X-Message-Number: 0035
Subject: The Cryobiological Case For Cryonics



             A.  Premises and their scientific evaluation
             B.  Short introductory summary of general conclusions
             C.  Detailed review of relevant current cryobiological
                  1.  General cryobiological background
                  2.  Living adult animal brains
                  3.  Living adult human and animal brain tissue
                  4.  Living fetal human and animal brain tissue
                  5.  Living human and animal isolated brain cells
                  6.  Post-mortem human and animal brains
                  7.  Post-mortem human spinal cord and outflowing nerves
             List of references cited

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Any casual newspaper reader will have decided quite confidently by now that 
cryonics  has  no chance whatever of success, due to the systematic
misinformation contained in  all  media  coverage of this subject to date. 
Not only has the scientific evidence  supportive  supportive  of  cryonics not
been presented, but the unchallenged,  supposedly  scientific  criticisms  of
cryonics presented in the media have been as harsh as they have been  vapid 
and  without  merit.   In reality, it seems that no  supposedly  scientific 
criticism  of  cryonics  has  ever addressed the real issues involved or ever
been based on  a  grasp  of  them.   The  purpose  of  this  discussion  is to
provide  a  summary  of  the  extensive  cryobiological  evidence  which 
exists  to support  cryonicists'  premise  that  existing  freezing techniques
preserve the molecular basis of human memory and personality and  thus  offer
a reasonable chance of allowing future restoration of cryonics patients to

Why  has  this  evidence  not been presented previously?   The  reasons  are 
largely  political.  Also it should be appreciated that even a neutral
position with respect to the  emotionally  charged  subject  of cryonics is
hazardous for  a  cryobiologist  because  of  hardened  opposition on the part
of many key scientists who control job  availability  and  grant support. 
This opposition is generally based on  a gut reaction and/or philosophical 
objections  that do not invite further consideration.  Unfortunately, almost 
no one  ever  seriously  asks  whether  anything  as seemingly outrageous as 
cryonics  could  have  any  compelling scientific foundation, despite the fact
that it does.  The problem is that  the  relevant  scientific  facts are far
from obvious or readily available, and that  no  well- established scientist
has ever dared or even been able to enunciate them.   

The  result  has  been the suppression of discussion, the creation  of 
anxiety,  the  propagation of gross misinformation among the general public,
and the censorship of  valid  scientific  observations: in short, the
antithesis of what science is supposed to  be  all  about.   It  is  time to
consider the scientific facts and to show  that  what  is  really  outrageous 
is not cryonics but the notion that there is no scientific basis for  cryonics
or that cryonics cannot possibly work.   

A.  Premises and their scientific evaluation 

What are the cryobiological issues?  Another way of asking this question is:
what  is  the  minimum cryobiological  requirement for "success" with the
cryonics endeavor?   Since  the  one  indispensable  goal of cryonics is
restoration of the brain, we  can  limit  our  attention to the cryobiological
requirements for the achievement of this goal.   Questions  concerning 
maintenance  of  the brain after restoration are not  cryobiological  and  can
therefore be neglected here. 

What then would be required for the brain to be restorable?  First, the brain
must  be  preserved  well  enough to repair, i.e., it must be possible today
to preserve  with  some  reasonable fidelity the basic biological components
of the brains of humans shortly  after  these  humans have clinically died. 
Second, repair technology must be available to  carry  out any repairs

The  two indispensable premises of cryonics, then, are reasonable brain 
preservation  and  the  development of advanced molecular scale 
(nanotechnological)  biological  repair  devices.   Both  premises  are  fully
open to scientific  scrutiny  and  falsification  by  experiment  or
calculation and, in fact, both seem at present to withstand such  scrutiny, 
as  the  experimental evidence which is presented in this paper as well as 
the  work   of  others  on  the  problems of biological repair (see K. Eric 
Drexler's  book,  Engines  of  Creation,  and  his technical papers) should
show.  If both premises are  valid  (assuming  cryonic  suspension is done
under reasonable conditions and nonscientific problems do  not  intervene),
then in principle cryonics should work to at least some extent.   

As  noted  above, this article is about the cryobiological basis of  cryonics 
rather  than  the cell repair aspect.   But because the cryobiological premise
of  cryonics  loses  significance  without  the  nanotechnological premise of
cryonics,   it  is  necessary  to  comment  at  least  briefly on
nanotechnology in order to clarify  the  relevance  of  the  evidence  to be
presented about cryobiology.   There appear to be no significant flaws  in  K.
Eric Drexler's concepts of molecular scale cell repair devices, and this 
judgment  is  supported  by  the  absence of even a single significant and 
coherent  objection  to  his  concepts.   The  concepts  involved are powerful
enough to make it  easy  to  imagine  the  technology not only for repairing
the fine structure of the brain but also the  technology  for  transplanting 
a brain into a new body.   It seems not only possible  but  inevitable  that 
such technologies will be developed, and a person waiting in liquid nitrogen 
should  remain changeless for centuries if need be while such developments

B.  Summary of general conclusions  

It  can  be  stated  quite  firmly that  cell  bodies,  cell  membranes, 
synapses,  mitochondria,  general axon and dendrite patterns, metabolites such
as  neurotransmitters,  chemical  constituents such as proteins and nucleic
acids, and general brain  architecture  are  preserved  reasonably  well or
excellently with current techniques.   The  brain  can  withstand severe
mechanical distortion by ice without impairment of subsequent  cognition,  and
a  glycerol  concentration of less than 4M -- a  concentration  achieved  in 
current  cryonics procedures -- can be shown to limit ice formation to
quantities currently thought  to be consistent with good functional recovery
of the intact brain.   

