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. 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. 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. 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. Freeze Concentration. 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.
Oxidative stress. Another common problem seen as a result of multiple freeze-thaw cycles is oxidative stress, which may be generated through different mechanisms.
Scientists have found that during the fall, wood frogs accumulate urea, and later glucose, to preserve their organs when the frogs freeze solid during the winter Water can make its way through a cell membrane unaided through the process of osmosis, but a quicker way into or out of a cell is through an aquaporin—a membrane protein that regulates the flow of water into and out of cells 5. Scientists have found that aquaporins help some freeze-tolerant frogs move not only water but glycerol into cells in preparation for freezing.
Aquaporins also help freeze-avoiding insects move water out of cells during cryoprotective dehydration. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration.
The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage.
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