Download Mechanisms of Cryoinjury in Living Cells

Mechanisms of Cryoinjury in Living Cells
Doyong Gao andJ. K. Critser
Abstract
B
iological metabolism in living cells dramatically
diminishes at low temperatures, a fact that permits the
long-term preservation of living cells and tissues for
either scientific research or many medical and industrial
applications (e.g., blood transfusion, bone marrow transplantation, artificial insemination, in vitro fertilization, food
storage). However, there is an apparent contradiction between
the concept of preservation and experimental findings that
living cells can be damaged by the cryopreservation process
itself. The challenge to cells during freezing is not their
ability to endure storage at very low temperatures (less than
-180°C); rather, it is the lethality of an intermediate zone of
temperature (-15 to -60°C) that a cell must traverse twice—
once during cooling and once during warming. Cryobiological research studies the underlying physical and biological
factors affecting survival of cells at low temperatures (during
the cooling and warming processes). These factors and
mechanisms (or hypotheses) of cryoinjury and its prevention
are reviewed and discussed, including the most famous "twofactor hypothesis" theory of Peter Mazur, concepts of cold
shock, vitrification, cryoprotective agents (CPAs), lethal intracellular ice formation, osmotic injury during the addition/
removal of CPAs and during the cooling/warming process,
as well as modeling/methods in the cryobiological research.
of cells and tissues for scientific research as well as many
medical applications such as blood transfusion, bone marrow
transplantation, artificial insemination, and in vitro fertilization. The major steps in the cryopreservation process can be
summarized as follows:
1. Add cryoprotective agents (CPAsl) to cells/tissues before
cooling.
2. Cool the cells/tissues toward a low temperature (e.g., at
-196°C, the liquid nitrogen temperature at pressure of
1 atm) at which the cells/tissues are stored.
3. Warm the cells/tissues.
4. Remove the CPAs from the cells/tissues after thawing.
Cryobiology is a multidisciplinary science to study the physical
and biological behaviors of cells and tissues (including their
interactions with environment) at low temperatures (especially less than the freezing point of water, 0°C). Biological
metabolism in living cells dramatically diminishes at low
temperatures, a fact that permits the long-term preservation
Major advantages of cryopreservation include the possible
banking of a large quantity of cells and tissues for human
leukocyte antigen typing and matching between recipients
and donors; facilitating the transport of cells/tissues between
different medical centers, and allowing sufficient time for
the screening of transmissible diseases (e.g., HIV) in the
donated cells and tissues before transplantation. However,
there is an apparent contradiction between the concept of
preservation and experimental findings that cells/tissues can
be damaged by the preservation process itself. Injury to cells
has been shown to be caused by each step or a combination
of the steps described above. An extremely important part of
the research in fundamental cryobiology is to reveal the
underlying physical and biological mechanisms related to
the injury of cells at low temperatures (so-called cryoinjury),
especially those associated with the phase change of water in
both extra- and intracellular environments. Understanding
the mechanisms of cell cryoinjury will make it possible to
establish biophysical/mathematical models describing cell
responses to environmental change during the cryopreservation process and to use these models to develop the optimal
procedures/devices for the long-term cryopreservation of
living cells, preventing cryoinjury. The fundamental cryobiology theories associated with the mechanisms of the cryoinjury in cells and its prevention are reviewed and discussed
below.
Dayong Gao, Ph.D., is Associate Professor in the Department of Mechanical
Engineering, Center for Biomedical Engineering, and the Department of
Physiology, University of Kentucky, Lexington, Kentucky. J. K. Critser,
Ph.D., is Director of the Cryobiology Institute and Professor in the Departments of Pediatrics and Immunology and Microbiology, Indiana University
Medical School, Indianapolis, Indiana.
'Abbreviations used in this article: CPA, cryoprotective agent; Ea, activation
energy; IIF, intracellular ice formation; L , permeability coefficient of a cell
to water; S/V, surface area to volume ratio.
