Cryobiology 50 (2005) 121–138

www.elsevier.com/locate/ycryo

q
The thermodynamic principles of isochoric cryopreservation
a,b,*
Boris Rubinsky , Pedro Alejandro Perez a, Morgan E. Carlson b

a
Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94720, USA
b
Department of Bioengineering, University of California at Berkeley, Berkeley, CA 94720, USA

Received 6 July 2004; accepted 13 December 2004

Abstract

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume)
cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed
in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient
experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage
during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably
causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been devel-
oped as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show
that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature
by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation
could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very
simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the the-
oretical thermodynamic predictions.

2005 Elsevier Inc. All rights reserved.

Keywords: Isochoric cryopreservation; Cryoprotectants; Hyperbaric cryopreservation

Introduction aspects of medicine, agriculture, veterinary medi-

The need to preserve biological materials for
extended periods of time is encountered in many
q
This work was supported by the Arnold and Barbara
Silverman Distinguished Chair in Bioengineering.
*
Corresponding author. Fax: +1 510 642 6163.
E-mail address: rubinsky@me.berkeley.edu (B. Rubinsky).
cine, and biotechnology. The principle of biolog-
ical material preservation is ingrained in the
observation that life processes are temperature
dependent chemical reactions, whose sum is the
metabolism. Similar to the kinetics of every
chemical reaction, the metabolism can be also re-
duced by lowering the temperature. Most en-
zymes of normothermic animals show a 1.5- to

