Two Essential Factors To Consider When Designing A Cryopreservation Protocol

Biobankers, along with the clinicians and research scientists who work with them, all want to maximize the viability of the cells and tissue they’re placing in cryopreservation. So, since I’m a global product manager of cold storage and liquid nitrogen cryopreservation for Thermo Fisher Scientific, I suppose it’s no surprise that I’m often asked very specific questions about preparing samples for long-term storage. 

However, if there’s anything I’ve learned from years of experience in the lab and working for Thermo, it’s that one size does NOT fit all when it comes to long-term storage at low temperatures. Every sample type has unique characteristics that require different methods of preparation for the cryopreservation process. 

Before I go into more detail, if you’re not familiar with the broader topic of cryopreservation, you may want to read Why Store Biological Samples At Low Temperatures, a post I wrote a few months back. As a quick recap: 

Cryopreservation means storing samples at very cold temperatures (usually below -130°C, the glass transition point of water). Virtually all biological activity is stopped at these temperatures, allowing samples to be preserved for months or years without much damage. But, choosing the right cryopreservation protocol is absolutely critical. Why? Because cryopreservation places an immense burden on living cells, and if you don’t optimize the process, your samples won’t be viable when they’re thawed. 

So, what’s the best way to go about designing a cryopreservation protocol? My advice is to start by considering these three essential factors: 

Cryoprotective agents (CPA) 

Choosing the right CPA helps minimize what’s called the solution effect: as ice forms in a freezing sample, solubilized chemicals are excluded and become concentrated. If toxic chemicals build up, the sample may be unviable after thawing. A carefully-selected CPA also helps prevent intracellular ice formation, one of the major causes of failed recovery. 

In other words, it’s essential to match the CPA to the type of cell you’re trying to preserve. Don’t simply rely on normal cell culture media. Instead, research the literature and learn which CPAs have been used successfully in projects similar to yours. 

Cooling Rate

This is another case of “one size does not fit all”—many scientists assume that the same rate of cooling can be applied to any sample type with equivalent results—this couldn’t be farther from the truth.

The optimal rate of cooling varies widely between sample types. For example, embryonic stem cells do best when cooled at 0.5°C/min, while red blood cells should be preserved in the range of 200-1,000°C/min. Sub-optimal cooling rates can have a negative effect on sample viability once cells are thawed. Once again, research the literature for recommendations before finalizing your protocol. 

Cooling rate and CPA optimization are critical for finding the point of maximized cell viability upon recovery. I recommend a review of Chapter 2 of Life in the Frozen State for anyone interested in reading more on this special relationship, but in short, as cooling rate increases, the solution effects of CPA decrease but the impact of intracellular ice formation (IIF) increases. Conversely, as cooling rate decreases, the impact of IIF decreases but the impact of solution effects increases. Thus at the intersection of IIF formation and solution effects lies the goal with is maximized viability post-thaw.

  . . . You may have noticed one glaring omission from this list: How do you go about actually freezing the sample? In other words, what methodology should you use for your sample type?

As it turns out, that’s a complicated topic . . . and I’ll discuss it in detail in my next blog post. I have also presented these topics and more in the Cell Culture Cafe webinar, “Avoid the Icebergs.” Check it out if you are interested in the finer points of cryopreservation.