370 
SURGERY AND TRANSPLANTATION 
minutes or hours, depending on the size of the 
specimen and the temperature differential be- 
tween the specimen and the cooling medium. 
In a majority of instances the cell death in 
mammalian tissues may be simply the result of 
failure to survive crystallization of freezing. 
Ice crystals may be formed both extracellularly 
and intracellularly. Whether ice crystal forma- 
tion is extracellular or intracellular is, at least 
in part, the function of rate of freezing. Ice 
formation is essentially extracellular when the 
drop in temperature is relatively slow. During 
ultrarapid freezing (many degrees per second), 
microscopic ice crystals form within cells and 
throughout the suspending fluids. Crystal size 
is also a function of the rate of cooling. How- 
ever, after the solid state has been reached, 
recrystallization or growth of crystals may oc- 
cur, depending on the temperature of storage.*^ 
To avoid lethal damage by freezing, tissues 
are exposed to cryoprotective compounds prior 
to freezing. Although the exact mechanism of 
action of agents in the prevention of freezing 
injury is not well understood, these agents have 
been successfully used in freezing of certain 
mammalian tissues. After thawing, the viabil- 
ity of such tissues has been adequately demon- 
strated both in vivo and in vitro. 
The first chemical agents which prevented 
freezing damage when added to tissues were 
ethylene glycol and glycerin glycerol. These 
were later supplemented by dimethyl sulfoxide. 
Since the introduction of ethylene glycol, 
glycerol and dimethyl sulfoxide (DMSO) as 
cryoprotective agents, polyvinyl pyrrolidone 
(PVP), dextran, hydroxyethyl starch and mag- 
nesium have been added to the list. However, no 
systematic screening, pharmacological testing 
and evaluation of cryoprotective properties of 
new potential cryoprotective compounds on dif- 
ferent biologic systems has been undertaken. 
Thus, old, familiar agents — glycerol and di- 
methyl sulfoxide — remain the most widely used 
and studied agents.*^ 
Toxicity of cryoprotective compounds has 
been customarily measured by alteration of cell 
structure and cell growth. Toxicity in whole 
organs can be reliably estimated only by meas- 
uring function on transplantation. The growth 
of HeLa cells in cultures was severely inhibited 
by exposure to DMSO in "cryoprotective" con- 
centrations, suggesting that a large proportion , 
of cells did not survive the treatment. Con- i 
comitantly, with the reduction of the growth : 
potential cytopathologic changes were noted in ; 
treated cells.** . 
In addition to direct toxic injury cryoprotec- | 
tive agents produce osmotic changes when in- | 
troduced in the vascular tree. Karow*^ states i 
that 2.IM glycerol or DMSO, when introduced | 
in the vasculature, exert approximately 2,100 « 
mOsm of osmotic force attracting water to the I 
vascular compartment. Such osmotic imbalance , 
exists as long as it takes the cryoprotective [ 
agent to equilibrate between the vascular and 
the interstitial fluid. i 
In whole hearts perfused with 2.IM DMSO | 
in Ringer's solution, initial "phasic contracture" | 
which relaxed gradually, presumably due to ; 
osmotic equilibration, was observed. No marked 
changes in the contractility of hearts perfused ' 
with media NCTC 135 prepared with hyper- 
tonic balanced salt solution, and up to 15% 
DMSO were noted by the author. However, 
when the concentration of DMSO exceeded 
15%, the heart stopped contracting abruptly 
(personal observations). 
Compared to isolated cell suspensions, whole 
organs are indeed very complex structures. 
When fluids of various tonicity are introduced 
in the vasculature of organs, equilibration takes 
place primarily between the interstitial fluid and 
the intravascular fluid, as is evident from per- 
fused organs in which edema can be produced 
by perfusion with hypotonic and "isotonic" solu- 
tions. In such organs the interstitial spaces are 
widely dilated and, if edema is severe, the deli- 
cate supportive connective tissue is disrupted. 
In the heart, the myocardial fibers themselves 
remain relatively unaffected. Edema forms 
quickly, as judged by an extremely rapid gain 
in weight of about 20-30% of either hearts or 
kidneys perfused with the so-called isotonic 
solutions. 
Edema in perfused organs is reduced rapidly 
when 2 to 3x "isotonic" concentration of inor- 
ganic salt is used in the perfusate. Autotrans- 
planted kidneys perfused with these solutions 
sustain life of rabbits and dogs in the presence 
of an immediate contralateral nephrectomy, and 
