VIABILI‘TY or FREEZE—DRŒD ORGANISMS
97
quite slow at 1 °C per minute. Thawing had to be as rapid as possible, and for stability
of the cell after thawing, the glycerol had to be removed slowly by dialysis.
A good account of Lovelock’s work appears in an article by Smith (1954) in which
states : “The dominant lethal factor during freezing and thawing appears, at the present
time, to be the increasing concentration of electrolytes.”
If a sodium chloride solution is frozen, at a temperature just below 0 °C pure water
freezes, thus concentrating the electrolyte to what is known as the eutectic concentration
with a freezing point at——— 21.6 °C. If glycerol were added to this electrolyte, depending
'
on the concentration, rst the point at which pure ice started to crystallise from the solution
would be lowered, but secondly the amount of ice would be reduced so that the salt would
never reach its eutectic concentration.
This can be shown by thermal analysis (see paper
by Davies in this book), but thermal analysis and resistivity measurements also show that
instead of the sudden freezing of eutectic mixtures in the absence of glycerol, there is a
slow hardening of a glass—like mixture in the presence of glycerol. Thus, the red cells
are suspended in a solution of glycerol and not too concentrated salt, which slowly hardens
without crystallisation.
If this ‘glass’ is formed too rapidly it may well be metastable
with a tendency to crystallise particularly on warming, and this may well account for the
superior results which follow a slow freeze.
Lovelock also showed that the protective action of glycerol was connected with the fact
that the glycerol rapidly penetrated to the inside of the cell. If the cell membrane was
made impermeable to glycerol by treating with copper, then the glycerol protective e‘ect
was lost.
Glycerol also failed to protect sheep red cells, whose membranes were impermeable to it, but the discovery that dimethyl sulphoxide (DMSO) would penetrate these
membranes and also had a greater protective effect than glycerol, solved this problem.
Since the discovery of glycerol and the more recent discovery of DMSO, it has been found
that many sugars and polymers such as polyvinylpyrrolidone (P.. V. P.) have some protective e‘ect, though as many of these substances do not penetrate the cells, the freezing
rates are much more critical.
Thermal analysis and resistivity measurement (Davies, this
book) have shown that all these substances have the same property as glycerol and DMSO
in preventing salt concentration and forming glass-like substances on freezing. But if the
additive will not enter the cell, salt concentration within the cell on freezing can only be
avoided by the diffusion of the salt into the less concentrated extracellular uid. This
must require time, and yet the longer time allowed for diffusion the longer the cell interior
will be subjected to high concentration. We would therefore expect that with an extra—
cellular additive the speed of freezing would be very critical and that the degree of protection would not be as good as that resulting from the use of an intracellular additive.
In a classical experiment, Meryman and Kag (1955) sprayed blood onto the surface
of liquid nitrogen. The spherical shape of the droplets meant that the rate of freezing
varied throughout the diameter of the drop so that there was a probability that some of
the cells would be subjected to the correct freezing rate. In fact, a
proportion,
in the region of 90 %, could be recovered intact.
More recently, Rinfret (1964) has adapted the Meryman technique for the freeze—preservation of large volumes of blood using extracellular additive. The most successful add1trves
have been P. V. P. and mixtures of glucose and lactose. By the use of suitably shaped
containers, the coating of the containers with insulators, controlled rate shaking
in the
liquid nitrogen bath and controlled rate shaking in a warm water bath for thawmg,
surv1val
rates better than 96 % have been achieved with volumes as great as one pint.
97
quite slow at 1 °C per minute. Thawing had to be as rapid as possible, and for stability
of the cell after thawing, the glycerol had to be removed slowly by dialysis.
A good account of Lovelock’s work appears in an article by Smith (1954) in which
states : “The dominant lethal factor during freezing and thawing appears, at the present
time, to be the increasing concentration of electrolytes.”
If a sodium chloride solution is frozen, at a temperature just below 0 °C pure water
freezes, thus concentrating the electrolyte to what is known as the eutectic concentration
with a freezing point at——— 21.6 °C. If glycerol were added to this electrolyte, depending
'
on the concentration, rst the point at which pure ice started to crystallise from the solution
would be lowered, but secondly the amount of ice would be reduced so that the salt would
never reach its eutectic concentration.
This can be shown by thermal analysis (see paper
by Davies in this book), but thermal analysis and resistivity measurements also show that
instead of the sudden freezing of eutectic mixtures in the absence of glycerol, there is a
slow hardening of a glass—like mixture in the presence of glycerol. Thus, the red cells
are suspended in a solution of glycerol and not too concentrated salt, which slowly hardens
without crystallisation.
If this ‘glass’ is formed too rapidly it may well be metastable
with a tendency to crystallise particularly on warming, and this may well account for the
superior results which follow a slow freeze.
Lovelock also showed that the protective action of glycerol was connected with the fact
that the glycerol rapidly penetrated to the inside of the cell. If the cell membrane was
made impermeable to glycerol by treating with copper, then the glycerol protective e‘ect
was lost.
Glycerol also failed to protect sheep red cells, whose membranes were impermeable to it, but the discovery that dimethyl sulphoxide (DMSO) would penetrate these
membranes and also had a greater protective effect than glycerol, solved this problem.
Since the discovery of glycerol and the more recent discovery of DMSO, it has been found
that many sugars and polymers such as polyvinylpyrrolidone (P.. V. P.) have some protective e‘ect, though as many of these substances do not penetrate the cells, the freezing
rates are much more critical.
Thermal analysis and resistivity measurement (Davies, this
book) have shown that all these substances have the same property as glycerol and DMSO
in preventing salt concentration and forming glass-like substances on freezing. But if the
additive will not enter the cell, salt concentration within the cell on freezing can only be
avoided by the diffusion of the salt into the less concentrated extracellular uid. This
must require time, and yet the longer time allowed for diffusion the longer the cell interior
will be subjected to high concentration. We would therefore expect that with an extra—
cellular additive the speed of freezing would be very critical and that the degree of protection would not be as good as that resulting from the use of an intracellular additive.
In a classical experiment, Meryman and Kag (1955) sprayed blood onto the surface
of liquid nitrogen. The spherical shape of the droplets meant that the rate of freezing
varied throughout the diameter of the drop so that there was a probability that some of
the cells would be subjected to the correct freezing rate. In fact, a
proportion,
in the region of 90 %, could be recovered intact.
More recently, Rinfret (1964) has adapted the Meryman technique for the freeze—preservation of large volumes of blood using extracellular additive. The most successful add1trves
have been P. V. P. and mixtures of glucose and lactose. By the use of suitably shaped
containers, the coating of the containers with insulators, controlled rate shaking
in the
liquid nitrogen bath and controlled rate shaking in a warm water bath for thawmg,
surv1val
rates better than 96 % have been achieved with volumes as great as one pint.