Information is lacking about the ultrastructure of frozen-thawed brains, but
much can  be  inferred  from  the customary observation of a high level of 
functional  recovery  of  frozen-thawed brains, brain tissue, or brain cells
which depends on a high degree of  both  local  and  long-range 
ultrastructural integrity.  Absolute proof is  lacking  about  the  quality 
of preservation in each and every brain region, since not all brain regions 
have  been  examined by neurobiologists to date.  However, in the experience
of those  who  have  histologically  examined  entire cross-sections through
the frozen-thawed  brain  at  many  different  levels, no clear differences in
preservation quality from one brain  region  to  another have ever been

A reasonable way of summarizing the world literature on this subject at
present is  to  say  that  wherever  either brain structure or brain function 
has  been  evaluated  after  freezing  to  low  temperatures and thawing,
robust preservation has  almost  always  been  demonstrable   provided   at 
least  some  minimal  attention  was   paid   to   providing  cryoprotection, 
and in some cases good preservation has been documented in  the  complete 
absence of reasonable cryobiological technique.  The implication of these
findings is that  structures  and  functions not examined to date will also
respond in a  favorable  way  to  freezing and thawing.   

C.  Detailed review of relevant current cryobiological knowledge  

1.  General cryobiological background 

Freezing is not a process of total destruction.  It is well known that human
embryos,  sperm, skin, bone, red and white blood cells, bone marrow, and
tissues such as parathyroid  tissue  survive  deep  freezing and thawing, and
the same is true for  systems  of  animal  origin.  In 1980 a table was
published listing three dozen mammalian organized tissues and  even  a few
mammalian organs which had been shown to survive cooling to  low  temperatures
(1),  and this list could now be expanded due to additional experiments on
other  systems.   Such  survival  could  not  occur if the  molecules 
comprising  biological  systems  were  generally  altered by freezing and
thawing.  In general, freezing does not cause  chemical  changes or protein

Contrary  to  popular imagination, cells do not burst as a  result  of 
intracellular  freezing.   The  expansion  of  water as it is converted to ice
causes  less  than  a  10%  increase in volume, whereas cells can withstand
far larger increases in volume, e.g.,  50- 100% increases.  But the primary
flaw in this concept is the idea that ice forms in  cells  at  all  under 
ordinary conditions of slow freezing: it does  not.   Instead,  ice  forms 
between cells, and water actually travels from the interior of the cell to the
ice outside  the cell, causing shrinkage rather than expansion of the cell. 

Cell  death  during  slow freezing may be related to changes  in  the  cell 
membrane  produced  by cell shrinkage, or to toxicity of cryoprotectants as
they  are  progressively  concentrated as a consequence of the formation of
pure ice in initially dilute  solutions.   Both of these putative causes of
death are relatively mild on the molecular level and  are  certainly not
irreversible in principle.  But whatever the cause of death, cells  examined 
in  the  frozen  state appear to be structurally intact even when they  are 
known  to  be  nonviable  upon thawing (with very few exceptions on the part
of nonmammalian systems  not  relevant to the brain).  This is true both for
single plant and animal cells and for cells  that  comprise  animal tissue. 
Hence, lack of functional recovery after  thawing  is  not  proof of lack of
structural preservation in the frozen state before thawing, and it is the 
latter that is relevant to cryonics. 

A truism of cryobiology is that different types of cells require different 
protocols  of  cryoprotectant  treatment, cooling and warming rates, and 
cryoprotectant  washout  in  order  to exhibit maximal survival.  All of these
differences can be minimized greatly  by  using  high  concentrations  of 
cryoprotectant,  provided  such  concentrations  can   be  tolerated. 
Nevertheless, other than a few generalizations such as those described  above,
it  is  difficult  to  extrapolate  from one biological system  to  another 
in  terms  of  predicting the details of its cryobiological behavior. 

For  this  reason,  if we wish to understand what happens to the  brain  when 
it  is  frozen,  we  can't argue on the basis of results obtained with kidneys
or plant  cells  or  embryos or granulocytes, but must, instead, focus
specifically on the brain.  Herein  lies  one of the largest errors
cryobiologists and other scientists have made in dismissing  the  prospects
for cryonics: making sweeping negative statements without knowing anything
about  the  cryobiology  of  the brain (or, for that matter, the primacy of 
the  brain,  or  the  concepts of nanotechnology). 

In  order  to  examine  the scientific evidence  bearing  on  the  only 
indispensable  cryobiological  premise of cryonics, then, the balance of this
article will be devoted  to  an extensive review of the contents of a large
number of scientific papers on the freezing  of  brains, brain tissue, and/or
brain cells.  As extensive as the following remarks  are,  it should be
understood that they are not exhaustive.  No attempt has been made to  obtain 
the  complete scientific literature describing the state of brains after
freezing in  ways  which  are  relevant to the issue of cryonics.  This review
simply reflects  all  relevant  information currently at hand.   

2.  Living adult animal brains 

Dr.  Robert J. White, the Chairman of the Dept. of Neurology at Case Western 
Reserve  University's  School of Medicine, has favorably discussed the
prospects for  the  eventual  successful  cryopreservation  of human brains
(2,3,4).  (Dr. White is also  an  expert  on  cephalic  transplantation  and
hypothermic brain preservation, and has  published  several  scientific 
papers  on these subjects.)  However, it is clearly impossible  to  experiment
with  entire living human brains, so the closest we can come to evaluating the
degree  of  total  brain  preservation  achieved in best-case cryonics
procedures  is  to  review  the  results of freezing the brains of animals. 

The earliest observations of this sort were made by Lovelock and Smith (5,6)
in 1956.   These investigators froze golden hamsters to colonic temperatures
between -0.5*C and  -1*C  and quantitated the amount of ice formed in the
brain, allowing them to determine how much  ice  formed  in  the  brains of
animals which made  full  neurological  recoveries.   They  determined that at
least 60% of the water in the brain could be converted into ice without 
damaging   the  ability  of  the  hamsters  to  regain  normal  behavior 
after   thawing.    Considerably  more  ice  was consistent with restoration
of breathing,  a  complex  neural  function.   However,  the  exact  quantity 
of  ice  (above  60%)  consistent  with   full  neurological recovery could
not be clearly determined, because of death due to intestinal,  pulmonary, 
and renal bleeding.  Nevertheless, tolerance of at least 60% ice by the  brain
shows that this organ is considerably more tolerant of freezing than is the

The  prospects for successfully avoiding damage due to the formation of ice 
at  much  lower  temperatures  can  be assessed to a first approximation based
on  this  finding  of  Lovelock  and  Smith.  The quantity of glycerol
required in theory to  prevent  mechanical  injury from ice (Cgr) can be
calculated from the equation (derivable from reference 7) 

Cgr = 9.3  - .093Vt 

where  Vt is the percentage of the liquid volume of the brain which can be
converted  into  ice without causing injury.  Assuming Vt = 60%, Cgr is 3.72M.