Keywords: cryobiology; cryopreservation; cryoprotective
agents; fundamental cryobiology; intracellular ice formation;
principles/mechanisms of cryoinjury
Introduction
Volume 4 1 , Number 4
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187
Cryoinjury Mechanisms during the
Freezing Processes
Contrary to popular belief, the challenge to cells during
cryopreservation is not their ability to endure storage at low
temperature; rather, it is the lethality of an intermediate zone
of temperature (-15 to -60°C) that cells must traverse twice—
once during cooling and once during warming (Mazur 1963).
As cells are cooled to approximately -5°C, both the cells and
surrounding medium remain unfrozen and supercooled.
Between -5 and approximately -15°C, ice forms (either
spontaneously or as a result of artificially introducing ice
crystals to the solution) in the external medium; however, the
cells' contents remain unfrozen and supercooled, presumably because the plasma membrane blocks the growth of ice
crystals into the cytoplasm. The supercooled water in the
cells has, by definition, a greater chemical potential than that
of water in the partially frozen extracellular solution; and
water thus flows out of the cells osmotically and freezes
externally (Figure 1). The subsequent physical events in the
cells depend on the cooling rate, as shown in Figure 1. If
cells are cooled too rapidly, intracellular water is not lost fast
enough to maintain equilibrium; the cells become increasingly
supercooled, eventually attaining equilibrium by freezing
intracellularly (Mazur 1963, 1990). Muldrew and McGann
(1994) invoked an osmotic rupture hypothesis and model of
intracellular freezing injury based on solid experimental data
and theoretical derivations, indicating that the water flux across
the cell membrane during the freezing/thawing can induce
intracellular ice formation causing injury. Diller and Cravalho
(1970) (also Diller 1982) developed novel microscopy to
observe the cell behavior as well as intracellular ice formation
(IIF1) temperatures and HF dynamics during the freezing
processes. More recently, based on heterogeneous nucleation
theory and thermodynamics as well as experimental results,
Toner and colleagues (1990, 1993) developed an IIF model
and equations describing the probability of IIF as a function
of cooling rate, temperature, and cryobiological characteristics of a specific cell type. The freezing behavior of the cells
can be modified by the addition of CPAs, which affect the
rates of water transport, nucleation, and crystal growth.
Karlsson and coworkers (1994) further developed a model of
diffusion-limited ice growth inside cells in the presence of
CPAs. They successfully predicted the IIF phenomena as a
function of cooling rate, temperature, and CPA concentration; and they also predicted optimum cooling protocols preventing IIF. In most cases, cells undergoing IIF are killed
(Farrant 1970, 1977; Fujikawa 1980; Mazur 1977, 1984;
Muldrew and McGann 1994; Rapatz et al. 1963; Steponkus
and Wiest 1979; Stowell et al. 1965; Toner et al. 1990,1993;
Trump et al. 1965).
If cooling is sufficiently slow, the cells will lose water
rapidly enough to concentrate the intracellular solutes sufficiently to eliminate supercooling. As a result, the cells
dehydrate and do not freeze intracellularly. However, if the
cells are cooled too slowly, they will experience a severe
volume shrinkage and long-term exposure to high-solute concentrations (composed mainly of electrolytes) before they
reach a eutectic temperature (when all components in a solution are solidified). Both volume shrinkage and long-term
exposure may cause cell injury (Figure 1). Lovelock (1957)
proposed that the increased concentration of solutes and cell
dehydration have deleterious effects on the lipid-protein complexes of cell membranes, weakening them and increasing
lipid and phospholipid losses. The cell is therefore rendered
permeable to electrolytes and swells, eventually rupturing.
Levitt (1962) proposed that loss of water from the protoplasm brings protein molecules into apposition, presenting
the opportunity for formation of new chemical bonds previously too distant and rigidly structured in hydrated form to
permit combination. Thawing has a disruptive force on the
new combination, permitting unfolding and denaturation.
-30°C
Slow Cooling
+22°C
-10°C
Optimal Cooling
Rapid Cooling
Figure 1 Schematic drawing of physical events in cells during freezing.
