How Do You Move Your -80ºC Samples to LN2 Storage?
November 23, 2015
We have focused on cryogenic temperatures and workflows (below -150ºC) on this blog thus far, but what happens upstream of cryo storage?
Obviously bio-samples start fresh and are later frozen to preserve them over time. Fresh cells specifically, should never be placed directly into an LN2 vapour freezer. They are normally prepared with a cryoprotectant and then frozen in a controlled manner to minimize cell damage during freezing and subsequent thawing. Without these precise freezing protocols, post thaw recovery would be poor.
The controlled freezing may be done in an automated device or a passive device (i.e. the CoolCell®) placed in a ULT mechanical freezer, but in both these cases the freezing device is separate (and may be far, far away) from the LN2 storage freezer.
So what happens between the freezing device and the LN2 freezer? How are the samples handled and moved? There will be transient warming during any sample movement outside of a controlled environment. And what are the variables that affect warming during this move?
At ESBB in London I presented a poster that demonstrated an example workflow of moving samples at -80ºC to an LN2 freezer. During the transfer we monitored the sample temperature and some variables that increased or decreased the rate of warming.
What we found is the rate of warming at -80ºC is less than half compared to the rate of warming at -190ºC, approx 0.5ºC/sec. We also found that transferring the -80C samples into a cryobox already at LN2 temperature stopped the transient warming (for how long though, we did not test). Lastly, the rate of cooling once inside the LN2 freezer is affected by the presence of surrounding samples.
The take-away from these experiments is if you are concerned with controlled rate freezing of your samples, you should also be concerned with how you transfer these samples to the LN2 freezer, lest you get uncontrolled warming.
Best practices that I recommend after performing these experiments are:
- Minimize the time the samples are outside of a controlled environment.
- Best case is to locate or take the -80ºC environment close to the LN2 freezer. Alternatively, bring the LN2 environment to the ULT freezer (i.e. CryoPod).
- Transfer the -80ºC sample(s) quickly to an already LN2-frozen cryobox.
- Don’t leave the LN2 freezer rack outside of the freezer long, as it will warm your innocent samples.
- After placing the -80ºC samples into the LN2 freezer, do not pull/expose that rack for at least 20mins (preferably several hours) to allow the -80ºC samples to cool down to near -180ºC.
- Write up a transfer procedure and follow it. At the very least, you will ensure this unavoidable transient exposure is controlled.