The  work of Lovelock and Smith was followed up by Suda and his associates 
(8,9,10),  who made a number of critical observations on frozen glycerolized
cat brains.  Their first  publication,  in  1966,  demonstrated  that cat
brains gradually  perfused  with  15%  v/v  glycerol  at  10*C  and  frozen 
very slowly for storage  for  45-203  days  at  the  very  unfavorable 
temperature  of -20*C regained normal histology,  vigorous  unit  (individual 
cell) activity in the cerebral cortex, hypothalamus, and cerebellar cortex,
and strong  if  somewhat slowed EEG activity (8) after very slow thawing. 

These  results are remarkable in a number of ways.  First, it is clear that no
other  organ would be capable of the same degree of activity after such
prolonged storage at such  a  high subfreezing temperature.  Second, Suda et
al. made no attempt to supplement  their  perfusion fluid (diluted cat blood)
with dextrose, which must have become depleted  fairly  rapidly, worsening the
EEG results.  Third, Suda and colleagues did not wash the  glycerol  from  the
brain  carefully, and this may have caused  injury  during  brain 
reperfusion.   Fourth, the presence of EEG activity implies preservation of
long-range neural connections  and  synaptic  transmission,  and unit activity
indicates preservation  of  cell  membrane  integrity,  energy  metabolism,
and sodium and potassium pumping  capability.   In  short,  these  brains 
appeared to be basically viable based both on function  and  on  structure.  
"Pial  oozing"  was noted (though not described adequately) after about an
hour  of  blood  reperfusion, but this defect seems minor.  

Their  second publication, in 1974 (9), went considerably farther.  After 7.25
years  of storage at -20*C, "well synchronized discharges of Purkinje cells
were observed" (i.e.,  normal  cerebellar unit activity) as well as
"spontaneous electrical  activity...from  the  thalamic  nuclei  and
cerebellar cortex", and short-lived EEG activity from  the  cerebral  cortex. 
Another brain stored for 777 days showed cortical EEG activity for 5 hours 
after  reperfusion.  In both cases, EEG activity was of lower quality than EEG
activity of  fresh  brains,  but the existence of any activity at all after
such extraordinary  conditions  is  amazing.  Cell loss after 7.25 years and
hemorrhage after reperfusion of brains stored for  5-7 years is not

More  important was a comparison of the frequency distribution of EEG activity
in  a  fresh brain before perfusion and then after storage at -20*C for 5
days.  The EEG  pattern  before  freezing and after thawing was very nearly
the same (9).  It should be noted  that  in  a  typical cryonics operation,
the time spent near -20*C is measured in  hours  rather  than  days  or years
and, based on the work of Suda et al., should not  therefore  involve 
appreciable deterioration of the brain. 

It  is noteworthy that in both reports of Suda's group, the brains were 
successfully  reperfused  with  diluted  cat blood after thawing.  The quality
of  reperfusion  was  not  documented in detail, but the autocorrelogram
comparing the EEG of the 5-day cryopreserved  brain  to the EEG of the same
brain before freezing could not have been as good as it  was  without 
relatively  complete restoration of cerebral circulation.  This is  an 
important  question not only with respect to viability and functional
recovery, but also with respect  to  the  accessibility  of the brain to
nanotechnological repair devices  which  might  be  administered via the
vascular system. 

Also  relevant were unpublished results mentioned in passing (9) on storage at
-60*C  and  -90*C  and  on  the effectiveness of other  cryoprotectants 
(dimethyl  sulfoxide  or  polymers).  Evidently, EEG activity could be
obtained after freezing to -60*C and  storage  for  weeks, but not after
freezing to -90*C, and dimethyl sulfoxide was effective but  not  as 
effective as glycerol.  This is confirmed in an unpublished manuscript by 
Suda  (10),  which reveals also that unit (single cell) activity can still be
recorded in brains frozen  to -90*C.  This unpublished paper (written in
Japanese) also shows that brain  reperfusion  was better after thawing when
glycerol rather than DMSO was used. 

These results can be evaluated with respect to the information obtained
previously by  Lovelock  and Smith.  For protection against mechanical injury
at -90*C, as  noted  above,  the results with hamsters suggest that 3.72 M
glycerol, or 27.2% glycerol by volume, might  be  required,  whereas Suda and
colleagues used only 15% glycerol by volume.   It  can  be  calculated (11)
that at Suda's storage temperature of -20*C, 62% of the liquid content  of 
the  brain was converted into ice, while at -60*C, 77% of the liquid volume of
the  brain  was  converted  to  ice,  a  quantity which equals or  exceeds 
the  tolerable  degree  of  distortion by ice in the hamster brain. 
Therefore, the finding by Suda and his colleagues  of  no injury at -20*C for
5 days but of injury after freezing to -60*C and especially  to  -90*C  is
entirely consistent with predictions from the work of Lovelock and Smith and 
is  also  entirely consistent with an absence of any such mechanical injury in
the  brains  of  cryonic suspension patients perfused with more than 3.72M

The  work with hamsters and with cat brains demonstrates that extensive 
freezing  of  the brain at high temperatures is compatible with its full
functional recovery and that at  least  partial  functional recovery from low
temperatures is a  reasonable  prospect,  but  these  studies  do  not
describe the histological effects of freezing brains  to  the  low 
temperatures required for truly long-term preservation.  This information was
provided  by  Fahy and colleagues (12-14a).  They reported that with either 3M
or 6M glycerol, excellent  histological  preservation of the cerebral cortex
and the hippocampus was  observed  after  slow  freezing  to  dry  ice
temperature (-79*C).  In fact, there  was  no  difference  in  structure 
between brains which had been perfused with glycerol only and brains which 
had  been perfused, frozen, and thawed.  Although Fahy et al. did not report
it formally,  this  finding was also true in every other region of the brain
examined, such as the  cerebellum  and  the  area  of the ventral brain
containing giant neurons  and  well-organized  axonal  bundles.   It  is of
interest that Fahy et al. observed brain shrinkage if  the  perfusion 
temperature  was held constant below room temperature (14a).  But Suda and his
colleagues  also  observed the same degree of brain shrinkage (10), yet this
did not prevent  apparent  survival of their frozen cat brains.   

