A major function of the carrier solution in addition to maintaining viability is to control cell volume. The principles of preparing carrier solutions to accomplish this in the presence and absence
of cryoprotectants are described in the next section.
3.4 Osmosis,
Osmotic Limits,
and Osmotic Protocols
Osmosis is the movement of water from a region of high water
concentration or vapor pressure to an area of low water concentration or vapor pressure. The importance of osmosis is particularly
high for vitrification protocols because of the much higher concentrations of cryoprotectants required in comparison to freezing
protocols, which necessarily reduce water concentration greatly,
even to the borderline of compatibility with life. Cavalier use of
both pCPAs and npCPAs without adequate avoidance of osmotic
shifts is a frequent preventable cause of injury associated with the
use of vitrifiable concentrations of cryoprotectants. More detailed
descriptions of osmotic effects during the introduction and removal
of cryoprotectants are available in many publications (e.g.,
[156, 161, 286, 287]), but here we will simply describe the basic
phenomena of relevance.
The rate of water movement across the cell membrane depends
on the transmembrane difference in water vapor pressure, or osmolality, without regard to the nature of the solutes whose presence
generates the transmembrane vapor pressure gradient. For pCPAs,
the effect of adding the pCPA on the transmembrane osmotic
gradient is transient. Initially, since water moves more rapidly
than the pCPA, extracellular pCPA raises extracellular osmolality
more than intracellular osmolality, and the cell loses water in
response. Later, as the pCPA enters the cell down its own transmembrane concentration gradient, water diffuses back into the cell
to maintain osmotic equilibrium. This sequence of events is often
referred to as the “shrink-swell” process.
The end result of this process depends on the carrier solution
[286]. When the pCPA concentration is (nominally) the same, per
unit liquid volume, on both sides of the membrane, the cell will
have returned to its original volume provided the osmotic effect of
the carrier solution, which was equal to the osmotic effect of
intracellular molecules before CPA addition, is also the same, per
unit solution volume, as it was prior to pCPA addition. Although
cells contain proteins and many other complex molecules whose
osmotic coefficient might be expected to differ from the osmotic
coefficient of the carrier solution, in practice, this difference is
small, and the cell can reasonably be modeled, to a first approximation, as a dilute salt solution having the osmolality of plasma
(~285 mOsm) [157, 161, 263]. If the osmotic coefficient of the
carrier is about the same as the lumped osmotic coefficient of
cytosolic solutes, then for the cell to return to its original volume,
the carrier must have the same concentration per unit volume of
Principles of Vitrification
61
of cryoprotectants are described in the next section.
3.4 Osmosis,
Osmotic Limits,
and Osmotic Protocols
Osmosis is the movement of water from a region of high water
concentration or vapor pressure to an area of low water concentration or vapor pressure. The importance of osmosis is particularly
high for vitrification protocols because of the much higher concentrations of cryoprotectants required in comparison to freezing
protocols, which necessarily reduce water concentration greatly,
even to the borderline of compatibility with life. Cavalier use of
both pCPAs and npCPAs without adequate avoidance of osmotic
shifts is a frequent preventable cause of injury associated with the
use of vitrifiable concentrations of cryoprotectants. More detailed
descriptions of osmotic effects during the introduction and removal
of cryoprotectants are available in many publications (e.g.,
[156, 161, 286, 287]), but here we will simply describe the basic
phenomena of relevance.
The rate of water movement across the cell membrane depends
on the transmembrane difference in water vapor pressure, or osmolality, without regard to the nature of the solutes whose presence
generates the transmembrane vapor pressure gradient. For pCPAs,
the effect of adding the pCPA on the transmembrane osmotic
gradient is transient. Initially, since water moves more rapidly
than the pCPA, extracellular pCPA raises extracellular osmolality
more than intracellular osmolality, and the cell loses water in
response. Later, as the pCPA enters the cell down its own transmembrane concentration gradient, water diffuses back into the cell
to maintain osmotic equilibrium. This sequence of events is often
referred to as the “shrink-swell” process.
The end result of this process depends on the carrier solution
[286]. When the pCPA concentration is (nominally) the same, per
unit liquid volume, on both sides of the membrane, the cell will
have returned to its original volume provided the osmotic effect of
the carrier solution, which was equal to the osmotic effect of
intracellular molecules before CPA addition, is also the same, per
unit solution volume, as it was prior to pCPA addition. Although
cells contain proteins and many other complex molecules whose
osmotic coefficient might be expected to differ from the osmotic
coefficient of the carrier solution, in practice, this difference is
small, and the cell can reasonably be modeled, to a first approximation, as a dilute salt solution having the osmolality of plasma
(~285 mOsm) [157, 161, 263]. If the osmotic coefficient of the
carrier is about the same as the lumped osmotic coefficient of
cytosolic solutes, then for the cell to return to its original volume,
the carrier must have the same concentration per unit volume of
Principles of Vitrification
61