5 Water and Solute Transport Across Cellular Membranes and Loading Cells
and Tissues with Protective Agents
5.1 Water Transport
Across Cellular
Membranes
Membrane permeability to water (i.e., hydraulic permeability or
Lp) is dependent on cell-specific properties including the membrane lipid composition. The rate at which water can pass or permeate the membrane determines the cell volume response when
exposed to molar concentrations of CPAs. Furthermore, Lp at
subzero temperatures determines the rate and extent of cellular
dehydration during freezing. Freezing-induced dehydration actually results in removal of water from the phospholipid head groups
and hence induces a sharp membrane fluid-to-gel-phase transition
[45, 56] (see Fig. 3a, b). Increased lipid packing in the presence of
ice causes water molecules to encounter a more hydrophobic environment when crossing the membrane. The change in membrane
phase state that is caused by ice formation affects Lp and the
Arrhenius behavior of Lp (i.e., the activation energy of water transport, E Lp ) (see Fig. 3c). Addition of CPAs does not prevent occurrence of the freezing-induced membrane phase transition. CPAs
counteract the effects of ice on water membrane permeability by
increasing Lp at a given temperature and decreasing E Lp . The
increased Lp in the presence of CPAs thus facilitates cellular dehydration to continue down to low subzero temperatures, which in
turn decreases the incidence of intracellular freezing.
At suprazero temperatures, Lp can be derived by analyzing cell
volume responses in anisotonic media, acquired microscopically or
via electronic particle sizing measurements [57, 58]. Assessment of
cell volume responses at subzero temperatures requires a special
cryomicroscope [26], but also other methods can be used to determine Lp under freezing conditions including differential scanning
calorimetry (DSC) [59] and Fourier transform infrared spectroscopy (FTIR) [45, 46, 56]. Subzero Lp measurements can be used
to extrapolate Lpg, i.e., Lp at a reference temperature of 0
C. Lpg
and the activation energy for water transport, E Lp , can be used to
model cell volume responses during freezing and predict optimal
cooling rates for cryopreservation [18, 59].
5.2 Loading Cells
with CPAs
In preparation for cryopreservation, the first step typically concerns
loading cells with CPAs. Permeating CPAs like ethylene glycol,
DMSO and propylene glycol can easily pass cellular membranes.
Transferring cells into a solution containing molar concentrations
of CPAs, however, needs to be done carefully since this creates an
osmotic gradient causing cells to respond via mass transport and
volume changes. Water can pass the cellular membrane faster as
permeating CPAs (see Fig. 4b) causing water to initially move out of
the cells. Thereafter, both the CPAs and water move into the cell
until equilibrium is reached between the extra- and intracellular
12
Willem F. Wolkers and Harrie ¨ tte Oldenhof
and Tissues with Protective Agents
5.1 Water Transport
Across Cellular
Membranes
Membrane permeability to water (i.e., hydraulic permeability or
Lp) is dependent on cell-specific properties including the membrane lipid composition. The rate at which water can pass or permeate the membrane determines the cell volume response when
exposed to molar concentrations of CPAs. Furthermore, Lp at
subzero temperatures determines the rate and extent of cellular
dehydration during freezing. Freezing-induced dehydration actually results in removal of water from the phospholipid head groups
and hence induces a sharp membrane fluid-to-gel-phase transition
[45, 56] (see Fig. 3a, b). Increased lipid packing in the presence of
ice causes water molecules to encounter a more hydrophobic environment when crossing the membrane. The change in membrane
phase state that is caused by ice formation affects Lp and the
Arrhenius behavior of Lp (i.e., the activation energy of water transport, E Lp ) (see Fig. 3c). Addition of CPAs does not prevent occurrence of the freezing-induced membrane phase transition. CPAs
counteract the effects of ice on water membrane permeability by
increasing Lp at a given temperature and decreasing E Lp . The
increased Lp in the presence of CPAs thus facilitates cellular dehydration to continue down to low subzero temperatures, which in
turn decreases the incidence of intracellular freezing.
At suprazero temperatures, Lp can be derived by analyzing cell
volume responses in anisotonic media, acquired microscopically or
via electronic particle sizing measurements [57, 58]. Assessment of
cell volume responses at subzero temperatures requires a special
cryomicroscope [26], but also other methods can be used to determine Lp under freezing conditions including differential scanning
calorimetry (DSC) [59] and Fourier transform infrared spectroscopy (FTIR) [45, 46, 56]. Subzero Lp measurements can be used
to extrapolate Lpg, i.e., Lp at a reference temperature of 0
C. Lpg
and the activation energy for water transport, E Lp , can be used to
model cell volume responses during freezing and predict optimal
cooling rates for cryopreservation [18, 59].
5.2 Loading Cells
with CPAs
In preparation for cryopreservation, the first step typically concerns
loading cells with CPAs. Permeating CPAs like ethylene glycol,
DMSO and propylene glycol can easily pass cellular membranes.
Transferring cells into a solution containing molar concentrations
of CPAs, however, needs to be done carefully since this creates an
osmotic gradient causing cells to respond via mass transport and
volume changes. Water can pass the cellular membrane faster as
permeating CPAs (see Fig. 4b) causing water to initially move out of
the cells. Thereafter, both the CPAs and water move into the cell
until equilibrium is reached between the extra- and intracellular
12
Willem F. Wolkers and Harrie ¨ tte Oldenhof