extracellular solution as it did prior to the addition of the cryoprotectant [286].
In other words, the correct way to prepare a cryoprotectant
solution, if the goal is no volume change after pCPA equilibration,
is to add all the carrier solutes needed for a given volume, add all the
CPA needed for the same given volume, and then bring the solution to the final desired volume by the addition of water. This
method essentially replaces water, volume for volume, with
pCPA, such that the molar concentration of carrier solution solutes
is unchanged.
This is important for three reasons. The first reason is that
making vitrification solutions on a % w/w basis, which may be
meaningful for physical reasons, is not meaningful for biological
purposes, because carrier solution and other impermeant solutes
included on this basis are not readily maintained at a molar concentration that can be evaluated with respect to the effect of the
solution on the volumes of cells that may be placed into it. A
procedure for preparing solutions on a % w/w basis while still
maintaining isotonicity has been described [238] and should be
employed, with suitable modifications if a defined tonicity other
than isotonic is desired. To maintain isotonicity in the presence of X
% w/w pCPA, (a) prepare a concentrate of the carrier solution, such
as dissolving the nonaqueous components of 1 l of carrier in water
so as to obtain a final total volume of 200 ml (this would be a 5X
carrier concentrate); (b) weigh the carrier solution concentrate;
(c) add a weight of pCPA equal to the weight of the carrier times
(X/(100 À X)) (thus creating a solution that has the correct % w/w
concentration of pCPA); and (d) q.s. to 1 l with X% w/w pCPA in
water (thus creating an isotonic solution with the correct final %
w/w).
The second reason to understand the principles of isotonicity is
that it is common for cell freezing labs to add a pure cryoprotectant
to a cell culture medium prior to freezing, which dilutes the culture
medium. Since cell freezing often employs, for example, 10% v/v
Me 2 SO, the error is not very significant in that case (a 10% dilution
of the carrier solution). But when composing a much more concentrated vitrification solution, diluting the carrier by, for example,
50% would require intracellular solutes to be diluted to the same
extent to maintain osmotic equilibrium at the end of pCPA equilibration, which means a doubling of cell liquid space [161]. Not
only might this be harmful for the cell, but it will also dilute the
intracellular solutes that also contribute to vitrification tendency of
the cytoplasm [105, 202], making the cell at greater risk of IIF
during both cooling and subsequent warming. In fact, it seems
desirable to vitrify cells when they are still in the shrinkage phase
of the shrink-swell process rather than after complete pCPA equilibrium, in part to reduce exposure time to the pCPA and in part
because it is at that time that the cytoplasm is the most
62
Gregory M. Fahy and Brian Wowk
In other words, the correct way to prepare a cryoprotectant
solution, if the goal is no volume change after pCPA equilibration,
is to add all the carrier solutes needed for a given volume, add all the
CPA needed for the same given volume, and then bring the solution to the final desired volume by the addition of water. This
method essentially replaces water, volume for volume, with
pCPA, such that the molar concentration of carrier solution solutes
is unchanged.
This is important for three reasons. The first reason is that
making vitrification solutions on a % w/w basis, which may be
meaningful for physical reasons, is not meaningful for biological
purposes, because carrier solution and other impermeant solutes
included on this basis are not readily maintained at a molar concentration that can be evaluated with respect to the effect of the
solution on the volumes of cells that may be placed into it. A
procedure for preparing solutions on a % w/w basis while still
maintaining isotonicity has been described [238] and should be
employed, with suitable modifications if a defined tonicity other
than isotonic is desired. To maintain isotonicity in the presence of X
% w/w pCPA, (a) prepare a concentrate of the carrier solution, such
as dissolving the nonaqueous components of 1 l of carrier in water
so as to obtain a final total volume of 200 ml (this would be a 5X
carrier concentrate); (b) weigh the carrier solution concentrate;
(c) add a weight of pCPA equal to the weight of the carrier times
(X/(100 À X)) (thus creating a solution that has the correct % w/w
concentration of pCPA); and (d) q.s. to 1 l with X% w/w pCPA in
water (thus creating an isotonic solution with the correct final %
w/w).
The second reason to understand the principles of isotonicity is
that it is common for cell freezing labs to add a pure cryoprotectant
to a cell culture medium prior to freezing, which dilutes the culture
medium. Since cell freezing often employs, for example, 10% v/v
Me 2 SO, the error is not very significant in that case (a 10% dilution
of the carrier solution). But when composing a much more concentrated vitrification solution, diluting the carrier by, for example,
50% would require intracellular solutes to be diluted to the same
extent to maintain osmotic equilibrium at the end of pCPA equilibration, which means a doubling of cell liquid space [161]. Not
only might this be harmful for the cell, but it will also dilute the
intracellular solutes that also contribute to vitrification tendency of
the cytoplasm [105, 202], making the cell at greater risk of IIF
during both cooling and subsequent warming. In fact, it seems
desirable to vitrify cells when they are still in the shrinkage phase
of the shrink-swell process rather than after complete pCPA equilibrium, in part to reduce exposure time to the pCPA and in part
because it is at that time that the cytoplasm is the most
62
Gregory M. Fahy and Brian Wowk