X-Message-Number: 16179
Date: Thu, 3 May 2001 05:30:48 -0400 (EDT)
From: Ben Best <>
Subject: HSCP -- Experiments and Accomplishments
The Hippocampal Slice Cryopreservation Project (HSCP) is
scheduled to terminate at the end of this June, but we are
still several thousand dollars short of necessary funds to keep
the project going that long. I need hardly stress the importance
of brain cryopreservation research to our survival. The project
has gathered a momentum of discovery that make it all the more
important to keep it running to the very end. It can take years
to get this kind of momentum started again -- and we have seen
such tragic interruptions before in the history of kidney
cryopreservation research.
To encourage others to contribute to keeping the HSCP going
as long as possible I have summarized the Experiments & Accomplishments
of the project to date. The summary is shown below the asterisks as
part of this message, and can also be found on my website:
http://www.benbest.com/cryonics/hscp.html
Please do contribute if you can to The Institute for Neural
Cryobiology (INC) which passes all contributions to the HSCP
research. INC is 501(c)3 and therefore all contributions are
tax-deductable.
Ben Best, President
The Institute for Neural Cryobiology
http://www.neurocryo.org/
****************************************************************************
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 -130C, 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 22C, 2C and -22C. Mannitol was added on washout to
buffer against osmotic jolts. Only in this initial experiment was MTT
(a measure of the ability of mitochondria to transport electrons) used as
an assay of viability. In all subsequent experiments the viability assay
was based on 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. The frozen tissues
(with no cryoprotectant) showed no better than 12% 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 --
while Veg was superior to glycerol for at least 2 of the 3 temperatures.
(Suda's experiments had also shown glycerol to be superior to DMSO.)
Decades ago the Japanese experimentalist I. Suda had exposed whole cat
brains to 15% glycerol, stored them for 5 days at -20C 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, Vol.70, p.527-531 (1974)]. In the spirit
of Suda, hippocampal slices were subjected to 30% glycerol
and stored for 12 hours at -20C, -40C and -76C. Although
the controls exposed to 30% glycerol showed 70% viability,those
cooled and stored at -20C were essentially dead. Those stored at
-40C and -76C had slightly better viability, but not more than
15% viability. 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 0C for one hour (without cryoprotectant) and then rewarmed &
assayed. The slices showed 30% of the viability of controls which had
been maintained at 37C. Hippocampal slices held at 0C 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 0C chilling injury.
The experiment was repeated with controls at 37C and 10C as well as
with 25% Veg slices at 10C and 0C. There was little significant difference
between the 37C controls, the 10C controls or the 10C 25% Veg slices, but
the 0C 25% Veg slices lost 30% of viability. This indicates that chilling
injury occurs between 10C and 0C. Worse results were obtained with Veg
addition at 15C -- indicating that 10C is the optimum temperature for
introduction of Veg. Moreover, at 10C introduction of Veg at 5-minute steps
of increasing concentration produced better 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
10C which was 50% *greater* than that of control slices at 37C after one hour
in the Oslo Chamber (although the difference disappeared after 2 hours). A
combination of 25% Veg with RPS-2 at 10C resulted in viability fully the
same as that of 37C controls -- although 25% Veg is insufficient concentration
for vitrification. Pushing the Veg concentration to 50% in RPS-2 at 10C
brought the slice viability back down to 50% that of the 37C 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 10C in
steps up to 25% concentration, and a final 25% Veg (bringing the total to 50%)
was added (and washed-out) at -10C. These 50% Veg slices were only slightly
less viable (and not statistically less viable) than the 37C controls. The
concentration of Veg required to vitrify is estimated to be 53%. Adding (and
later washing-out) 30% rather than 25% Veg at -10C (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
-10C while the other group with 53% Veg was cooled to vitrification
temperature (-130C) and then rewarmed. The non-vitrified 53% Veg slices had
60% viability and the vitrified 53% Veg slices had 56% viability when
compared with controls -- a difference that is 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 10C (as
before), but the final addition (and washout) at -10C was given 10 minutes
for diffusion. Viability of both the vitrified and the non-vitrified
53% Veg groups increased, but the vitrified group was still significantly
less viable than the non-vitrified group -- and the vitrified group was
just under half the viability of 35C controls. Using 10-minute steps
throughout all phases of the addition and washout procedure protected completely
against vitrification/devitrification injury, but was associated with increased
toxicity. Nonetheless, viability was increased to 56% that of controls.
Subsequent experiments (still in progress) have made use of ice blockers
and cryoprotectant cocktails less toxic than Veg. The results of these
experiments have shown promise of achieving over 90% viability -- and
perhaps even complete viability of hippocampal slices which have been
vitrified at -130C.
SUMMARY OF DISCOVERIES FROM THE HSCP
(1) Chilling injury can be minimized by minimizing time spent at 0C.
Chilling injury is confined to a window between +10C and -10C,
being absent at both of these temperatures.
(2) Chilling injury can be moderated by cryoprotectants, but not eliminated.
Veg moderates chilling injury better than glycerol.
(3) RPS-2 can be used with Veg to significantly increase the
viability of hippocampal slices.
(4) Loading (and unloading) half the Veg at 5-minute intervals at 10C
and the other half in a 10-minute interval at -10C can be used to
vitrify hippocampal slices at -130C with resulting viability that is
50% that of 35C controls. Using 10-minute steps throughout all phases
of the addition and washout procedure protected completely
against vitrification/devitrification injury, but was associated with
increased toxicity that prevented net viability from increasing above
56% of controls. Thus, less toxic cryoprotectants than Veg are
necessary for vitrification with viability significantly higher than
56% of untreated controls.
By using ice-blockers and cryoprotectant cocktails less toxic than
Veg, it may be possible to vitrify hippocampal slices at -130C with complete,
or nearly complete, viability. The project only has a couple of months before
it must end when Yuri leaves to do research elsewhere. However, HSCP has
insufficient funds to go until the end of June. The work being done now is
no mere footnote to the work done previously -- viability of over 90% has
been obtained, but not consistently. Only a few thousand dollars are
required to take advantage of the current momentum and keep the project
running until the end of June. The importance of brain cryopreservation
technology can hardly be overstated.
Contributions to The Institite For Neural Cryobiology are
501(c)3 tax-deductable. Please send donations to:
The Institute for Neural Cryobiology
238 Davenport Road #240
Toronto, Ontario
M5R 1J6 CANADA
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