#7 Common mistakes in cryopreservation

Despite your care and attention to your sample, cells can sometimes show poor recovery or unsatisfactory viability after freezing. If you're struggling to improve your results, explore our quick troubleshooting guide to cryopreservation. It’s designed to help you pinpoint common issues and offer practical, solutions.

#1 Check cell growth phase and viability before freezing

It is good practice to check your cell viability before freezing. Ensure your cells have high viability and are in the log phase of growth before proceeding with cryopreservation. Freezing cells that have entered the plateau phase carries the risk that apoptosis may have begun, leading to difficulties in resuscitation and reduced post-thaw viability. You can use CytoSMART™ to streamline the process of determining cell viability.

#2 Use a freezing medium free of serum and animal components

Serum is used to lessen ice crystal formation and at the same time to supply the cells with nutrients during the thawing process. This so thawing is as low-risk as possible and the cell can regenerate optimally. Depending on the origin of the animal or due to impurities, conventional serum can severely impair quality and is therefore not optimally reproducible. For ethical reasons, the use of a serum-free medium is also recommended. FREEZin1 has animal component-free composition, and has excellent performance in terms of surface adhesion, cell survival rate and cell proliferation after thawing.

#3 Do not fill sample tubes to capacity

Due to the expansion of liquids during freezing, a sample tube can burst if overfilled. To prevent this, you should never fill the tube more than 2/3 or up to the upper graduation mark. This ensures that the samples can be frozen safely. Need bigger cryogenic tubes? Check them out here.

#4 Exercise caution with freezer boxes that use alcohols

Cryoboxes using alcohols such as isopropanol to guarantee the -1 °C cooling rate, can lose effectiveness over time as the alcohol can become contaminated with water, compromising its cooling/isolating performance. To maintain optimal freezing, regular replacement of e.g. isopropanol is essential.

If you want to avoid this hassle, we recommend the CellCamper freezer boxes! They are build using a thermally conductive alloy and highly insulating outer materials that ensure a uniform heat dissipation from each individual sample, providing constant and reproducible freezing conditions.

#5 Store your samples correctly

Cell are often kept at -80 °C, however storing them long-term at this temperature is not recommended. At -80 C a decline in viability can be observed after a few months. This decline is dependent on cell type, exposure to thermal cycling, and transient warming events such as repeated opening of the freezer door. If possible, store your samples between -150 and -196 °C, this ensures viability for many years. For this, choose to store your samples in either a cryogenic freezer or liquid nitrogen tanks.

#6 Avoid storing cells in the liquid phase of nitrogen

Storing samples in a liquid nitrogen tank is a reliable method, but certain precautions are necessary. Avoid submerging vials in the liquid phase due to the risk of cross-contamination between samples. If you choose to store your vials in the liquid phase, be aware that improperly sealed vials may trap nitrogen, posing an explosion hazard during thawing.

Generally, storage in the gas phase is preferred, as it reduces these risks. However, this approach requires strict monitoring of nitrogen levels to maintain a stable temperature, in the higher portion of the tank, making it more labour-intensive. Tanks equipped with monitoring of temperature and level indicators, can help ensuring the integrity of your samples.

#7 Minimize freeze-thawing cycles

Cells are highly sensitive to thawing and freezing cycles. Instead of freezing them in large batches, divide your cells into small, ready-to-use aliquots that can be thawed as needed. To keep track of your samples and ensure you know which aliquots are available, use inventory systems like Fluics Connect or 1D barcodes.