each additional cycle.

When low post-thaw viability is observed, several factors should be reviewed. The health and density of the cells at the time of freezing are critical, and cells should not remain in dissociation reagents, CPAs, or at room temperature for prolonged periods during collection. Cooling must occur at a controlled rate, and the samples should be protected from unintended warming during transfer to long-term storage. During thawing, rapid warming and proper removal of the CPA are essential to minimize cytotoxic and osmotic damage. As noted earlier, careful control of these steps can significantly improve recovery.

Before refreezing, it is advisable to assess the culture under a microscope to check for dead or damaged cells. PBMCs in particular often accumulate non-viable cells after thawing, and gentle low-speed centrifugation during washing can be used to remove them. Keep in mind that PBMC viability may naturally decline with prolonged storage, and donor-related factors can also play a role. For example, studies have shown that PBMCs from individuals with certain infections—such as dengue virus—may exhibit lower viability after cryopreservation compared with PBMCs collected from healthy donors.

If you need to refreeze cells, apply the same principles used in the initial cryopreservation: start with healthy cells at an appropriate density, use the correct concentrations of serum and DMSO, follow proper PBMC centrifugation steps, cool at a controlled rate, and ensure that the samples reach the appropriate long-term storage temperature. Following these guidelines will help minimize the viability loss associated with a second freeze.

There are several key principles to follow when preparing tissues for cryopreservation, especially when high-quality imaging will be performed after thawing. Fresh tissues should be handled quickly—kept on ice and frozen or fixed without delay—to minimize morphological changes. Smaller tissue pieces are preferred because they freeze more uniformly and reduce ice-crystal formation. Before freezing, excess surface liquid should be removed to avoid artifacts that can interfere with imaging.

For optimal preservation, tissues are often embedded in a suitable cryomedium such as OCT or snap-frozen using liquid nitrogen–cooled isopentane to limit ice damage. During thawing, samples should be warmed rapidly, typically in a 37°C water bath, to reduce the time spent at intermediate temperatures where structural degradation can occur.

GenFollower provides reliable cryopreservation labware designed to support consistent and safe long-term cell storage. Our cryo tubes ensure secure sample containment during freezing and liquid-nitrogen storage, while our durable cryo boxes offer organized, temperature-stable storage for laboratory cell banks. If you have additional questions about cell cryopreservation protocols or need assistance selecting the right consumables for your workflow, our team is always available to help.