Optimizing Cryopreservation Protocols

Cryopreservation, a technique that uses low temperatures to stabilize biological materials, has utility for applications spanning basic research to personalized medicine. Here, we take a look at what’s involved and share some cryopreservation best practices.

The only viable long-term preservation technique

Samples requiring cryopreservation include cell lines, primary cells, and solid tissue samples; semen, oocytes, and embryos; and apheresis-derived material such as that used for stem cell or T cell therapies. Consequently, the methods involved can vary, along with the degree of flexibility.

“In academic settings, cells can usually be allowed to recover upon thawing by being grown in culture,” reports Peter Kilbride, Ph.D., Senior Research Scientist, Cryobiology at Cytiva. “However, in clinical settings, cell therapies are typically infused immediately after thawing, meaning cryopreservation protocols must be tightly controlled to give optimal post-thaw cell outcomes.”

Importantly, no other technique comes close to cryopreservation for storing precious sample material. “Cryopreservation is the only viable long-term preservation technique that has been successfully applied to preserve biological materials for research and therapeutic use,” reports Nilay Chakraborty, Ph.D., MBA, BioNexus Foundation Principal Scientist at ATCC. “By stopping any enzymatic or chemical activity that might damage or degrade the biomaterial in question, cryopreservation safeguards the integrity and functionality of many different sample types.”

A wealth of benefits

One of the best-known applications of cryopreservation is its use for maintaining backup stocks of cell lines for in vitro research, which is critical to prevent data from becoming skewed over time and avoid accidental cell loss. “Living cells are constantly exposed to factors that can reduce their long-term viability, cause morphological or genetic changes, or present contamination issues—and the associated risk increases each time the cells are handled,” explains Markus Markowich, Technical Support Specialist at Corning Life Sciences. “Cryopreservation mitigates these effects, as well as prevents cells from showing signs of aging and differentiation, and it also reduces the time and expense of performing daily care.”

According to Chris Creasey, Ph.D., Manager Engineering Operations, Cryopreservation at Cytiva, further advantages of cryopreservation are that it enables the safe, convenient transport of biological material and allows for storage of samples that cannot be used straight away. “These benefits can especially be important for therapeutic applications to circumvent the inherent challenges of on-demand manufacture,” he says. “Not only does cryopreservation help to ensure that a treatment is administered only when the patient and clinical team are ready to proceed, but it also offers treatment consistency by providing repeated access to the same batch of cells.”

Key protocol steps

A typical cryopreservation protocol involves several main steps, each of which requires optimization for the type of sample being handled. First, samples must be transferred into a suitable cryopreservation medium—traditionally a nutrient-rich medium containing DMSO as a cryoprotectant, which can be supplemented with serum. “Serum functions to provide a protective environment for the cells, but it should be noted that it has some drawbacks,” cautions Benjamin Theek, Ph.D., Senior Scientist at Miltenyi Biotec. “These include lot-to-lot variations that could alter the recovery rate after thawing, and the potential for inducing differentiation of stem cells or basal activation of primary cells that may lead to increased background responses in downstream functional assays.”

Next, samples must undergo controlled-rate cooling. “Cooling rate is cell type dependent, but for somatic mammalian cells a rate of around -1°C/minute is optimal, with slower rates required for larger and more complex cellular structures such as spheroids or organoids,” reports Kilbride. “Passive freezing containers that can be placed in a -80^oC freezer overnight are usually sufficient to freeze samples in standard cryovials for academic research where cells are being banked for future use, but lack of control and variability in cooling rates makes such vessels unsuitable for cell therapy work. For these types of applications, Cytiva’s VIA Freeze Controlled-Rate Freezers provide a GMP-compliant and liquid nitrogen-free solution.”

