What factors can impact your cryopreservation quality?

by Lana Mogilyanskiy, Manager, Cell Banking Services

Proper cryopreservation of cells is an extremely important part of cell bank preparation. The quality of cryopreservation depends on many factors with the three most important being: 1) the biology of the cell line being frozen, 2) the freeze media used, and 3) the method of freezing and the freeze profile that are used. Cell bank size (e.g. a 20 vial bank vs a 500 vial bank) can also have a significant impact on cryopreservation quality as the size change may result in significant vial to vial variability due to temperature fluctuations, handling nuances and difference in timing with a small vs a larger bank.

Certain cell lines freeze well in general regardless of what point in the growth curve the freezing process occurs, what freeze media or what freeze profile is used. These cells have a high viability when thawed (≥90%) with viability remaining high through the period of recovery (the first few days after the cells are thawed). These cell lines typically demonstrate minimal vial-to-vial variability regardless of bank size. However, not all cell lines are created equal and if thawing of cells after cryopreservation results in poor viability and recovery, an alternative freeze profile can sometimes improve the quality of the bank. Controlled rate freezers are typically used for the production of cell banks in a GMP environment. Freezing of the cells is controlled by two probes – one probe monitors the temperature of the chamber within the controlled rate freezer and a second probe is used to monitor an actual vial of cells. Controlled rate freezers usually come with the manufacturer’s recommended freeze profile, and this profile typically works on the majority of cell lines, but freezing parameters such as the speed and timing of each freezing phase can be modified to improve cryopreservation quality for the particular cell line of interest. Sometimes a standard 1 degree C/minute freeze (“Mr. Frosty”) provides better results than a more sophisticated freeze profile with multiple cycles, including accommodation for the heat of fusion.

Often our clients have a particular freeze profile that they have worked out and want us to use for their cell bank. If there are any doubts about that profile, we will evaluate it in a mock freeze study prior to actual bank preparation. During this process, we may see red flags that need to be discussed with the client. For example, we had a situation when the probe inserted into the vial showed that the heat of fusion was bringing the sample dangerously close to 0 degrees C. The freeze profile was modified and the bank was frozen successfully.

We recommend that mock freezing studies be performed to determine whether cell bank viability and recovery will be as expected. If we observe poor cell viability during the post-freeze thaw, inadequate recovery, or significant variability between vials, an attempt should be made to address this issue.