THEODORE I. MALININ 
369 
temperature (87° C) contracted for about 24 
hours. 
The temperature dependence of the amplitude 
of contractions of isolated hearts was noted by 
Nawrocki in 1896.^^ The validity of his ob- 
servations was easily confirmed by Gollan in a 
perfused dog.^o He noted myocardial weakness 
in the region of 20°C. Above 20°C the heart 
contracted with sufficient frequency to eject the 
coronary thebesion flow from the left ventricle. 
At the time of highest contractility, the coro- 
nary flow and the myocardial oxygen consump- 
tion have undergone temperature-dependent 
decreases. 
Some controversy still exists over the im- 
portance of pulsatile versus non-pulsatile flow. 
Little difference is observed in results between 
the two during the first eight-to-ten hours of 
perfusion of the kidney. However, kidneys 
which must be preserved for longer periods 
require pulsatile flow. Contractile ability of the 
heart is lost after non-pulsatile perfusion of 
over six to eight hours (personal observations). 
Thus, the necessity for pulsatile flow for long- 
term perfusion appears to be established. 
To complete the discussion of asanguinous 
perfusion as a means of organ preservation, it 
is necessary to compare the results obtained by 
this method to those obtained by other meth- 
ods. Preservation of functional dog kidneys for 
72 hours by pulsatile perfusion at hypothermia 
has been achieved. ^2 other method of stor- 
age has offered equivalent-or-better results. In 
fact, when the functions of autografted canine 
kidneys preserved for 24 hours by perfusion or 
by simple hypothermia alone were compared 
under identical experimental conditions, per- 
fused kidneys functioned satisfactorily while 
kidneys preserved by hypothermia alone did 
not.^^ The experience with simian hearts is 
similar.ii 
With animal kidneys, perfusion flow char- 
acteristics indicate the patency of vascular bed 
and have been relied upon by many investiga- 
tors. More precise tests of physiologic function 
can also be performed.^'^ 
FREEZING AND CRYOPROTECTIVE AGENTS 
Ideally, long-term preservation methods 
should be simple and should provide for an in- 
definite storage of tissues. Freeze-preservation, 
when possible, meets these criteria. It has made 
a marked impact on tissue culture, on experi- 
mental hematology and on the animal breeding 
industry. It has also provided for indefinite 
storage of non-viable tissues. 
Since the advent of refrigeration, biologic 
materials have been preserved at temperatures 
below 0°C. 
The use of refrigeration for preservation of 
biochemical entities has created a general feel- 
ing that the lower the temperature, the longer 
a specific biological activity will be maintained. 
This is not the case when physiologic integrity 
of organs must be preserved. In practice, all too 
often specimens are frozen at random with tem- 
perature selection being based not on the knowl- 
edge of phenomena associated with freezing, 
but simply on the availability of a given type 
of refrigeration. Changes which are produced 
in tissues, cells and biologic fluids as a conse- 
quence of crude freezing methods are signifi- 
cant. 
Lowered temperatures will decrease meta- 
bolic rate, but cold, by itself, usually does not kill 
cells. However, irreversible injury is produced 
by freezing and is caused by a multiplicity of 
factors which may include chemical injury, 
mechanical injury, dehydration, and metabolic 
aberrations resulting from storage at tempera- 
tures where some enzymes continue to function. 
To achieve the near cessation of biochemical 
activity, it is necessary to store the tissues well 
below 0°. Many investigators have for years 
stored their material at — 79°C, the melting 
point of easily-obtainable solid carbon dioxide. 
However, for long-term storage of tissues, it is 
necessary to maintain temperature below 
— 120°C. Only at such temperatures can the 
damage caused by the gradual growth of ice 
crystals be avoided. These temperatures are 
easily obtained by the use of liquidified gases, 
such as nitrogen and helium. 
A review of events associated with cooling, 
freezing, storage at sub-zero temperatures, and 
thawing of tissues places emphasis on the sig- 
nificance not only of heat transfer, but also of 
various associated phenomena. The change from 
a colloidal to a solid state does not occur in- 
stantaneously but takes place within a few 
