With the above concepts in mind, it is possible to explore
unusual osmotic protocols that are intended to enable more rapid
cryoprotectant addition and removal without exceeding but while
taking advantage of the osmotic limits of the cell. For example, in a
scheme by Meryman [287], a cell is exposed to a concentration of
pCPA that brings the cell transiently to its hypertonic osmotic limit
(about four times isotonic) but contains a carrier solution that is at
only half of its isotonic concentration. After equilibration, the cell
swells to twice its isotonic volume (neglecting b), which then allows
the osmotic concentration of the medium to be raised by a factor of
8 in the next step rather than 4: an increase of twofold would
merely bring the cell back to its isotonic volume, so an increase of
fourfold in addition to that is needed to once again bring the cell to
its hypertonic osmotic limit (again, neglecting the b value of the
cell, which would allow the fold change to be even greater). Upon
washout, the cell is placed into a solution containing a four-times
isotonic carrier plus whatever pCPA is needed to avoid the hypotonic transient osmotic limit of the cell during swelling, after which
the cell comes to its hypertonic osmotic limit prior to the next
dilution step. The next dilution step can then reduce total extracellular osmolality by a factor of 8. In principle, the steps involved in
this scheme could be even greater, even beyond accounting for the
b value, because in practice significant permeation of the pCPA will
usually take place during the shrinkage or swelling phases of the
process, thus limiting volume extremes.
Although the above guidelines are helpful for determining the
final equilibrium state after concentration changes and boundary
conditions on volume excursions during transient shrinking or
swelling, the design of a cryoprotectant addition and removal protocol also generally requires some knowledge of the permeability of
the system to the cryoprotectants employed. The timing, magnitude, and temperature of concentration steps can be considerably
improved by using computer modeling of the shrink-swell and
swell-shrink processes under a variety of virtual conditions (e.g.,
[289–292]), and this approach is recommended whenever possible.
When this is not possible, our experience has been that a protocol in
which pCPA concentration is doubled on every addition step and
halved on every dilution step is effective in avoiding osmotic injury
during the preparation of rabbit renal cortical slices [227].
Clever computer-modeled schemes have been proposed to
exploit known permeation rates and the osmotic limits of the cell
so as to maximally accelerate CPA addition and removal
[293, 294], though most have not been tried.
The problem of introducing and removing cryoprotectants is
more difficult when the pCPA and npCPA must be administered by
perfusion. In a kidney, for example, exponential addition of pCPA is
counterproductive because lags in distribution of the CPA to the
medulla result in little benefit of the exponential addition rate,
Principles of Vitrification
65
unusual osmotic protocols that are intended to enable more rapid
cryoprotectant addition and removal without exceeding but while
taking advantage of the osmotic limits of the cell. For example, in a
scheme by Meryman [287], a cell is exposed to a concentration of
pCPA that brings the cell transiently to its hypertonic osmotic limit
(about four times isotonic) but contains a carrier solution that is at
only half of its isotonic concentration. After equilibration, the cell
swells to twice its isotonic volume (neglecting b), which then allows
the osmotic concentration of the medium to be raised by a factor of
8 in the next step rather than 4: an increase of twofold would
merely bring the cell back to its isotonic volume, so an increase of
fourfold in addition to that is needed to once again bring the cell to
its hypertonic osmotic limit (again, neglecting the b value of the
cell, which would allow the fold change to be even greater). Upon
washout, the cell is placed into a solution containing a four-times
isotonic carrier plus whatever pCPA is needed to avoid the hypotonic transient osmotic limit of the cell during swelling, after which
the cell comes to its hypertonic osmotic limit prior to the next
dilution step. The next dilution step can then reduce total extracellular osmolality by a factor of 8. In principle, the steps involved in
this scheme could be even greater, even beyond accounting for the
b value, because in practice significant permeation of the pCPA will
usually take place during the shrinkage or swelling phases of the
process, thus limiting volume extremes.
Although the above guidelines are helpful for determining the
final equilibrium state after concentration changes and boundary
conditions on volume excursions during transient shrinking or
swelling, the design of a cryoprotectant addition and removal protocol also generally requires some knowledge of the permeability of
the system to the cryoprotectants employed. The timing, magnitude, and temperature of concentration steps can be considerably
improved by using computer modeling of the shrink-swell and
swell-shrink processes under a variety of virtual conditions (e.g.,
[289–292]), and this approach is recommended whenever possible.
When this is not possible, our experience has been that a protocol in
which pCPA concentration is doubled on every addition step and
halved on every dilution step is effective in avoiding osmotic injury
during the preparation of rabbit renal cortical slices [227].
Clever computer-modeled schemes have been proposed to
exploit known permeation rates and the osmotic limits of the cell
so as to maximally accelerate CPA addition and removal
[293, 294], though most have not been tried.
The problem of introducing and removing cryoprotectants is
more difficult when the pCPA and npCPA must be administered by
perfusion. In a kidney, for example, exponential addition of pCPA is
counterproductive because lags in distribution of the CPA to the
medulla result in little benefit of the exponential addition rate,
Principles of Vitrification
65