188
WAR Journal
Karow and Webb (1965) explained slow freezing injury as a
consequence of the extraction of bound water from cellular
structures for incorporation into ice crystals, denuding proteins
of lattice-arranged bound water essential to cell integrity.
The effects that may cause cell injury as a result of concentration of solutes, termed "solution effects," have been
characterized collectively by Mazur and colleagues (1972),
who suggest that these effects on cells are greatly enhanced
in a slow cooling process when the exposure of cells to a
highly concentrated solution is prolonged. They also indicated that hyperosmotic stress may cause a net leak/influx of
nonpermeating solutes (mainly electrolytes); when cells are
returned to isosmotic conditions, they swell beyond their
normal isotonic volume and lyse. Meryman (1970, 1974)
proposed that the slow freezing results in the decrease in
intracellular volume beyond a critical volume. As the cell
reduces in size in response to increasing extracellular osmolality, the compression of contents enhances the resistance to
further shrinking. This compression results in a hydrostatic
pressure difference across the cell membrane, incurring cell
membrane damage.
Steponkus and Wiest (1979) invoked a maximum cell
surface area hypothesis: The cell shrinkage induces irreversible membrane fusion, hence the effective area of cell membrane is reduced; when returned to isotonic condition, the
cells lyse before their normal volume is recovered. Some
cells have been shown to exhibit a reversible inhibition of
shrinkage at some point during a progressive increase in
extracellular solute concentration, implying the development
of membrane stress (Williams 1979). Alternate theories have
embraced such mechanisms of slow-freezing injury as
macromolecule denaturation from dehydration or phase transition of membrane lipids (Fishbein et al. 1978). Pegg and
Diaper (1988) invoked a "packing effect," which is an inverse
dependence of cell survival on the proportion of the initial
sample volume occupied by the cells. Mazur and Cole (1985),
however, have proposed that a considerable portion of the
damage, at least in human erythrocytes, is due to the reduction in the size of the unfrozen channels, which results in
increased cell-ice or cell-cell contacts or interactions.
In general, a cooling rate that is either too high or too low
can kill cells, although the mechanisms underlying cell damage are different. These cell responses to freezing were first
expressed quantitatively by Mazur (1963) and directly linked
with cell cryoinjury by the following two-factor hypothesis
(Mazur et al. 1972): (1) At slow cooling rates, the cryoinjury
occurs due to the solution effects (i.e., the solute/electrolyte
concentration, severe cell dehydration, and the reduction of
unfrozen fraction in the extracellular space); and (2) at high
cooling rates, cryoinjury occurs due to the lethal IIF. The
optimal cooling rate for cell survival should be low enough
to avoid HF but high enough to minimize the solution effects.
Based on these criteria, an optimal cooling rate for cell
cryosurvival should exist between high and low rates, as
shown in Figure 1. This scheme has been confirmed experimentally. As shown in Figure 2, cell survival plotted as a
function of cooling rate produces a characteristic inverted
U-shaped curve. There is an optimal cooling rate for a
specific cell type. For a given cell type, a low cooling rate
(relative to the optimal cooling rate) results in solution effects
cryoinjury, whereas a high cooling rate (relative to the optimal cooling rate) results in lethal IIF.
It is clear from the cryobiology theory described above
that whether a given cooling rate is too high or low for a
given cell type depends on the ability of water to move across
the cell membrane (i.e., the water permeability of the cell
70
60 *?
W
5 0
"
20 10 -
0.1
1
10
100
1000
10000
COOLING RATE (C/min)
Figure 2 Relation (inverted "U" shape) between percentage of survival and cooling rate in three types of cells. Stem cells refer to mouse
bone marrow stem cells, and hamster cells refer to hamster oocytes. RBC refers to human red blood cells. Adapted from Mazur P. 1976.
Freezing and low temperature storage of living cells. In: Muhlbock O, ed. Proceedings of the 1974 Workshop on Basic Aspects of Freeze
Preservation of Mouse Strains, Jackson Laboratory, Bar Harbor, ME. Stuttgart: Gustav Fisher Verlag. p 1-12.