One report (14b) has appeared which briefly documented the ultrastructural
effects of  a  now-obsolete cryonics procedure on the brain.  A single dog was
perfused directly  with  15%  DMSO for 55 minutes at 10-17*C.  The head was
then cooled at 0.1*C/min to  -14*C  and  then cooled at 0.5*C/min to lower
temperatures.  The brain was estimated to have reached - 79*C  after 3 hours,
after which it was shipped cross-country for thawing,  fixation,  and 
examination by light and electron microscopy.  Histochemical staining of
undefined  nature  showed evidence for appreciable enzymatic activity and
cellular retention of histochemical  reaction product, i.e., intact cell
membranes.  Ultrastructure, as documented in a  single  electron  micrograph, 
revealed  intact cell bodies, an intact  double  nuclear  membrane,  intact  
myelin   sheaths  around  small  myelinated   fibers,   recognizable  
organelles  (mitochondria and endoplasmic reticulum), and recognizable
synapses.  Extensive damage was  also  apparent,  but  it  was not clear
whether this was  due  to  freezing  and  thawing,  perfusion with DMSO in one
step as opposed to gradual addition, or abrupt dilution of DMSO  upon 
fixation.   No  details were provided as to DMSO washout  and  fixation 
procedures.   Significantly, the concentration of DMSO employed was not
sufficient to prevent mechanical  damage  according  to "the Smith criterion"
mentioned earlier.  The presumption  would  be  that  current  cryonics 
procedures, employing the preferred  cryoprotectant  glycerol  in  higher 
concentrations, better preserve ultrastructure.  Nevertheless, it is  not 
obvious  from the published micrograph that the original brain structure could
not be inferred. 

3.  Living adult human and animal brain tissue 

In  1981, Haan and Bowen (15) reported that they had collected sections  of 
cerebral  cortex from living human patients (during brain operations requiring
removal of cortex  to  allow  access to deep tumors), and frozen them using
10% v/v dimethyl sulfoxide (DMSO)  as  the  cryoprotectant.   The DMSO was
added and removed essentially in one step  each,  with  some  agitation of
tissue samples to promote equilibration in the short times allowed  for 
equilibration at 4*C.  Freezing was accomplished by a two-step method in which
the  tissue  was placed at -30*C for 15 min (5 min required to reach -30*C,
for a cooling rate of about  6*C/min,  and  10 min of equilibration at -30*C)
and then transferred directly  to  liquid  nitrogen.   Thawing  was  rapid. 
For  comparison,  rat  brain  tissue  was  obtained   by  decapitating rats
and removing their brains (probably involving a warm ischemic insult  of  5-10
min), and this rat brain tissue was equilibrated with dimethyl sulfoxide and 
frozen  in the same way. 

The  results?  Norepinephrine uptake was 94-95% of control uptake for both 
rats  and  humans.  Incorporation of glucose-derived carbon into acetylcholine
was 89-100% of control  incorporation  for rats and 85% of control for humans.
Incorporation  of  glucose-derived  carbon into CO2 was 86-100% of control for
rats, 78% of control for humans.   

Haan  and Bowen noted that their tissue prisms are mostly synapses, so their 
results  imply  that  synapses of both rats and humans survive freezing by
their  technique.   This  agrees  with  inferences  noted above that synapses
survive in whole  brains  frozen  with  completely  different  techniques.  
Although  not strictly  brain  tissue,  the  superior  cervical  ganglion,
considered part of the central nervous system, also demonstrated  100% 
recovery  of  synaptic  function after freezing to dry ice temperature  in 
15%  glycerol,  according  to Pascoe's report in 1957 (16).  It was noteworthy
that Pascoe's ganglia  also  showed  100% recovery of action potential
amplitude and conduction velocity after  thawing  from dry ice temperature

In  1983, Hardy et al. (17) confirmed the extreme survivability of synapses in
human  brain tissue beyond any doubt.  Once again, normal living adult human
cerebral cortex  was  removed  during operations on deep brain structures and
compared to viable rat  forebrains  in  terms  of freeze-thaw recovery.  The
best results were obtained by freezing  1-5  gram  pieces  of  human brain (or
1 gram rat forebrains), as opposed  to  freezing  homogenates.     The 
cooling rate to -70*C was slow but was not measured or controlled; the 
thawing  rate  was  fast but not measured or controlled; the sole
cryoprotectant was 0.32 M sucrose  (Far  from  an  optimal regimen!).  After
thawing, synaptosomes were prepared  from  the  tissue  samples and tested for
functional recovery.  Here is a summary of the results: 

                  Measurement                                 human    rat
          number of synaptosomes recovered                 not done     80
          number of mitochondria recovered                    133**     67 
          increase in number of unidentifiable
             (damaged) structures                              29       24
          amount of protein recovered                          91       70
          oxygen uptake/100 mg of protein                      78       59
          stimulation of oxygen uptake by veratrine            86       86
          potassium accumulated/100 mg protein                 86       70
          loss of potassium stimulated by veratrine            39       85
          retention of neurotransmitters (aspartate,          good     good
             glutamate, GABA)
          stimulated transmitter release (amount,             good     good
            selectivity, and drug modulation
          * recovery compared to unfrozen control samples.
          ** suboptimal technique

As  Hardy  et al. stated, it is apparent that both human and rat brain
tissue  frozen  to  -70*C with almost no cryoprotection has synapses
"closely comparable to [those from]. .  .  fresh tissue". 

As  if  this  were  not demonstration enough, Walder (18) has  shown  that 
not  even  cryosurgery  destroys synapses.  He applied a -60*C cryoprobe to
the brain of cats  for  5  min  and  examined the resulting lesions in the
electron microscope.  Not only  were  well  preserved  synapses found, but
also cell bodies, organelles, and neuronal processes  could  be  identified, 
despite considerable damage to the organization of the  neuropil  and  to 
astrocyte cell membranes. 

