A.  Premises and their scientific evaluation 
B.  Short introductory summary of general conclusions 
C.  Detailed review of relevant current cryobiological knowledge  
    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 

*    *    *    *    *    *    *    *    *    *    *    *    *    *    *  


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 required. 

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 proceed. 

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 apparent. 

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

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 denaturation.   

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

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 kidney.   

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

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 surprising. 

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 glycerol.  

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 (16). 

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: 

Percent recovery* 

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

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 nitrogen." 

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 undertaking. 

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
Edition, A.M.  Karow, Jr. and D.E. Pegg, Eds., Marcel Dekker, New York,
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.
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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 

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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

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.

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 
transplantation in the rat brain, Brain Res, 192, 570-574 (1980). 

22. Houle, J.D., and G.D. Das, Freezing and transplantation of brain tissue
in rats,  Experientia, 36, 1114-1115 (1980). 

23. Das, G.D., J.D. Houle, J. Brasko, and K.G. Das, Freezing of neural
tissues and their  transplantation in the brain of rats: technical details
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24. Jensen, S., T. Sorensen, A.G. Moller, and J. Zimmer, Intraocular grafts
of fresh and  freeze-stored rat hippocampal tissue:  a comparison of
survivability and histological  and connective organization, J Comp Neurol,
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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

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

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 (1973). 

42. Tower, D.B., and O.M. Young, Interspecies correlations of cerebral
cortical oxygen  consumption, acetylcholinesterase activity and chloride
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Spinal cord and spinal nerves 

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