Volume 4 1 , Number 4
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189
membrane, which is cell type dependent). Differences in
water permeability account largely for the magnitude of
difference in optimal cooling rates for different cell types
(Figure 2). Numerous investigators (Diller 1982; Gao et al.
1994,1996; Levin et al. 1976; McGrath 1985; McGrath and
Sherban 1989) have developed devices/modeling to determine cell membrane permeabilities. Devireddy and colleagues
(1998) developed a novel differential scanning calorimeter
method to determine cell permeabilities during the freezing
processes. The rate of water transport across a cell membrane during the freezing process with different cooling rates
can be described by four simultaneous equations (Mazur
1963). The first equation relates the loss of cytoplasmic water
to the chemical potential gradient between intracellular supercooled water and external ice, expressed as a vapor pressure
ratio:
dV_LpART
—
dt
P
,
in
V»
R
(1)
where V is the volume of cell water (|am3/cell), t is time; Lp is
the permeability coefficient for water ((irn/min/atm) (hydraulic
conductivity); A is the cell surface area (|Jm2); R is the
universal gas constant; T is temperature (K); v is the molar
volume of water; and P e and P4 are the vapor pressures of
extracellular and intracellular water, respectively. The
change in this vapor pressure ratio with temperature can be
calculated from the Clausius-Clapeyron equation and
Raoult's law, as follows:
dln(P c /P i ) = L f
dT
RT 2
Nv
dV
(V + N v o ) V d T '
Here, N is osmoles of intracellular solute, and Lf is the latent
heat of fusion of ice. Time and temperature are related by the
cooling rate (B), which, if linear, is given by:
(3)
dt
Finally, L is related to temperature by the Arrhenius relation,
= Lpo exp
5s.
R IT
T.
(4)
where L is the permeability coefficient of water at a known
temperature, To. Ea is the activation energy of L , and R is
the universal gas constant.
To avoid intracellular freezing, the water content of the
cell given by the four equations above must, before reaching
the intracellular ice-nucleation temperature (usually between
190
-5 and -40°C), have approached the equilibrium water content given by:
V=
Y+Nv.
(5)
RT
273RJ
where W{ is the initial cell water volume and M; is the initial
osmolality of the extracellular solution.
The quantitative expressions above permit one to calculate the extent of supercooling in cells as a function of the
cooling rate, provided that one knows or can estimate the
permeability coefficient of the cell to water (L *), its temperature coefficient or activation energy (EaJ), the osmoles
of solute initially in the cell, and the ratio of the cell surface
area to volume. The results of such calculations are commonly expressed as plots of the water content of a cell as a
function of temperature relative to the normal or isotonic
water volume. In Figure 3, for example, computed water loss
curves for mouse ova are shown. The calculated extent to
which a cell becomes supercooled is the number of degrees
that any given curve is displaced to the right of the equilibrium curve at a given subzero temperature. In the example
shown, mouse ova cooled at 4°C/min will be supercooled by
15°C as the temperature passes through -20°C. Three biological parameters have special influence on the position and
shape of these curves: the L ; its Ea; and the size of the cell
or, more properly, its surface area to volume ratio (S/V1). An
increase in L produces an effect similar to a decrease in
cooling rate. The effect of cell size is the opposite. An
increase in size reduces the cooling rate required to produce
a given probability of intracellular freezing. Changes in L
and S/V shift the positions of the curves. Changes in the Ea
have a major effect on the shape of the curves. For instance,
the dashed line in Figure 3 represents the effect of changing
the E a from 14 to 17 kcal/mol at a cooling rate of 4°C/min.
All of the parameters needed to compute the curves can be
estimated from permeability measurements. Once computed,
these curves can be used to estimate the probability of intracellular freezing as a function of cooling rate. Cells that have
dehydrated close to equilibrium before reaching their icenucleation temperature will have a zero probability of undergoing intracellular freezing. Cells that are still extensively
supercooled when cooled to their nucleation temperature,
and therefore still hydrated, have a high probability of undergoing intracellular freezing.
