Cryopreservation of mammalian cell lines video protocol
Learn about cryopreservation of mammalian cell lines for long term storage in liquid nitrogen.
Cryopreservation is a vital technique for long-term storage of mammalian cell lines, preserving their viability and genetic integrity. Proper cell freezing methods and the use of appropriate cell freezing media are essential to maintain cell viability and functionality during the cryopreservation process. This protocol outlines a step-by-step method for freezing cells using cryoprotectants like DMSO or glycerol, followed by storage in liquid nitrogen. It includes guidance for both adherent and suspension cells, ensuring optimal cell density and viability. The use of controlled-rate freezing devices like CoolCell® enhances reproducibility and minimizes damage. This protocol is ideal for researchers seeking reliable methods to bank cell lines for future experiments, reduce contamination risks, and maintain consistent biological characteristics across studies.
Introduction
Cryopreservation allows researchers to store mammalian cell lines for extended periods without compromising viability or genetic stability. This protocol provides a standardized approach to freezing cells at optimal growth phases using cryoprotective agents. Whether working with serum-containing, serum-free, or chemically defined media for sensitive applications, the method ensures cells are preserved in a way that supports high recovery rates upon thawing. By following this protocol, scientists can maintain a consistent supply of cell stocks, reduce variability in experiments, and safeguard against contamination or genetic drift during prolonged culture.
Background and principles
Cryopreservation relies on the principle of minimizing ice crystal formation within cells during freezing. This is achieved by using cryoprotectants such as DMSO or glycerol, which reduce intracellular ice damage. The protocol emphasizes controlled cooling at a rate of 1°C per minute, typically using devices like CoolCell®, followed by storage in liquid nitrogen. Cells should ideally be frozen at high viability (>75%) and optimal density (1 million cells/mL), ensuring successful recovery. The method is applicable to various cell types and media conditions, with adjustments for serum-free cultures or glycerol-sensitive lines. It is important to select an appropriate growth medium for cell passaging, washing, and resuspension prior to cryopreservation to maintain cell viability. Proper labeling and sterile, aseptic handling are essential throughout the process.
Cryopreservation is a method whereby cultured cells are frozen, maintaining their viability, until they are defrosted months or years later. Cells are cryopreserved to minimize genetic change and avoid loss through contamination. This process also allows for preserving and reviving cultured cells as needed. It is best to cryopreserve cells when they are at their optimal rate of growth (typically the logarithmic growth phase).
When preparing for cryopreservation, it is critical to passage and prepare cells prior to freezing to ensure optimal viability. Following a systematic procedure is essential for consistent and reliable results.
In summary, proper cryopreservation techniques are vital for maintaining the viability and genetic stability of each cell line, ensuring their unique characteristics are preserved for future research and applications.
Materials and reagents
- 1–2 mL cryovials
- CoolCell® freezing unit (BioCision)
- Freezing media
Stage 1 - Cryopreservation
Materials required
Use the supernatant from the centrifuge step (step 7)
Mix well and warm to 37°C before use
Steps
Label cryovials with the date, name of researcher, cell number, passage number and cell type (and any other useful information, for example genetic modifications).
If cells are adherent, remove the cell culture media, wash in PBS, add enough trypsin to cover the cells and incubate for approximately 2 min in a 37°C incubator.
- Resuspend in cell culture media and transfer into a 50 mL Falcon tube.
If cells are in suspension, just transfer the desired volume directly into a 50 mL Falcon tube.
Count cells using a hemocytometer to determine their viability. Cell viability should be at least 75% for cryopreservation.
Centrifuge for 5 min at 300 x g at room temperature.
Prepare freezing media (see Table 1).
Remove the supernatant (keep this; it is needed for the freezing media, see Table 1) and loosen the pellet gently.
Add freezing media to the required cell density.
- For mammalian cells this is usually 1,000,000/mL of freezing media. Cells should not be at room temperature in freezing media for more than 10 min.
Aliquot 1 mL into cryovials and secure the lids.
Transfer the cryovials into a CoolCell® at room temperature.
- Put into a -80°C freezer.
After approximately 24 h, remove the cryovials from the CoolCell® and transfer into liquid nitrogen for long-term storage.
Stage 2 - Thawing frozen cell lines
Steps
Remove the cryovial from liquid nitrogen storage.
- Place in a 37°C water bath until only about 80% defrosted (do not thaw at room temperature). This should take no longer than 1 minute.
Cells should generally be thawed as quickly as possible, above room temperature as a slow thawing process can damage cells.
