X-Message-Number: 16394
Date: Thu, 31 May 2001 07:31:46 -0400 (EDT)
From: Ben Best <>
Subject: HSCP -- Summary and Update

    Below I am posting a revised summary of experiments and accomplishments 
of the Hippocampal Slice Cryopreservation Project (HSCP). Dr. Fahy has 
proofread this document very carefully and corrected errors that I had in 
the version I originally posted. Please ignore the previous version. We
have also updated the final sections somewhat to reflect the current 
status of the project. 

     The project is definitely ending at the end of June. There will be
no extension for another month as I had hoped. At least this means there
are no more funding worries -- the month of June has been fully paid-for
-- including one set of electron micrographs. THANK YOU to all of you 
who have contributed to this project. I actually have received checks in
excess of what was needed. I was planning to return these, but Dr. Pichugin
suggested -- and Dr. Fahy agreed -- that another set of electron micrographs
taken later in June might be very useful. So all of the money will probably 
end-up being spent. I will give a final report at the end of the project. 
                   -- Ben Best, President
                      The Institute For Neural Cryobiology
            Ben Best ()


    The Hippocampal Slice Cryopreservation Project (HSCP)
is a research project being conducted by cryobiologists Dr. Gregory
Fahy and Dr. Yuri Pichugin at a major California university 
with funds provided by The Institute for Neural Cryobiology (INC)
and the university. The objective of the project is to cryopreserve 
hippocampal slices through vitrification at -130oC, with complete 
viability upon rewarming.

    The hippocampus is the area of the brain thought to be most critical
in learning new information through a process known as LTP (Long-Term
Potentiation). More neurophysiological experiments are performed on 
hippocampal slices than on any other brain tissue.The hippocampus is 
also the brain tissue most easily damaged by oxygen deprivation 
(ischemia) and is one of the first areas of the brain affected by 
Alzheimer's Disease. In addition to the medical and neurophysiological
significance of the hippocampus is the fact that hippocampal slices are 
not only easy to study & manipulate, but are so widely studied by others
that considerable literature & equipment exists to assist the study.

     The HSCP uses hippocampal slices which Dr. Pichugin prepares from
rat brains and evaluates for viability in an Oslo Chamber. An Oslo Chamber
is a plastic container that looks like a casserole dish with 
aerated partitions where hippocampal slices can be maintained in an 
environment controlled for temperature, oxygenation and humidity. 
Hippocampal slices from an adult rat can live in Oslo Chambers for 
12 hours or longer. Injuries to slices occurring during surgical removal 
from a rat brain may even heal in the Oslo Chamber. 

    An initial experiment compared introduction of various cryoprotectants
with stepped increases (and washouts) of cryoprotectants so as to reach
peak concentration at 22oC, 2oC and -22oC. Mannitol was added on washout
to buffer against osmotic jolts. In this initial experiment the dye MTT 
(which measures the ability of mitochondria to transport electrons) was
used to indicate viability. 

    The cryoprotectants used in this initial experiment were ethylene glycol
(used in automobile antifreeze), DMSO, glycerol and Veg (pronounced "Vee Ee 
Gee", not "veg"). Veg is a modification of the cryoprotectant cocktail VS41A 
(VS41A is a Vitrification Solution which is a mixture of DMSO, formamide and 
propylene glycol) in which the propylene glycol is replaced with an equal 
weight of ethylene glycol.  [Veg is patent pending,  21st Century Medicine 
(21CM).]  DMSO showed the worst viability, ethylene glycol the next worst, 
and glycerol the third worst at two of the 3 temperatures studied, with Veg
being the best overall.  The superiority of glycerol to DMSO was reassuring 
in view of unpublished experimental results of Isamu Suda showing the same 
superiority for whole brains (see next paragraph).

    Decades ago the Japanese experimentalist I. Suda had exposed whole cat
brains to 15% glycerol, stored them for 5 days at -20oC and then 
demonstrated that the cat brains had EEG patterns similar to those of cat
brains that had not been subject to cooling [NATURE 212:268-270 (1966) 
and BRAIN RESEARCH 70:527-531 (1974)]. In the spirit of Suda, hippocampal 
slices were subjected to 30% glycerol and then either stored for 12 hours 
at -20oC, -40oC and -76oC or cooled to these temperatures and rewarmed
without storage.  In these and all subsequent experiments, the viability 
assay was based on the potassium/sodium (K+/Na+) ratio. For neural tissue, 
especially, the ability of cells to maintain membrane potential with the 
sodium pump is an easily-measured & reliable indicator of viability.  Based 
on this assay, although the controls exposed to 30% glycerol showed 
70% viability, those cooled and stored at -20oC were essentially dead.
Those stored at -40oC and -76oC had slightly better viability, but not
more than 15% viability, and the effects of cooling to -76oC were not 
improved by warming immediately from that temperature (no storage). Using 
30% Veg rather than glycerol produced even worse results -- possibly because 
the temperature of introduction of Veg was too high. Veg needs to be added 
at a *lower* temperature than glycerol. 