4.  Living fetal human and animal brain tissue 

In  1986,  Groscurth  et al. reported the successful freezing of  human  fetal
brain  tissue (19).  1x2x2 mm brain fragments from a 9-14 week abortus were
treated with 10% DMSO  and  20%  fetal calf serum and placed into a -30*C
environment for 3 hours  or  overnight,  then  stored  at  -80*C for several
weeks, then finally transferred  to  liquid  nitrogen.   After storage for
3-12 months, the samples were "thawed at room temperature", trypsinized,  and
seeded on glass cover slips for 2-4 weeks of tissue culture at 37*C.   The
brain cells  were  found to be alive and to grow in culture:  "Twenty-four
hours  after  trypsinization  the cells formed clusters of variable size.... 
During further cultivation numerous  fiber  bundles  were  found  to  grow
from the margin of  the  clusters.   Single  fibers  showed  varicosities  as
well as growth cones at the terminal projection.  Bipolar  spindle-shaped 
cells with a smooth surface were regularly apposed along the bundles." 

The first reports of attempts to freeze fetal animal brain tissue seem to be
those of  Houle  and Das in 1980 (20-22).  These attempts were fully
successful,  the  frozen-thawed  transplanted  cerebral  cortex  being 
indistinguishable  from  non-frozen  brain   tissue  transplants  in  every
way.  Das et al. have more recently described  their  technique  in  finer 
detail  (23).  Briefly, they use 10% DMSO, a cooling rate of  1*C/min, 
storage  at  -90*C,  and rapid thawing.  Survival was best if the tissue was
not dissociated or  minced  before freezing.   

Although  a  variety of conditions allowed for 100% success rates for 16  and 
17-day  neocortex,  brainstem tissue from 16-day fetuses showed at best a 50%
survival  rate,  and  Das  et  al. suggested that these more differentiated
cells, which have a  low  transplant  survival  rate  even  in the absence of
freezing and thawing, might  be  more  damaged  by  freezing  and  thawing. 
On the other hand, it should be kept in mind that, as  should  be  clear  from
the  earlier  discussion  of  cryoprotectant  concentrations  necessary   for 
protection  at  low  temperatures, 10% DMSO is a rather low concentration  of 
a  possibly  suboptimal  cryoprotectant (Suda indicated that glycerol was
superior to DMSO for  brain),  and  better survival might well have been
obtained using the more gentle  freezing/thawing  conditions employed in
cryonics procedures. 

Jensen and colleagues (24)  reported their work on freezing fetal hippocampal 
tissue  in 1984, again using 10% DMSO, a cooling rate of 1*C/min, storage in
liquid nitrogen,  and  rapid  thawing.   Treatment  with DMSO at 4*C was for 2
hr, with  rapid  washout  at  room  temperature  (not necessarily an innocuous
approach; unfortunately, no DMSO controls  were  done).  Although 21% of the
cryopreserved hippocampi showed ideal structural  preservation  after 
development in oculo, in general there was some structural alteration 
compared  to  nonfrozen control hippocampal transplants.  It was felt that
this may have been due to the  extra  manipulations  of  the  cryopreserved
tissue (controls  were  not  washed  in  DMSO  solutions,  etc.).   Only half
of the cryopreserved transplants at most were found  to  be  present  after
20-68 days in oculo, survival rate being dependent upon fetal age.  It  was 
felt  that this once again may have been due to loosening of the hippocampal
structure  by  the experimental manipulations. 

This  tended to be confirmed by transplants into the brain rather than into
the  eye:   the  brain  provides  more  confinement to transplanted 
hippocampi,  helping  to  prevent  disintegration  of the grafts, and, in
fact, 100% of hippocampi transplanted to the  brain  survived.   (It  should
be obvious that the hippocampus of a frozen intact brain  will  of  course
receive support from all surrounding structures and will thus be more
analogous  to  the intracerebral transplants noted by Jensen et al. than to
the intraocular  transplants,  in addition to being spared from disruptive
manipulations in vitro.)   

Frozen-thawed hippocampi grown in oculo were smaller than control grafts, and
frozen- thawed hippocampi transplanted either to the eye or to the brain
showed a loss of  dentate  granule  cells (a 35% loss was seen in oculo).  In
several other ways, this complex  brain  structure  important  for  encoding
and decoding memories appeared  to  be  unaffected  by  freezing  and 
thawing.  Moreover, freezing in 10% DMSO, as noted above, might not  be  an 
ideal  procedure.  It should be noted that Fahy et al. were not impressed by
any  loss  of  dentate cells in whole adult rabbit brains after freezing and
thawing (12-14a). 

Jensen's  group  followed up this work with more extensive work on  many 
different  subregions  of  the  fetal  rat brain, i.e., the neocortex, 
habenula,  septum  and  basal  forebrain,  cerebellum,  and retina (25).  All
of these regions showed good  survival  and  preservation  of  normal 
structural  organization after  transplantation  into  an  adult  recipient's 
cerebral  cortex, despite wide, uncontrolled variations in  cooling  protocol 
from run to run.  The only exception was the cerebellum: only 2 of 7 grafts
were found  at  the  time of sacrifice, although they were structurally
normal.  The numbers involved  are  too  small  for  adequate  statistical
analysis, and no  control  cerebellar  grafts  were  performed to determine if
this rate of takes is normal for this tissue.  All in all, then,  this paper
tends to confirm the impression from other studies that tissue from many 
quite  different brain areas survives freezing and thawing quite well. 

5.  Living human and animal isolated brain cells 

Silani  et al. (26) dissociated human fetal cerebral cortex into cells and
froze  the  cells  at  1*C/min  in 7% DMSO plus 20% fetal calf serum.  After
more than  12  months  in  liquid  nitrogen,  the  cells were thawed rapidly. 
Immediately after  thawing,  the  cell  recovery  was  96.5+/-2.1%,  showing 
that brain cells are  not  physically  destroyed  by  freezing even under
rather severe conditions.  After 72 hours of culture, 53% of the total  cell
population was alive, but only 24% of the neurons were alive.  The surviving 
neurons  were, however, morphologically and functionally normal, as were
astrocytes.  Silani et al.  considered their yield of human neurons to be
high.  These results show unequivocally that  human  brain cells can survive
freezing and thawing and imply that, as was the  experience  of Hardy et al.
(17) and Das et al. (23) (and as is suggested by the experience of  Jensen  et
al. (24)), it is best to use undissociated tissues (analogous to the intact 
brain  in  cryonics procedures) rather than dissociated cells to obtain
optimal results. 