It should be mentioned that in the text above, Mazur's
theory and method have been introduced as an example to
describe how to predict an optimal cooling rate to prevent
IIF. Different parameters and cryobiological properties of
cells will be needed to predict optimal cooling conditions to
prevent IIF if different physical models are used, as described
by Toner and coworkers (1990, 1993) and Muldrew and
McGann (1994).
WAR Journal
8C7min
[2C7min;Ea=17kcal/mol]
-10
-20
-30
-40
-50
-60
TEMPERATURE (°C)
Figure 3 Computed kinetics of water loss from mouse ova cooled at 2 to 8°C/min in 1 M dimethylsulfoxide. The curve labeled EQ represents
the intracellular water content that the cell must achieve to maintain chemical potential between intra- and extracellular ice, as a function of
temperature. The other solid curves, labeled 2, 4, or 8°C/min, were computed assuming the activation energy (Ea) of L to be 14 kcal/mol.
Dashed curve reflects the effect of changing Ea to 17 kcal/mol. Adapted from Mazur P. 1984. Freezing of living cells: Mechanisms and
implications. Am J Physiol M7(Cell Physiol 16):C125-C142.
Preventing Cryoinjury during Slow Freezing
As described above, we know that a "slow" cooling process
should be used to avoid IIF. Although the avoidance of
numerous and large intracellular ice crystals is necessary for
cell survival, it is not sufficient. Most cells also require the
presence of CPAs. The role of glycerol as an effective CPA
for sperm and human erythrocytes was discovered approximately 50 yr ago by a group in Mill Hill, England (Polge and
Lovelock 1952; Polge et al. 1949). Glycerol remains one of
the more effective and commonly used CPAs to date,
although others (e.g., dimethylsulfoxide, ethylene glycol,
methanol, propylene glycol, and dimethylacetamide) have
emerged since that time and are, in some cases, more effective. The most important characteristics of all of these solutes
are that they are, for the most part, readily able to permeate
the cells and they are relatively nontoxic to cells in concentrations approaching 1 M or more. The primary basis for
their protective action is that they decrease the concentration
of electrolytes during freezing and decrease the extent of
osmotic shrinkage at a given low temperature (Mazur 1984;
Woods et al. 1999). The extent of protection depends primarily on the molar ratio of the CPA to endogenous solutes
inside and outside the cells, and the general protective mechanism of action is thus colligative (i.e., dependent only on the
concentration of dissolved substances and not on their nature).
The effectiveness of a given CPA for a given cell type usually
depends on the permeability of that cell to that CPA and its
toxicity. Although this class of CPAs must permeate to
protect, their permeation before freezing and their removal
after thawing generate osmotic volume changes, which alone
can be damaging (Gao et al. 1995; Mazur and Schneider
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2000
1986; Schneider and Mazur 1988). This topic is discussed
further in the following text (Critser and Mobraaten 2000).
The second class of CPAs is composed of nonpermeating
solutes. This class includes sugars and higher molecular weight
compounds such as polyvinylpyrrolidone, hydroxyethyl
starch, polyethylene glycols, and dextrans. In most cases,
these solutes will not protect in the absence of a permeating
CPA but will often substantially augment the effectiveness
of a permeating CPA or permit the use of a lower concentration of permeating CPA. These nonpermeating CPAs may
contribute to enhance vitrification of the solutions, stabilize
proteins and membranes, and prevent progressive ice formation (Fahy 1986; Fahy et al. 1984; Takahashi et al. 1985).
