Scientists
have made an important step forward in their understanding of
cryoprotectants—compounds that act as natural ‘antifreeze’ to protect
drugs, food and tissues stored at sub-zero temperatures.
Researchers
from the Universities of Leeds and Illinois, and Columbia Univ. in
New York, studied a particular type of cryoprotectants known as
osmolytes. They found that small osmolyte molecules are better at
protecting proteins than larger ones.
The
findings, published in Proceedings of the National Academy of Sciences,
could help scientists develop better storage techniques for a range of
materials, including human reproductive tissue used in IVF.
Biological
systems can usually only operate within a small range of temperatures.
If they get too hot or too cold, the molecules within the system can
become damaged (denatured), which affects their structure and stops them
from functioning.
But
certain species of fish, reptiles, and amphibians can survive for months
below freezing by entering into a kind of suspended animation. They are
able to survive these extreme conditions thanks to osmolytes—small
molecules within their blood that act like antifreeze—preventing damage
to their vital organs.
These
properties have made osmolytes attractive to scientists. They are used
widely in the storage and testing of drugs and other pharmaceuticals; in
food production; and to store human tissue like egg and sperm cells at
very low temperatures (below -40 C) for a long period of time.
“If
you put something like human tissue straight in the freezer, ice
crystals start to grow in the freezing water and solutes—solid
particles dissolved in the water—get forced out into the remaining
liquid.
This
can result in unwanted high concentrations of solutes, such as salt,
which can be very damaging to the tissue,” said Dr Lorna Dougan from the
Univ. of Leeds, who led the study. “The addition of
cryoprotectants, such as glycerol, lowers the freezing temperature of
water and prevents crystallisation by producing a ‘syrupy’ semi-solid
state. The challenge is to know which cryoprotectant molecule to use and
how much of it is necessary.
“We
want to get this right so that we recover as much of the biological
material as possible after re-thawing. This has massive cost
implications, particularly for the pharmaceutical industry because at
present they lose a large proportion of their viable drug every time
they freeze it.”
Dougan
and her team tested a range of different osmolytes to find out which
ones are most effective at protecting the 3D structure of a protein.
They used an atomic force microscope to unravel a test protein in a
range of different osmolyte environments to find out which ones were
most protective. They discovered that smaller molecules, such as
glycerol, are more effective than larger ones like sorbitol and sucrose.
Dougan
said: “We’ve been able to show that if you want to really stabilise a
protein, it makes sense to use small protecting osmolytes. We hope to
use this discovery and future research to develop a simple set of rules
that will allow scientists and industry to use the best process
parameters for their system and in doing so dramatically increase the
amount of material they recover from the freeze-thaw cycle.”
Study abstract: “Probing osmolyte participation in the unfolding transition state of a protein”