The freezing media contains essential cryoprotection agents, such as DMSO, to prevent the formation of intracellular and extracellular ice crystals that could damage the cell membrane and components. DMSO does have the drawback of being cytotoxic, and therefore, cells should be thawed at a speed faster than is possible at room temperature in order to facilitate the quick dilution and removal of DMSO from the immediate environment of the cells.
The slow freezing process (-1ºC per minute) also helps prevent the formation of intracellular ice crystals by allowing sufficient water efflux before freezing. Thawing the cells quickly also prevents any crystals that have formed during the freezing process from damaging the cell membrane or components.
Using a pipette transfer the contents of the vial into a 15 mL Falcon tube containing about 10 mL of pre-warmed culture media.
Centrifuge at 300 x g for 5 min, discard the supernatant, and resuspend in the appropriate amount of cell culture media.
Transfer the cells into a culture vessel.
- Then transfer into a 37°C incubator.
Importance of cryopreservation
Cryopreservation is a cornerstone technique in cell culture, essential for maintaining the integrity and viability of cell lines over extended periods. By freezing cells and storing them at very low temperatures, typically in liquid nitrogen, researchers can preserve living cells and tissues for future use without compromising their biological characteristics. This process is critical for ensuring a stable supply of high-quality cell lines, reducing the risk of contamination, and preventing genetic drift that can occur during prolonged culture.
The primary goal of cryopreservation of cells is to protect them from ice crystal formation, which can cause irreversible cell death. Using a suitable freezing medium helps shield mammalian cells from damage during the freezing process. The choice of cryopreservation media is crucial, as it must be compatible with the cell type, whether working with adherent cells, suspension cells, embryonic stem cells, or pluripotent stem cells, to maintain optimal cell viability.
A controlled cooling rate, typically 1°C per minute, is vital for successful freezing. This gradual decrease in temperature allows water to exit the cells, minimizing the risk of intracellular ice crystal formation and preserving the structural integrity of the cell membrane. Once frozen, cells are stored in liquid nitrogen, where the extremely low temperatures halt all metabolic activity, enabling long-term storage of frozen cells for months or even years.
Cryopreservation offers significant benefits beyond simple storage. It enables researchers to bank cell lines for reproducible results across experiments, supports the sharing and transportation of valuable cell samples between laboratories, and provides a safeguard against unexpected contamination or loss of cultures. For sensitive cell types, such as stem cells and primary cells, using the right cryopreservation media and following a standardized protocol is essential for maintaining their unique properties and ensuring high post-thaw viability.
Comparison to other methods
Compared to uncontrolled freezing or storage at -80°C, this protocol offers superior cell viability and long-term stability. While some labs use passive freezing methods or store cells at suboptimal temperatures, this approach ensures consistent cooling and rapid transfer to liquid nitrogen and is generally preferred. Unlike flash freezing or cryopreservation without cryoprotectants, this method minimizes cellular damage and improves post-thaw recovery. It also accommodates different cell types and media formulations, making it more versatile.
Applications
This cryopreservation protocol is widely applicable in biomedical research, biotechnology, and pharmaceutical development. It supports long-term storage of cell lines used in drug screening, genetic studies, and cell-based assays. Researchers can bank primary cells, immortalized lines, or genetically modified cells for future use, ensuring reproducibility across experiments. The method is also valuable for maintaining backup stocks, sharing cell lines between labs, and preserving rare or sensitive cell types. Its adaptability to different media and cell types makes it suitable for academic, clinical, and industrial settings.
Limitations
While effective, this protocol has limitations. DMSO, a common cryoprotectant, may not be suitable for all cell types due to toxicity. Glycerol can be used as an alternative, but requires specific handling. The protocol assumes access to controlled-rate freezing equipment and liquid nitrogen storage, which may not be available in all labs. Additionally, cells stored at -80°C for extended periods may suffer reduced viability. The method also requires careful timing and aseptic technique to prevent contamination. Variability in cell response to freezing may necessitate optimization for specific lines.
Troubleshooting
If post-thaw viability is low, check that cells were frozen at optimal density and viability (>75%). Ensure freezing media was properly prepared and warmed to 37°C before use. Avoid leaving cells in freezing media at room temperature for more than 10 minutes. If using DMSO, confirm it is compatible with your cell type; switch to glycerol if needed. Inconsistent cooling rates can damage cells; verify that the CoolCell® or equivalent device is functioning correctly. Contamination or poor labeling can also affect outcomes, so maintain aseptic conditions and detailed records throughout the process.