Preventing Cryoinjury by Vitrification
All discussions above refer to the role of CPAs during slow
equilibrium freezing at low enough cooling rates to prevent
IIF. A diametrically different approach to cryopreservation
is to use either high concentrations of certain CPAs or ultrarapid cooling rates (>106° C/min) to induce the cell cytoplasm
to form a glass (i.e., to vitrify cells/tissues) rather than to
crystallize (Rail and Fahy 1985). However, ultrarapid cooling rates are technically difficult to achieve. Several of the
CPAs that are effective in ameliorating slow freeze injury
also act to promote glass formation, but the required concentrations are so high (e.g., 4-8 M [Fahy et al. 1984]) that they
can be very toxic to the cells/tissues (Fahy 1986; Fahy et al.
1984). Jackson and coworkers (1997) have demonstrated
the significant effect of interaction between the microwave
and CPA concentration on vitrification of aqueous solutions,
191
and they are developing a novel single-mode microwave
cavity to enhance the vitrification with relatively low CPA
concentrations and relatively low cooling rates.
Cryoinjury during the Storage Period
Usually, the cryopreserved samples are stored at or below
-70°C. Although the question is often asked as to the length
of time cells can be kept in the frozen state without damage,
the question is probably moot if the storage temperature is
less than -120°C and is certainly moot at -196°C (liquid
nitrogen). Less than -120°C, chemical reactions cannot occur
in human-relevant times. At -196°C, no thermally driven
reactions can occur in less than geologically relevant times.
The reactions that can occur are the slow accumulation of
direct damage from ionizing radiation, but this accumulation
would probably become significant only after centuries of
storage (Mazur 1984).
Cryoinjury Associated with the
Warming Process
A cell that has survived cooling to low subzero temperatures
is still challenged during warming and thawing, which can
exert effects on survival comparable with those of cooling
(Mazur 1984). These effects depend on whether the prior
rate of cooling has induced intracellular freezing or cell
dehydration. In the former case, rapid thawing can rescue
many cells, possibly because it can prevent the growth of
small intracellular ice crystals into harmful large ice crystals
(i.e., so-called recrystallization). Even when cells are cooled
slowly enough to preclude intracellular freezing, the response
to warming rate is often highly dependent on the freezing
conditions and cell type and is difficult to predict a priori.
For human sperm, previous reports used a wide variety of
imprecise methods yielding a wide variety of warming rates.
These methods include thawing in ambient air, in water baths
with temperatures ranging between 5 and 37°C, in coat
pockets, and thawing the samples in the investigator's hands.
Mahadevan (1980) conducted a more comprehensive study
in which warming rates between 9.2 and 2140°C/ min utilizing coat pocket, hands, air (5, 20, or 37°C), or water baths
(5, 20, 37, 55, or 75°C) were examined in combination with
a standard freezing rate of 10.0°C/min from +5 to -80°C. The
results of that study indicated that slower warming in 20 or
35°C air resulted in more optimal cryosurvival of human
sperm in terms of both motility and supravital staining;
although there were significant differences in postthaw motilities only between the fastest two rates (1837 and 2140°C/min)
and the other 12 rates. Additionally, Mahadevan (1980)
examined the interaction between cooling and wanning rates.
Slow warming was reported optimal for samples frozen
slowly; however, there was little effect of warming rate when
a fast cooling rate was used, probably because the cells had
192
been killed by the IIF during the fast cooling process before
the warming process started.
Osmotic and Potential Toxic Effects of
CPAs on the Cell Injury
Survival of cells subjected to cryopreservation depends not
only on the presence of CPAs but also on the concentration
of the CPAs. For example, the percentage of mouse bone
marrow stem cells that survive freezing after cooling at the
optimal rate increases dramatically as the concentration of
glycerol increases from 0.4 to 1.25 M (Leibo et al. 1970).
The results are similar for other cells (Mazur 1984). Two
important procedures related to the use of permeating CPAs
in the cryopreservation of cells are (1) the addition of a CPA
to the cells before freezing, and (2) the removal of the CPA
from the cells after thawing. Cells transiently shrink when a
CPA is added and then return to near-normal volume as the
CPA permeates. They undergo transient volume expansion
during the removal of the CPA, the magnitude of which
depends on how the removal is effected and on the inherent
permeability of the cell to water and CPA. In several cell
types (e.g., oocytes), the addition of cryoprotectants
(dimethylsulfoxide and 1,2-propanediol) has been shown to
cause disruption of the cytoskeleton chemically (Johnson
and Pickering 1987; Joly et al. 1992).
