Freeze-thaw—you know it’s bad for your samples, don’t you? While working in the lab, you have most likely heard someone say ‘aliquot your protein/cells/DNA/RNA to avoid too many freeze-thaw cycles.’ But do you actually understand why?
You probably thought that avoiding freeze-thaw cycles had something to do with damaging cell structure as well as proteins or DNA/RNA—and you would be right. But you might be surprised to know that freeze-thaw cycles can damage your samples in several other ways, and we don’t quite understand how all of them work.
Damaging your samples during freeze-thaw cycles can cause problems with downstream processes. For example, multiple rounds of freezing and thawing can damage protein structures, which can interfere with study protein kinetics using surface plasmon resonance. Even minor DNA damage can result in uninterpretable data from PCR.
Your samples are not the only concern when it comes to freeze-thaw cycles. At the moment, a lot of research is going into the cryopreservation of embryos and gametes. Humans aren’t the only ones using assisted reproductive technology. Animal husbandry professionals, zoologists, and others are using assisted reproductive technology to increase farming production or aid in the preservation of endangered species. Many studies have shown that the freeze-thaw process can affect DNA integrity in sperm and also hinder embryo development.
Through this research and years of experimentation in the lab, we’ve learned that a variety of factors are responsible for damage caused by freeze-thaw cycles.
Different Mechanisms Cause Instability During Freeze-Thaw Cycles
Ice crystals that are formed during the freeze-thaw process can cause cell membranes to rupture. Rapid freezing results in ice crystal formation in the outer parts of cells, which causes the interior of the cells to expand, pushing against the plasma membrane until the cell bursts. While slow cooling allows water to leach out and reduce ice crystal formation, slow cooling still leads to cell rupture due to an imbalance in osmotic pressure. If you are freezing live cells or microorganisms, both of these processes can greatly decrease viability.
In addition to mechanically damaging cells, ice crystals can also cause the salts and proteins in the buffer to become concentrated. This problem is known as freeze concentration and can cause significant stress on the stability of proteins. Although the exact mechanism of ice-induced protein denaturation is not fully understood we do know that changes in the physical environment of the protein lead to stresses that can impact stability. For example, freeze concentration has been shown to cause protein unfolding at the ice:aqueous interface for several proteins, including, azurin, liver alcohol dehydrogenase and alkaline phosphatase.
Another common problem seen as a result of multiple freeze-thaw cycles is oxidative stress, which may be generated through different mechanisms. Ice crystal-induced damage to organelle structures could lead to activation of rescue systems that are associated with energy generation. This results in a subsequent increase in oxidative stress and production of reactive oxygen species (ROS, free radicals produced as by-products of reduction-oxidation, or redox, reactions). When the balance between ROS and antioxidants is lost, oxidative stress results in molecular damage to DNA, proteins, and lipids in the cell. Some studies have shown that thawed cells contain an increase in phosphorylated H2AX,a marker of double strand breaks in DNA.
Tips to Minimize the Damage Caused by Freeze-Thaw Cycles
There are two main ways to avoid the changes seen after freeze-thaw cycles:
Don’t do it. The easiest and most obvious solution is to prevent freeze-thaw cycles. As I said at the very beginning, one of the best ways to avoid multiple freeze-thaw cycles is to aliquot everything – your samples, your antibodies, your cells, and anything else you can think of.
Use cryoprotectants. In addition to aliquoting, add a cryprotectant to your cells/samples/etc. to help prevent the stresses caused by freezing.
Cryoprotectants, which are an important addition to samples on their freezing journey, were first discovered in the UK by Christopher Polge in 1949. He inadvertently supplemented an experimental freezing solution with glycerol, resulting in the unexpected survival of his experimentally frozen cells. I’m sure you’ve all used something in the lab that has had glycerol added for this purpose (e.g., antibodies or RNase). Even the antifreeze for your car has glycerol added.
There are two main classes of cryoprotectants:
Intracellular agents.These agents penetrate the cell to prevent the formation of ice crystals and, thereby, membrane rupture. There are several common reagents, including dimethylsulfoxide (DMSO), glycerol, and ethylene glycol. The most common agent used in the lab is DMSO, which provides a high rate of cell survival. However, some groups have shown that it can promote stem cell differentiation in neuronal cells. Also, DMSO can be cytotoxic at room temperature. Even though DMSO may have some drawbacks, it works well enough for most applications.
Extracellular agents. These agents do not penetrate the cell membrane but act by reducing the hyperosmotic effect in the freezing procedure. Common extracellular agents include sucrose, dextrose, and polyvinylpyrrolidone. Cells preserved in extracellular agents (e.g., sucrose) tend to have a lower viability after thawing than DMSO-preserved cells. This may be because extracellular agents don’t prevent ice crystal formation.
Whatever method you use, you should always be mindful of the changes you are causing in your samples during freezing and thawing. Especially those that cannot be seen—they could affect your results.