    In addition to the ice crystal damage of freezing and the toxicity of
cryoprotectant, attempts to cryopreserve organs & tissues are also hampered
by a mysterious phenomenon of unknown mechanism known as "chilling 
injury". To investigate this phenomenon hippocampal slices were cooled 
to 0oC for one hour (without cryoprotectant) and then rewarmed & 
assayed. The slices showed 30% of the viability of controls which had 
been maintained at 37oC. Hippocampal slices held at 0oC in artificial
CerebroSpinal Fluid (aCSF) for 50 minutes also showed 30% viability. 
This result was not changed by using modified aCSF containing
mannitol (maCSF) instead of the standard aCSF, at 50 min of exposure to 0oC.
But adding 10% glycerol seemed to reduce the chilling injury somewhat -- 
and adding 10% Veg resulted in a 40% viability. 25% Veg (with 
& without mannitol) resulted in 50% viability. These results indicate 
a protective effect of Veg against the 0oC chilling injury. 

     The experiment was repeated with controls at 37oC and 10oC as well as 
with 25% Veg slices at 10oC and 0oC. There was little significant
difference between the 37oC controls, the 10oC controls or the 10oC 25%
Veg slices, but the 0oC 25% Veg slices lost 30% of viability. This
indicates that chilling injury occurs between 10oC and 0oC. Worse results
were obtained with Veg addition at 15oC -- indicating that 10oC is the
optimum temperature for introduction of Veg. Moreover, at 10oC
introduction of Veg at 5-minute steps of increasing concentration produced b
etter viability than introduction of Veg at 10-minute steps -- indicating 
that toxicity damage is more important than osmotic damage at this temperature
and for these times & concentrations. Experiments also indicated that 
300mM mannitol is the optimum concentration for buffering osmotic damage 
during removal of Veg. 

    Carrier solutions can enhance cryoprotection. RPS-2 (Renal Preservation
Solution number 2) was developed by Dr. Fahy in 1981 as a result of studies
on kidney slices. RPS-2 actually resulted in hippocampal slice viability at
10oC which was 50% *greater* than that of control slices at 37oC after one
hour in the Oslo Chamber (although the difference disappeared after 2 hours). 
A combination of 25% Veg with RPS-2 at 10oC resulted in viability fully
the same as that of 37oC controls -- although 25% Veg is insufficient
concentration for vitrification.  Pushing the Veg concentration to 50% in 
RPS-2 at 10oC brought the slice viability back down to 50% that of 
the 37oC controls. 

     The next experiment was based on the idea that both chilling injury and 
toxicity should be reduced at sub-zero temperatures. Veg was added at 10oC
in steps up to 25% concentration, and a final 25% Veg (bringing the total 
to 50%) was added (and washed-out) at -10oC. These 50% Veg slices were
only slightly less viable (and not statistically less viable) than the
37oC controls. The concentration of Veg required to vitrify is estimated 
to be 53%. Adding (and later washing-out) 30% rather than 25% Veg at -10oC
(bringing the total to 55%) resulted in viability 77% of that of controls. 

    In the next experiment one group of slices with 53% Veg was held at 
-10oC while the other group with 53% Veg was cooled to vitrification 
temperature (-130oC) and then rewarmed.  The non-vitrified 53% Veg slices
had 60% viability and the vitrified 53% Veg slices had 56% viability as 
compared to untreated controls.  The difference between the Veg-treated
slices and the slices that were exposed to Veg and then vitrified 
was not statistically significant. But when the experiment was repeated, 
the vitrified group was significantly less viable. 

     The following experiment added Veg at 5-minute intervals at 10oC, but 
the final addition (and washout) at -10oC was given 10 minutes for diffusion.
Viability of the non-vitrified slices exposed to 53% Veg was 65%, but when 
slices treated in this way were vitrified, the K/Na ratio was only 42% of 
control K/Na, perhaps due to inadequate equilibration.  In conclusion, using 
10-minute steps throughout all phases of the addition and washout procedure 
was able to protect completely against vitrification/devitrification injury, 
but was associated with increased toxicity. Nonetheless, the viability after 
vitrification was up to 56% of untreated control viability.   

    Subsequent experiments (still in progress) are making use of ice blockers

and cryoprotectant cocktails less toxic than Veg as well as longer equilibration
times prior to vitrification.  The worst of these newer experiments has given

results equal to the best results obtained with Veg, and the best results of the
newer experiments have indicated no difference between vitrified slices and
untreated controls.  However,  the variability of the results has been very 
high -- up to plus or minus 25%, with an average of 75% viability of 
hippocampal slices which have been vitrified at -130oC.  Current efforts are 
being devoted to reducing this variability and moving the average viability 
closer to 100%.


(1)   Vitrification currently results in at least five times higher recovery 
      of K/Na ratio than could be obtained after freezing, even though 
      freezing involved higher concentrations of glycerol than were used by 

(2)   Chilling injury can be minimized by minimizing time spent at 0oC.
      Chilling injury is confined to a window between +10oC and -10oC,
      being absent at both of these temperatures. 

(3)   Chilling injury can be moderated but not eliminated by cryoprotectants.
      Veg moderates chilling injury better than glycerol.

(4)   RPS-2 can be used with Veg to significantly increase the 
      viability of hippocampal slices.

(5)   Although it is apparently possible to achieve 100% recovery of K/Na
      ratio after vitrification and warming, it is presently not possible to
      do this on a consistent basis.  Perhaps by eliminating sources of 
      experimental variation and continuing to refine procedures and 
      cryoprotectants, this final barrier can be overcome.

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