Sperm of many species (e.g., bull, boar, and human) are
damaged by exposure to glycerol (Watson 1979). Sherman
(1964) reported that glycerol exposure caused a reduction in
human sperm motility. Critser et al. (1988b) have demonstrated that exposure of human sperm to 7.5% glycerol for as
little as 15 min causes a dramatic reduction in sperm motility,
accounting for approximately 50% of the motility loss observed
24 hr after thawing. Historically, bull spermatozoa were
exposed to medium containing glycerol for an extended
period of time, which was referred to as "equilibration time."
However, Berndtson and Foote (1972a,b) reported that
glycerol was able to permeate bull sperm at either 25 or 5°C
within 3 to 4 min, and they further found that maximum
postthaw motility occurred when the sperm were exposed to
glycerol for the shortest time measured (10 sec). These data
suggest that bull spermatozoa are either highly permeable to
glycerol or, unlike other cells, permeation is not required for
protection.
What then is the role of "equilibration time"? Is it, as
some have proposed, a matter of allowing for protective
membrane alterations (Watson 1979)? It is important to note
that human sperm differ from the sperm of most domestic
species and do not show enhanced cryosurvival with increasing delay between addition of CPA and freezing (Sherman
1964). Karow and colleagues (1992) found that the survival
of human sperm in vitro at 3°C was significantly enhanced
by 20 mM K+ in Tyrode's solution relative to Tyrode's solution with less K+. A metabolizable sugar such as glucose was
essential to maintain sperm viability in K+-free media. The
WAR Journal
addition of raffinose to media containing glucose improved
motility of sperm stored at 3°C for 6 hr. The detrimental
effect of glycerol on sperm can be alleviated to a certain
extent by reducing the temperature at which the cryoprotectant is added in both bull and human sperm cells
(Critser et al. 1988a,b; Sherman 1973). To what extent the
sensitivity of sperm to glycerol reflects the osmotic consequence of its addition and removal, or to what extent it
reflects chemical toxicity, is a current subject of investigation. Recently, it has been shown that for human sperm, the
adverse effect of glycerol is attributable mainly to the osmotic
volume changes induced during addition and removal of
glycerol rather than specific chemical action (Gao et al.
1995). In this study, a systematic investigation was also conducted to determine the human sperm volume excursion
(shrinkage or expansion) tolerance limits beyond which the
cells will be irreversibly injured. The tolerance limits determined for human sperm in combination with the permeability
data of cell membrane to water and CPA have been used to
develop the optimal multistep procedures for addition and
removal of glycerol in sperm to prevent cell osmotic injury
(Gao et al. 1995).
Cold Shock Injury to Sperm at
Low Temperatures Higher Than the
Water Freezing Point
Currently, most procedures for cryopreservation of sperm,
across species, utilize an initial slow cooling rate between
body temperature or the temperature at which the sample is
collected (often ambient temperature) and +5°C (Foote 1975;
Graham 1978; Sherman 1973; Watson 1979). In general, this
choice is due to the fact that some species of mammalian
spermatozoa are sensitive to temperature changes in this
range, with abrupt cooling (a high cooling rate) causing a
high frequency of the cells to become irreversibly damaged.
This phenomenon has been termed "cold shock" or "chilling" injury (Watson 1979, 1981, 1995). The sensitivity of
sperm to cold shock varies among species: Bull, ram, boar,
and stallion sperm are highly sensitive; dog and cat sperm
are moderately sensitive; and rabbit and human sperm are
relatively resistant (Critser etal. 1988b; Watson 1979,1995).
The resistance of human sperm to cold shock is of particular
importance in the context of developing new approaches to
human sperm freezing. It is also an important example of the
differences in fundamental cryobiology of cell types among
species, highlighting the fact that simple modifications of
procedures developed for freezing of domestic animal sperm
are unlikely to yield optimal results in other species. Currently, the mechanism of cold shock is unclear.