Kim et al. (27) isolated living oligodendrocytes and astrocytes from the white
matter  of  brains  of   human  cadavers aged 62, 86, and 93 years after 5,
14,  and  6  hours  of  clinical death, respectively.  These cells were
cultured for 2-28 days, then scraped  from  their  substratum,  exposed 
abruptly  to 10% DMSO, frozen to  -70*C  at  an  unknown  and  uncontrolled, 
exponentially decreasing rate, immersed in liquid nitrogen for  1-3  weeks, 
thawed  rapidly,  and abruptly diluted to 1% DMSO, further washed,  and 
recultured.   The  excellent  morphology  of  the cultured cells after thawing
and  the  robust  presence  of  membrane  markers was not different from what
existed before freezing.  70%, 60%, and  55%  survival was obtained after 2,
7, and 28 days of culture before freezing, respectively. 

Kim  et al. (27) also reported informally the following.  "Recently, we  have 
frozen  various types of neural tissue cultures and found that the recovery of
frozen neurons  and  glial cells was excellent.  The neural cultures tested
were: (a) dissociated chick  embryo  spinal  cord and dorsal root ganglia; (b)
dissociated newborn mouse cerebellum and  dorsal  root  ganglia;  (c)
dissociated adult mouse dorsal root ganglia, and; (d)  dissociated  or 
explant fetal human brain cultures." 

Kawamoto and Barrett (28) froze rat fetus striatal (including overlying
cortical) and  spinal  cord  cells  by dissociating these tissues in 5-10%
DMSO  and  placing  them  into  uninsulated boxes in a -90*C freezer and
leaving them there for up to 88 days.  They  were  then  thawed  rapidly  and
exposed immediately to DMSO-free solution,  a  procedure  these  scientists 
found  to be damaging.  Nevertheless, they observed "neuronal  survival  rates
comparable  to those of brain tissues plated immediately after  dissection".  
Preliminary  results  indicated  similar survival of neuroglia frozen in the
same  way.   Survival  was  roughly independent of DMSO concentration above
5%.  Increased sensitivity of the cells to  mechanical  forces was observed
after thawing or after simple cold storage, but  this  was  reduced by using
cryoprotectant carrier solutions low in sodium.  Beautiful morphology was 
seen after thawing, and vigorous regrowth of cellular processes occurred after
thawing, to  yield  mature cultures indistinguishable from controls. 
Surprisingly,  dissociated  cells  survived freezing and thawing better than
cells embedded in undissociated tissue. 

Scott  and  Lew (29) gradually exposed undisturbed cultured adult mouse 
dorsal  root  ganglion  cells  to 10% DMSO, placed them in a -15*C environment
for 30 min,  then  placed  them  in  liquid  nitrogen vapor.  Thawing took 5
min, after which the  DMSO  was  removed  gradually.   Other cultured neurons
were dissociated and frozen and thawed similarly as  a  cell  suspension.  
The relative number of surviving neurons was not quantitated  in  this  study,
although  there was evidently considerable cell death (probably due  to  the 
high  cooling  rate  below -15*C, which would be expected to induce
intracellular  freezing  and  cell  death).   Nevertheless, many neurons
survived and were capable of  basically  normal  electrical activity as well
as regeneration of new nerve fibers. 

6.  Post-mortem human and animal brains 

Human brain banks are now in existence for investigators interested in 
understanding  human  brain  biochemistry and pathology (30-33).  Sections or
subregions  of  post-mortem  human  brains, frozen rapidly several hours after
death, are sent to  medical  researchers  who analyze these brains for
neurotransmitters, proteins, enzyme activity, lipids, nucleic  acids,  and 
even histology.  There would be no reason for such banks if no  molecular  or 
structural preservation were achieved by freezing. 

Haberland  et al. (34) isolated synaptosomes after freezing the nucleus
accumbens  of  rats  and  of 72 (plus or minus 5) year old humans.  The humans
were dead 15 +/-  5  hours  before  this  brain  structure was removed and
frozen.  Previous  studies  indicated  that  dopamine uptake by synaptosomes
could still achieve 55% of the values of fresh brains even  24  hours  after
death.  In this study, the humans were not refrigerated until  3-5  hours 
after  death.  Freezing was done with varying concentrations up to 10% DMSO,
1.2*C/min  to  -25*C, and subsequent immersion in liquid nitrogen. 
Experiments on rat nucleus  accumbens  (NA)  removed  5-10  min after
decapitation of the rat indicated that  freezing  to  -25*C  caused  no 
measurable reduction of dopamine uptake.  When rat NA was  frozen  to  -196*C,
survival  ranged  from 96% of control using 0.07 M DMSO to 99.7% of control 
using  0.7  M  DMSO.   Human  NA  frozen to -196*C as described in the
presence of 0.7 M  DMSO  (5%  v/v)  yielded dopamine uptakes equaling
102.9+/-5.2% of unfrozen control uptakes.   

Stahl and Swanson (35) looked at the fidelity of subcellular localization of 6
brain  enzymes  and total brain protein after guinea pig or post-mortem human
brain tissues  were  frozen  to  -70*C without a cryoprotectant simply by
being placed into a  freezer.   Their  conclusion: "subcellular fractionation
of brain material is possible even with post-mortem  tissues  removed from the
cranial cavity some hours after death."  Two other  groups  have  subsequently
fractionated human post-mortem brain and have come to a similar  conclusion: 
"Our present study further shows that even after freezing and prolonged
storage, human and  guinea  pig  brains  can  be  separated  into 
biochemically  distinguishable  subcellular  fractions....Frozen storage for
several months did not strikingly modify the fractionation  characteristics of
freshly homogenized cerebral cortex." 

