Hippocampal Slice Cryopreservation Project Status Report and Plans

From: Paul Antonik Wakfer (paulwakfer@internet.look.ca)
Date: Thu Jan 11 2001 - 13:26:24 MST

The Project is looking for a small amount of funding to enable it to
continue until at least March 31, 2001, by which time Dr Pichugin
expects to have a visa which will enable him to leave the project to
pursue research elsewhere.

Those wishing to support this endeavor with a donation which might be
used for US federal and California state tax relief (possibly still
for the year 2000, if done quickly) may do so by sending a monetary
instrument to:

The Institute for Neural Cryobiology
238 Davenport Rd, #240
Toronto, ON M5R 1J6

Thank you for your attention,

Paul Antonik Wakfer

HSCP Progress Report and Plans

     By the time of the Asilomar meeting, the HSCP had
managed to achieve a more than 10 times improvement in the
state of the art of cryopreservation of large, integrated
neural systems, the cryopreservation of which had not
previously been reported in the formal scientific literature
to our knowledge. Hippocampal brain slices frozen in a
variety of ways using the standard glycerol originally found
by Suda to be optimal for cryopreservation of brains were
found to yield only about 5% plus or minus 5% viability after
thawing from dry ice temperature. Hippocampal slices
vitrified with the simplest and most elementary exemplar
of the new 21CM vitrification solutions (essentially Veg)
recovered with 53% of the potassium and sodium transport
capacity of fresh, untreated slices. This accomplishment
greatly understates the magnitude of the advance over the
prior state of the art in that it is safe to assume that frozen-
thawed slices sustain major structural damage as a result of
the formation and dissolution of ice, whereas such damage
is presumably absent after vitrification and warming. Therefore,
the new methods are believed to achieve both immensely
greater functional recovery and immensely greater structural
preservation than has been possible with more conventional

     Since the Asilomar meeting, an attempt has been made to
further improve upon these results. The bottom line is that,
at the time of this report (January 9, 2001), the viability of
vitrified/rewarmed rat hippocampal brain slices has been
increased to about 66%. Progress was slower than hoped for
because the thrust of the experiments was to exploit more
sophisticated vitrification solutions, and this required a
re-optimization of the method for adding and removing the
cryoprotectant. The damage sustained by the slices is very
sensitive to factors such as osmotic stress, temperature, and
time, and the previous method of addition and washout was no
longer the best. Also, due to the presence of non-penetrating
cryoprotectants that require time to diffuse into the slices,
we found that it was necessary to expose the slices to the
highest concentrations of cryoprotectant for longer times in
order to allow vitrification to occur.
When the previous equilibration time of 10 min was used, slices
that were cooled to below -130oC and rewarmed gave variable and
usually suboptimal results, but when equilibration time was
extended to 20 min, then vitrification yielded the 66% recovery
just noted. It is possible this can be improved slightly by
even longer exposure to the cryoprotectant prior to
vitrification, or by modifications of the solution that will
allow for faster equilibration.

      At the moment, 66% recovery of brain slice function is
equivalent to the recovery of K/Na ratio typically achieved for
vitrified/rewarmed rabbit kidney slices by 21CM, a remarkable
coincidence. There is indirect evidence that this may be
sufficient for long term cellular survival after transplantation
and in vivo recovery. We attempted to improve the K/Na ratio of
rat hippocampal slices by culturing them for longer times after
exposure to cryoprotectant to see if self-repair would be
apparent, but at incubation times at least as long as 8 hours,
there was no apparent improvement. On the other hand, we believe
that 66% recovery implies 66% recovery of the function of each
individual cell, rather than outright loss of 34% of the cells
with full recovery of the remaining 66%. Several brain slices
have been fixed using an electron microscopy grade fixative and
will be examined structurally to verify general integrity of all
cells and the preservation of brain slice structure after
vitrification and rewarming. Unfortunately, as of the time of
this writing, the samples have not yet been processed and the
structural results are not available. If the project continues,
this gap will be closed by processing these samples as well as
additional samples that will further probe the issue of
structural preservation after vitrification.

     In January, we plan to use a vitrification solution that
we believe is an improvement over the one adopted following the
Asilomar meeting. In February, we expect to have a further
improvement ready for trial on brain slices. The 3rd
vitrification solution has already been shipped to Dr. Pichugin
and has been received, along with more of the 2nd solution for
direct head-to-head comparison. At present, we have no reason
to believe that we cannot exceed the 66% functional recovery
level achieved so far.

-- G.M. Fahy, Director of Research, INC

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