CryoTip™: How to troubleshoot your cell cryopreservation process

Want to improve your cryopreservation process and cell health outcomes, but don’t know where to start? One way to approach the issue without wasting time, effort, or resources is to find out where your major losses are coming from before making any changes.

There are many different steps in the cryopreservation process that could be contributing to variability or poor outcomes. These steps include, but are not limited to:

– Cryopreservation media introduction
– Incubation

During controlled rate freezing protocol:
– Initial hold (step 1)
– Ice nucleation (step S2a)
– Main freezing rate (step S2)
– Rapid freezing (step S3)

– Transient warming events during storage

Time Freeze Chart

As Evia Bio Founder & CSO Dr. Allison Hubel likes to say, “Once your cells are dead, they stay dead.” This may seem like common sense, but this concept can become an effective tool in determining which steps of your cryopreservation process are introducing high rates of variability in post-thaw outcomes, or are outright killing your cells.

To find out which step is causing issues, stop the cryopreservation process after each of the steps above and assess the cells according to your usual metrics. Once you have pinpointed the issue, it’s easier to make targeted changes to improve cell health outcomes.

For example, if your issue is related to the introduction of your cryopreservation media, you do not have to perform the entire cryopreservation process before assessing cell health. Simply assess your starting cell material, introduce your cryopreservation media using your typical method, and then immediately stop to assess cell health. You can also use this strategy when changing methods for individual steps to ensure cell health is not damaged.

It’s important to remember that both the cryopreservation process and cells are complex. A single assessment method is typically not enough to obtain a full picture of cell health. We recommend looking at some combination of cell identity, viability, recovery, and functionality (attachment, differentiation potential, signaling, etc.) for a more complete assessment of cell health.

Some characteristics of the freezing process, like the formulation of the cryopreservation media itself or the container used for freezing, are involved in the entire process and may not be able to be pinpointed using this method. However, if no step within your protocol shows significant losses, it may be a sign to investigate other factors.

The thawing process is another step that could be significantly impacting the process. If thawing is a potential issue within your process, resolve this first by keeping solution introduction and freezing processes consistent and altering certain thawing parameters (e.g., thawing rate). If certain thawing rates provide consistently better outcomes, move forward using those. If changing thawing parameters doesn’t seem to have much of an effect on outcomes, or any changes result in poorer outcomes, then start to investigate some of the other steps of the process.

When manufacturing cell therapies, it’s critical to retain as many cells as possible during the cryopreservation and storage process so the resulting therapy retains its potency. This strategy can be used to pinpoint which steps in the process result in the most losses, so they can be targeted for improvement. Small, specific changes can often result in significant post-thaw improvements.

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