Following controlled-rate cooling, the samples must be moved to the vapor phase of liquid nitrogen (196^oC) for long-term storage. Here, Markowich stresses the importance of recording detailed information in a shared repository. “For cells, this should include the culture identity, passage number, date frozen, freezing medium and method used, and the number of cells per vial,” he says. “It is also important to record the total number of vials initially frozen, and their locations, and to update these values each time a vial is removed. Other useful information covers the expected viability, the results of any quality control tests—such as sterility, mycoplasma, and karyotype—and details of cell-specific growth parameters or characteristics.”

A typical cryopreservation protocol for an adherent mammalian cell line

1. Check the cells using a microscope; when they are approximately 85–95% confluent, aspirate the old culture medium using proper aseptic technique

2. Carefully rinse the cell monolayer with calcium- and magnesium-free PBS

3. Add the pre-warmed cell dissociation reagent (e.g., trypsin) to the culture vessel and allow the cells to incubate at 37^oC for at least 1 minute, depending on cell type

4. Add fresh culture medium to the vessel to inactivate the proteolytic enzyme reaction within the cell dissociation reagent

5. Transfer the cells to a 15 mL centrifuge tube placed on ice

6. Take a sample for counting from the centrifuge tube then spin at 100 x g for 5 minutes to obtain a cell pellet using a centrifuge; while the cells are spinning, calculate the total cell number and viability (e.g., using Trypan Blue)

7. Following centrifugation, discard the supernatant and resuspend the cell pellet at a density of 4 x 10⁶ –1 x 10⁷ cells/mL in pre-chilled freezing medium

8. Aliquot the cells into labelled cryovials

9. Place the vials into a freezing container at -80^oC overnight

10. Transfer the vials to the vapor phase of liquid nitrogen for long-term storage, remembering to update the cell repository records accordingly

Information provided by Corning Life Sciences

Best practice recommendations

Cryopreservation must be carefully optimized on a sample-specific basis. For cryopreserving cultured mammalian cells, the following suggestions (based on the combined feedback of all five contributors to this editorial) are recommended:

• Make sure cultured cells are in log phase growth, at 85–95% confluency, and exhibit high viabilities
• Remember to test for contaminants such as bacteria, yeast, and mycoplasma; using a separate liquid nitrogen tank for quarantine purposes will help prevent the spread of contamination while waiting for test results to be returned
• Freeze cells at a density of 4 x 10⁶ – 1 x 10⁷ cells/mL to preserve viabilities
• Ensure the freezing medium has been pre-chilled; consider using a freezing medium that is optimized for your particular cell type
• Use a suitable container to ensure controlled-rate freezing at a rate of -1°C/minute; Corning’s CoolCell® container is an alcohol-free freezing container that avoids the problem of alcohol degradation over time
• Transfer cells to the -80^oC freezer as quickly as possible; cryoprotectants such as DMSO can be cytotoxic if left at room temperature for extended periods
• Avoid prolonged storage at -80^oC; this can compromise viabilities
• Always place vials in the vapor phase of liquid nitrogen; placing vials in the liquid phase risks contamination and could cause vials to explode upon thawing

When working with other sample types, cryopreservation protocols will need to be optimized accordingly. For example, when cryopreserving tissue samples, Theek recommends cutting these into small pieces and allowing them to settle in the freezing medium for a few minutes before transferring them to the -80^oC freezer. “This will increase the surface area exposed to the freezing medium for improved penetration into the tissue,” he says. “In this situation, and also when working with primary mammalian cells, it can be worth using a specialized freezing medium such as MACS^® Freezing Solution to better preserve the sample and avoid the use of animal components which can impact cellular function and complicate downstream analyses.”

Future perspectives

While established best practices in cryopreservation center on optimizing post-thaw viability, this is just one measure of cryopreservation success. “At ATCC, we are working on cryopreservation solutions that not only focus on cellular viability but also use advanced transcriptomics, proteomics, and metabolomics technologies for post-thaw cellular functional characterization,” reports Chakraborty. “Through these efforts, we aim to address customer pain points related to post-thaw recovery times, viability, and reliability of functional responses, which will be critical to inform the development of next generation cryopreservation techniques.”