Towards Better Cryoprotectants, With an Eye on Thawing

Below find an interesting discussion of one research program aimed at producing a better class of cryoprotectant that enables tissue thawing without damage. The organ storage and cryonics industries have many of the same technical goals: how to preserve complex tissues for the long term at low temperatures while enabling a safe thaw at the far side of storage. Some research companies straddle both industries, such as 21st Century Medicine. The enemy here is ice, as it is crystallization that destroys cells and structures in straight freezing. If near-future thawing is not a concern, then many varieties of cryoprotectant compounds are useful. When infused into tissue the result is vitrification rather than freezing, with minimal ice crystal formation and preservation of even very fine-scale cellular structures, such as synapses and other aspects of brain structure thought to hold the data of the mind.

To date there are very few examples of the successful thaw and use of a vitrified organ, even in the laboratory. It is research programs such as the one noted here that may help to change this state of affairs. Given better cryoprotectants and significant use of long-term organ storage in medicine, one would hope that the public will become more accepting of cryonics as an end of life choice, a shot at living again to see a better future for those who will age to death prior to the advent of rejuvenation therapies.

Researchers have synthesised a polymer that limits ice crystal growth in frozen red blood cells as they thaw. The polymer is set to pave the way for similar synthetic structures that mimic the properties of natural antifreeze proteins. During cryopreservation, cells and tissues are stored at sub-zero temperatures and thawed before use. However, frozen cells can be damaged as they defrost. When ice melts, it can refreeze into larger crystals that puncture cells from the outside. This process, called recrystallisation, is especially damaging for organs and blood bags, which defrost over a long time. "'If you directly freeze cells they don't survive due to ice-induced damage, and the traditional solution is to add antifreeze solvents. Although these work, they involve complex preparation procedures, and transfusing large volumes of solvent is not desirable. Alternatives to the conventional cryoprotectants are urgently required as the fields of regenerative medicine and tissue engineering continue to advance."

Unlike proteins, which need to be extracted or expressed in microorganisms, polymers are more accessible, processable, tunable and cheaper. Researchers modified an already available polymer called a polyampholyte, which is composed of monomers with both positively and negatively charged groups. The polymer functions outside the cells, so it can be washed-off after thawing. This may explain its good compatibility with red blood cells. Up to 60% red blood cell recovery after freezing was observed during slow thawing when the new polymer was used, and this increased to 80% when the cells were thawed quickly. Notably, the polymer was capable of inhibiting ice recrystallisation by 50%.

The mechanism by which the synthesised polymer inhibits ice recrystallisation is still not clear. Although it has been assumed for many years that macromolecules had to bind directly to ice crystal faces to inhibit growth, their work supports the idea that binding to ice crystals is not essential. "It seems that they somehow disrupt the rate exchange of water molecules between ice crystals, via the quasi liquid layer, although we do not have direct evidence for this at the moment. As to why the ampholyte structure works, we are not sure, but we are thinking that it might be a semi-rigid polymer due to charged interactions along the backbone, which helps. Cheap, non-toxic, degradable polymers that inhibit ice recrystallisation may become attractive non-permeating additives for cryopreservation of red blood cells if these boost cell recovery by more than 80% and allow for prolonged post-thaw storage."



80% of the cells survived. Only 20% died. Seems like this technology is a long way from being practical.

Posted by: Jim at July 29th, 2015 3:49 PM

Agreed Jim. Besides it is about blood cells. I guess this ratio will drop in soft tissues, where cryoprotectant hardly reaches every cell.

Posted by: Martin S. at July 30th, 2015 1:07 AM

While getting good cryoprotectants is important, I hope there's as much research going on with the other aspects of cryopreservation, namely, methods to better infuse all important tissues with cryoprotectants, and to defrost tissue in better/faster ways that don't cause as much damage. These things can be just as important.

I am curious what an "acceptable" percentage of cell destruction would be that would still allow the organism to survive thawing. IE Humans often lose some brain tissue in strokes but can still survive and recover.

Posted by: KC at August 11th, 2015 4:18 PM
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