Other Potential Mechanisms of Cryoinjury
In many previous studies to date, cells have generally been
considered simple compartments of cytoplasmic solution
Volume 4 1 , Number 4
2000
enclosed by a semipermeable plasma membrane. The
emphasis has been on how cell survival is related to the
physical responses of the whole cell to the physical-chemical
events involved in the cryopreservation process. Survival
has generally been defined in terms of its viability (e.g., the
intactness of cell membrane) and/or the ability of the cells to
undergo subsequent growth and development (e.g., hematopoietic stem cells or embryos). Intactness of the plasma membrane is obviously necessary for functional cell survival.
However, in many cases (e.g., granulocytes [Armitage and
Mazur 1984]), an intact plasma membrane is not sufficient.
Findings like the latter emphasized that cells are not simply
bags of cytoplasm bound by a plasma membrane. Rather, within
the bounds of the plasma membrane are other membranebound structures and organelles essential to cell functions.
Little is known about how these intracellular structures and
organelles respond to freezing, partly because it is difficult to
assay their state and function in situ in an unambiguous
fashion (McGann et al. 1988).
The spermatozoon has been used as an especially good
model for investigating the cryobiological role of both intracellular structures and the plasma membrane because sperm
possess two clearly defined and measurable characteristics,
motility and the acrosome reaction phenomenon, both of
which depend on both functional integrity of organelles and
the plasma membrane. An especially attractive feature of the
acrosome reaction endpoint in cryobiology is that it involves
membrane fusional events (Yudin et al. 1988). Investigation
of the effects of freezing on such a reaction may provide an
experimental model for the growing view that membrane
fusion plays a major role in cryobiological injury (Mazur and
Cole 1985, 1989). To be fully functional, sperm must reach
the site of fertilization and the oocyte; they must capacitate,
undergo the acrosome reaction, penetrate the zona pellucida,
and fuse with the plasma membrane of the oocyte. Freezing
could interfere with or ablate the capacity of the sperm to
undergo one or more of these steps (Critser et al. 1987a,b).
Although a motile spermatozoon is not necessarily a fully
functional cell, a nonmotile sperm or one exhibiting a damaged plasma membrane is almost certainly nonfunctional (in
the context of unassisted fertilization). Consequently, multiple levels of biological function must be considered in
assessing the role of the biophysical parameters that have
been shown to be critical to cell survival.
Summary
Although there is great diversity in the cryobiological
response of different cell types or given cells among different mammalian species, cryosurvival requires that cell
freezing and thawing be carried out within certain biophysical and biological limits defined by the following cryobiology principles:
•
Cells must be frozen in such a way that little or none of
their water freezes intracellularly.
193
•
•
Cells must be warmed in such a way that any unfrozen
intracellular water remains unfrozen during warming, or
that small ice crystals formed during cooling remain
small during warming (Mazur 1984).
Even when the conditions described above are met, most
cells will not survive unless substantial concentrations of
CPAs are present. These CPAs must be introduced before
freezing and removed after thawing in ways that do not
exceed osmotically tolerable limits. Their concentrations
also must not be toxic.
Acknowledgments
This work was supported, in part, by grants from the American
Heart Association of the Ohio Valley (9960401V), American Cancer Society (RPG-00-092-01-LBC), National Science Foundation (NSF 9760239), Whitaker Foundation (to
D.Y.G), and National Institutes of Health (R24-RR1 3195
and U01-RR 15012 to J.K.C.).
References
Although these general limits are necessary, they may not be
sufficient for one or more possible reasons. Cells may be
injured by factors such as cold shock that have nothing to do
with ice formation or CPA damage. Another reason is that
cell viability limits are defined primarily in terms of an intact
plasma membrane that retains normal, semipermeable properties. It is possible that conditions that allow the plasma
membrane to "survive" may not allow the "survival" of critical organelles inside cells.
As reviewed above, with the progress in understanding
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