Transient Warming Does Matter!
August 3, 2016
There is good understanding that colder temperature storage yields better preservation of biological material. Generally, with proper cryo preservatives, freezing and thawing protocols, what you put into a LN2 vapour freezer is what you will get back out – including high recovery, viability and functionality of your cells.
But what if you do everything right and still have poor recovery post thaw? Many experts have pointed to the concern of transient warming – that brief time all innocent vials are exposed while the rack is out of the freezer. Even though just 1 vial may be retrieved, 1,000+ others are exposed to 200°C+ warmer temperatures, possibly daily/weekly over months or years of storage.
What is the effect on the cells due to this transient warming?
We have already published data on the rate of warming of different individual vials, vials in cryoboxes and even that vials in cryoboxes still warm when back in the freezer! But we did not have any (and could not find any published) about the effect of transient warming on cells in a typical LN2 freezer workflow (ie. exposed few times a week, always allowed time to re-equilibrate).
We partnered up with Cook Regentec who are focused on developing research and clinical tools for Cell Therapy. They sourced and cryo preserved mesenchymal stem cells and shipped to us in LN2 dry shippers. We divided them into four batches and stored two of the batches in a -190°C LN2 vapour freezer. One batch was left in the freezer and never touched, the other was pulled up in the rack 20 times over 67 days for just long enough to allow the cells to cross Tg (glass transition). After the 67 days we sent back three samples from each batch to Cook Regentec who performed viability and recovery testing. The results were surprising…
Comparing the control batch (never warmed to Tg) vs. the variable batch (warmed above Tg), the variable batch’s viability was only about 6% lower, but its recovery was approximately 50% lower. This means that approximately 50% of the cells exposed to transient warming above Tg failed to live and even be detectable after thawing/washing.
This study is ongoing and we plan to pull more samples and run the same tests after one year of storage.
One thing is clear thus far, no matter how you store and retrieve your samples from LN2 storage (automated or manual), be sure all exposures are quick and controlled enough to avoid crossing Tg.