Schwarcz  (36)  subjected rat brains to post-mortem conditions  comparable  to
those  experienced  generally by humans: 4 hours of storage in situ at room
temperature  followed  by  24 hours of storage in situ at 4*C followed by
brain isolation and freezing  of  brain  regions  by  placement  in  a -80*C
freezer for 5  days.   Glutamate  uptake  by  striatal  synaptosomes prepared
from striata frozen in this way amounted to 26% of control uptake by  fresh 
tissue synaptosomes, an amazing degree of preservation.  (Schwarcz noted, 
however,  that  glutamate uptake processes may be more resistant than
serotoninergic,  dopaminergic,  and cholinergic uptake mechanisms.) 

Brammer and Ray (37) confirmed that it is possible to isolate intact, if not 
living,  oligodendroglial cells from bovine brain white matter after freezing
to -30*C without  any  cryoprotective  agent,  more than 1 hour after the
slaughter of the  cow.   (The  original  paper describing isolation of human
oligodendroglia under similar circumstances is that of  Iqbal  et  al.  (38)) 
If the white matter was treated with  polyvinyl  pyrollidone  (PVP)  before 
freezing, cytoplasmic enzyme activities were not different from enzyme 
activities  in  unfrozen cells (without PVP, enzyme activities were one half
to one fourth of  control  values, which demonstrates significant preservation
of enzyme structure and function  even  under  these  highly adverse
circumstances.)  Although no data were shown  concerning  the  effects  of 
glycerol  or DMSO, it was stated that these agents  did  not  improve  enzyme 
activity.  Nevertheless, it should be recalled that Kim (27) isolated the same
cells  from  post-mortem human brains before freezing and found that
pretreatment with 10% DMSO allowed  them to survive freezing to liquid
nitrogen temperature. 

Morrison and Griffin (39) isolated undegraded messenger RNA from human brains
after 4  or  16 hours of death, with or without freezing in liquid nitrogen. 
The mRNA was used  to  direct  protein  synthesis  in  vitro,  which was  then
analyzed  by  2-D  O'Farrell  gel  electrophoresis.  Normal protein
populations were observed, causing them to conclude "that  post-mortem 
storage  for  4 and 16 hours at room temperature had  little  effect  on  the 
spectrum  of  isolated  mRNAs"  and  "the  profile  of  proteins 
synthesized.....was  not  changed....when the tissues were stored in liquid

Many  similar reports exist in the literature.  Tower et al. showed 
preservation  of  oxygen  consumption  and  enzyme activities in brains of
many  species,  including  whales  subject to many hours of warm ischemia,
after isolation from the dead animal and  freezing  (40-42).  Hopefully, the
point is clear that brain structure and even some brain functions  and 
enzymatic  activity  survive  freezing even when freezing  is  done  after 
hours  of  unprotected clinical death and even with minimal or no

7.  Post-mortem human spinal cord and outflowing nerves 

One  report (43) is available documenting the effects of cryonics procedures 
on  the  spinal  cord, which is part of the central nervous system.  A human
cryopreserved by  now- obsolete cryonics procedures was decapitated while
frozen, the body thawed, and the spinal  cord  and  spinal nerves examined
histologically after aldehyde fixation  and  osmication.   The basic finding
was that myelin sheaths were intact, and shrunken axoplasm could be seen 
within  the myelin sheaths, conceivably indicating intact axolemmas.  Large
neuronal  cell  bodies  were  observed  which  appeared  intact and normal  in
shape.   In  general,  the  histological  preservation was impressive. 
Apparently intact blood vessels were  observed  within the spinal cord. 
(Other, non-neuronal tissues were also examined and were found to  be 
surprisingly  intact,  with the exception of the liver and, to a  lesser 
extent,  the  kidney.) 


The  scientific literature allows no conclusion other than that brain 
structure  and  even  many brain functions are likely to be reasonably well
preserved by freezing  in  the  presence  of  cryoprotective agents,
especially glycerol in  high  concentrations.   Thus,  cryonics'   premise  of
preservation  would  seem  to  be  well  supported  by   existing 
cryobiological knowledge.  This is not to say that cryonics will inevitably
work.  But  it  is to say that cryonics may work and that it is a reasonable

List of references cited 

General cryobiological background 

1.  Fahy, G.M., Analysis of "solution effects" injury: rabbit renal cortex
    frozen in the presence of dimethyl sulfoxide.,  Cryobiology, 17, 371-388

         Living adult animal brains

2.  White, R.J., Brain, In: Organ Preservation for Transplantation, A.M.
    Karow, Jr., G.J.M. Abouna, and A.L. Humphries, Jr., Eds., Little, Brown, &
    Company, Boston, 1974. pp. 395-407.
3.  White, R.J., Brain In: Organ Preservation for Transplantation, Second
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    1981. pp.  655-674.
4.  White, R.J., Cryopreservation of the mammalian brain, Cryobiology, 16,
    582 (1979).
5.  Smith, A.U., Revival of mammals from body temperatures below zero.  In:
    Biological Effects of Freezing and Supercooling, A.U. Smith, Ed.
    Edward Arnold, London, 1961. pp. 304-368.
6.  Lovelock, J.E., and A.U. Smith, Studies on golden hamsters during
    cooling to and rewarming from body temperatures below 0*C.  III.
    Biophysical aspects and general discussion,  Proc. Roy. Soc. B, 145,
    427-442 (1956).
7.  Fahy, G.M., D.I. Levy, and S.E. Ali, Some emerging principles
    underlying the physical properties, biological actions, and utility
    of vitrification solutions,  Cryobiology, 24, 196-213 (1987).
8.  Suda, I., K. Kito, and C. Adachi, Viability of long term frozen cat
    brain in vitro, Nature (London), 212, 268-270 (1966).
9.  Suda, I., K. Kito, and C. Adachi, Bioelectric discharges of isolated
    cat brain after revival from years of frozen storage,  Brain Res, 70,
    527-531 (1974).

10. Suda, I., Unpublished Japanese language manuscript (including figures)
    based on a talk given by Dr. Suda (President of Kobe University) in
    Japan and reportedly being prepared for publication in English.
11. Fahy, G.M., Analysis of "solution effects" injury: Equations for
    calculating phase diagram information for the ternary systems
    NaCl-dimethylsulfoxide-water and NaCl-glycerol-water, Biophys J, 32,
    837-850 (1980).
12. Fahy, G.M., T. Takahashi, A.M. Crane, and L. Sokoloff, Cryoprotection
    of the mammalian brain, Cryobiology, 18, 618 (1981).
13. Fahy, G.M., T. Takahashi, and A.M. Crane, Histological cryoprotection
    of rat and rabbit brains, Cryo-Letters, 5, 33-46 (1984).
14a. Fahy, G.M., and A.M. Crane, Histological cryoprotection of rabbit
    brain with 3M glycerol, Cryobiology, 21, 704 (1984).
14b. Gale, L., Alcor experiment: Surviving the cold, Long Life Magazine, 2,
    58-60 (1978).

          Living adult human and animal brain tissue

15. Haan, E.A., and D.M. Bowen, Protection of neocortical tissue prisms
    from freeze-thaw     injury by dimethyl sulphoxide, J Neurochem, 37,
    243-246 (1981).
16. Pascoe, J.E., The survival of the rat's superior cervical ganglion
    after cooling to -76*C, Proc. Roy. Soc. (London) B, 147, 510-519 (1957).
17. Hardy, J.A., P.R. Dodd, A.E. Oakley, R.H. Perry, J.A. Edwardson, and
    A.M. Kidd,    Metabolically active synaptosomes can be prepared from
    frozen rat and human brain, J Neurochem, 40, 608-614 (1983).
18. Walder, H.A.D., The effect of freezing and rewarming on feline brain
    tissue: an electron microscope study  In: The Frozen Cell,  G.E.W.
    Wolstenholme and M. O'Connor, Eds., J. & A. Churchill, London, 1970.
    pp. 251-266.

          Living fetal human and animal brain tissue

19. Groscurth, P., M. Erni, M. Balzer, H.-J. Peter, and G. Haselbacher,
    Cryopreservation of human fetal organs, Anat Embryol, 174, 105-113 (1986).
20. Houle, J.D., and G.D. Das, Cryopreservation of embryonic neural tissue
    and its successful transplantation in the rat brain, Anat Rec, 196, 81A
21. Houle, J.D., and G.D. Das, Freezing of embryonic neural tissue and its 
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22. Houle, J.D., and G.D. Das, Freezing and transplantation of brain tissue
    in rats, Experientia, 36, 1114-1115 (1980).
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    tissues and their transplantation in the brain of rats: technical
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    of fresh and freeze-stored rat hippocampal tissue:  a comparison of
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25. Jensen, S., T. Sorensen, and J. Zimmer, Cryopreservation of fetal rat
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          Living human and animal isolated brain cells

26. Silani, V., A. Pizzuti, O. Strada, A. Falini, et al, Human neuronal
    cell cryopreservation, (abstract from unidentified literature source)
27. Kim, S.U., G. Moretto, B. Ruff, and D.H. Shin, Culture and
    cryopreservation of adult human oligodendrocytes and astrocytes,
    Acta Neuropathol (Berlin), 64, 172-175 (1984).
28. Kawamoto, J.C., and J.N. Barrett, Cryopreservation of primary neurons
    for tissue culture,  Brain Res, 384, 84-93 (1986).
29. Scott, B., and L. Lew, Neurons in cell culture survive freezing, Exp
    Cell Res, 162, 566-573 (1986).

            Post-mortem human and animal brains

30. Itabashi, H.H., W.W. Tourtellotte, B. Baral, and M. Dang, A freezing
    method for the preservation of nervous tissue for concomitant molecular
    biological research and histopathological evaluation, J Neuropath Exp
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31. Tourtellotte, W.W., R.C. Cohenour, J. Raj, A. Morgan, R. Warwick, J.
    Sweeder, et al, The NINCDS/NIMH human neurospecimen bank,
    Neuro-Psychopharmacol, 2, 1593-1595 (1978).
32. Bird, E.D., Brain tissue banks, Trends in Neurosci, 1(5), I-II (1978).
33. Tourtellotte, W.W., H.H. Itabashi, I. Rosario, and K. Berman, National
    neurological research bank: A collection of cryopreserved human
    neurological specimens for neuroscientists, Ann Neurol, 14, 154 (1983).
34. Haberland, N., L. Hetey, H.A. Hackensellner, and G. Matthes,
    Characterization of the synaptosomal dopamine uptake from rat and
    human brain tissue after low temperature preservation, Cryo-Letters,
    6, 319-328 (1985).
35. Stahl, W.L., and P.D. Swanson, Effects of freezing and storage on
    subcellular fractionation of guinea pig and human brain, Neurobiology,
    5, 393-400 (1975).
36. Schwarcz, R., Effects of tissue storage and freezing on brain glutamate
    uptake, Life Sci, 28, 1147-1154 (1981).
37. Brammer, M.J., and P. Ray, Preservation of oligodendroglial cytoplasm
    in cryopreservative-pretreated frozen white matter, J Neurochem, 38,
    1493-1497 (1982).
38. Iqbal, K., et al., Oligodendroglia from human autopsied brain.  Bulk
    isolation and some chemical properties, J Neurochem, 28, 707-716 (1977).
39. Morrison, M.R., and W.S.T. Griffin, The isolation and in vitro
    translation of undegraded messenger RNAs from human post-mortem brain,
    Anal.  Biochem, 113, 318-324 (1981).
40. Tower, D.B., S.S Goldman, and O.M. Young, Oxygen consumption by frozen
    and thawed cerebrocortical slices from warm-adapted or hibernating
    hamsters: the protective effects of hibernation, J Neurochem, 27,
    285-287 (1976).
41. Tower, D.B., and O.M. Young, The activities of butyrylcholinesterase
    and carbonic anhydrase, the rate of anaerobic glycolysis, and the
    question of a constant density of glial cells in cerebral cortices of
    various mammalian species from mouse to whale, J Neurochem, 20, 269-278
42. Tower, D.B., and O.M. Young, Interspecies correlations of cerebral
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          Spinal cord and spinal nerves

43. Anonymous, Histological study of a temporarily cryopreserved human,
    Cryonics, #52, 13-32 (Nov, 1984).

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