0011-2240/$ - see front matter
2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cryobiol.2004.12.002
122 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
2-fold decrease in metabolic activity for every
10 C decrease in temperature, purely from ki-
netic predictions [1]. Since the reduction of meta-
bolic rates is a strong function of temperature, it
would be obviously most desirable to preserve
biological materials close to absolute zero, where
the chemical reactions cease and they can be pre-
served indefinitely. However, biological materials
are mostly water in a physiological saline solu-
tion. Freezing removes the water from solution,
in the inert form of ice crystals and completely
modifies the biophysical environment the cells
experience. The process of freezing and the dam-
age that this induces to cells and tissues will be
discussed next, briefly.
Because we exist in an environment at a con-
stant (isobaric) pressure of 1 atm most of the re-
search on cryopreservation was done under
isobaric atmospheric conditions. At constant pres-
sure, when the temperature becomes lower than
the solution freezing point temperature at that
pressure, freezing may occur. In pure water at
atmospheric temperatures, this is 0 C. In physio-
logical saline, this is
0.57 C. However, freezing
is a probabilistic event. For ice to form, water mol-
ecules must assemble, during their random move-
ment, into an ice-like structure (nucleus) with a
critical size, or assemble around an impurity in a
critical nucleus size [7]. Pure water, in the absence
of nucleation sites can supercool to temperatures
as low as
40 C [6]. Preservation of biological
materials at a very low temperature, in a super-
cooled state, could be an ideal means for signifi-
cantly lowering the cell metabolism without
inducing freezing damage. Unfortunately, super-
cooling and ice nucleation is a probabilistic event,
and in reality solutions of cells usually freeze at
temperatures higher than
5 C in an unpredict-
able manner.
The probability of ice nucleation is a function
of the volume of the solution. Therefore, ice nucle-
ation usually occurs first in the much larger extra-
cellular space, when cells are frozen in a cellular
suspension, or in the vascular and interstitial space
when tissues are frozen [16]. The probability for
cells to freeze intracellularly is much lower, be-
cause of their smaller volume. Furthermore, even
when a cell occasionally freezes, the ice will not
trigger ice formation in other cells. During freez-
ing of biological materials, the ice and solution
in the extracellular space are in thermodynamic
equilibrium. However, the unfrozen cells are ther-
modynamically supercooled and have a higher
Gibbs free-energy and Helmoltz potential from
the extracellular solution. It is important to recog-
nize, in the context of this paper, that to equili-
brate the difference in chemical potential between
the interior of the cell, where the solution is super-
cooled, and the exterior of the cell, in which ice is
in thermodynamic equilibrium with the extracellu-
lar solution, water will leave the cell through the
water-permeable cell membrane. This is the only
possible mechanism. Consequently, the intracellu-
lar solution will become hypertonic. It was origi-
nally proposed by Lovelock [10] and later
incorporated in his comprehensive theory by Ma-
zur [11] that the increased hypertonic intracellular
solution damages the cells. It is not entirely clear
what is the mechanism of hypertonic damage;
chemical denaturation of proteins and other mol-
ecules, or changes in the cell structure or both.
Nevertheless, it appears that the damage to cells
increases with increasing extracellular concentra-
tion and with time of exposure—which validates
the concept of chemical damage, e.g., [20,21].
However, experiments have also shown that it is
not only the temperature which affects the survival
of cryopreserved cells. In fact, the rate with which
the cells are brought to cryogenic temperatures is
probably the most important thermal parameter
of cryopreservation. This parameter is known as
the cooling rate and is usually given in C/min.
The effect of cooling rates is explained to be re-
lated to the process of mass transfer (primarily
water transport) across the cell membrane during
cooling in the high subzero freezing zone of tem-
peratures. The intracellular composition does not
become instantaneously equal to the extracellular
composition. The mass transfer across the cell
membrane and the cell shrinkage are a rate depen-
dent function of time, cell membrane permeability,
and driving force, i.e., extracellular solution osmo-
lality. Since the chemical damage is a function of
temperature, and since the mass transfer and cell
dehydration are a function of time, it should be
anticipated that if cells are brought rapidly to
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
cryogenic temperature the chemical mode of dam-
age during freezing could be eliminated. Indeed, it
has been observed that increasing the cooling rates
during freezing improves cell survival [11,12].
However, experiments have shown that this in-
crease in viability suddenly reverses when the cool-
ing rates exceed a certain optimal value, and cell
survival decreases precipitously with a further in-
crease in cooling rates. When cell survival is plot-
ted as a function of cooling rates, the survival
signature has an inverse U-shape, with survival
gradually increasing with an increase in cooling
rates to an optimal cooling rate, and than decreas-
ing with a further increase in rate. While, this is an
ubiquitous behavior of every cell, the values of
each so-called inverse U-shaped survival curve is
cell type specific and the optimal cooling rate
can vary even by orders of magnitude from cell
type to cell type. Experiments have correlated
the decrease in cell survival with the sudden for-
mation of intracellular ice [11,12,20,21,4]. The
sudden formation of intracellular ice is also re-
lated to the dynamics of mass transfer across the
cell membrane during freezing. When cells are
cooled too rapidly for the intracellular solution
to equilibrate osmotically with the extracellular
solution, the intracellular solution is thermody-
namically supercooled. The probability for intra-
cellular ice nucleation increases with the extent
of thermodynamic supercooling. It is not clear if
the nucleation site for intracellular ice formation
is intracellular, extracellular or on the membrane
[22]. However, whatever the cause of intracellular
ice may be, it is almost always lethal to the cells,
and it is avoided in designing cryopreservation
protocols.
We will show later, in our thermodynamic
studies, that in isochoric cryopreservation the
cells will be exposed to an intracellular chemical
composition that is almost an order of magnitude
lower than that during isobaric cryopreservation
at the same temperature. This is the basis of
our hypothesis that isochoric cryopreservation
could improve the survival of biological materials
at low temperatures. It should be emphasized that
this paper deals with the thermodynamics of iso-
choric cryopreservation and not the proof of the
hypothesis.
123
Isobaric cryopreservation
The underlining principle in cryopreservation
techniques is to avoid intracellular chemical dam-
age in the high subzero temperatures range, during
the biological materials excursion to lower temper-
atures. Cryopreservation, as practiced today, is
mostly an isobaric (constant pressure) process,
which takes place at a pressure of 1 atm. It is nat-
ural that this type of process would have evolved,
because it is convenient to implement on Earth.
Working at constant atmospheric pressure, the
most obvious (in hindsight) method to reduce
chemical damage at high subzero temperatures is
by adding compounds that depress the freezing
temperature of the solution, can penetrate the cell
membrane and do not harm the cell and its constit-
uents. Again, it is relevant to this study to recog-
nize that most cryoprotective agents need to
easily penetrate the cell membrane to be effective.
Smith and co-workers [14] found that the addition
of glycerol allows cattle sperm to survive freezing
at the dry ice temperature of
79 C. Subse-
quently, it was found that other polyhydric alco-
hols and other compounds, such as dimethyl
sulfoxide, which penetrate the cell membrane,
facilitate frozen cell survival. The protective effect
of these cryoprotectants is probably through their
colligative effect. Cryoprotectants such as glycerol,
ethylene glycol and DMSO penetrate the cell and
depress the freezing temperature of the solution.
Among other effects they cause a reduction in the
intracellular and extracellular ionic concentration
and they reduce potentially lethal shrinkage during
slow freezing, at any temperature. This paper sug-
gests that in isochoric cryopreservation, pressure
could produce a similar effect to a cryoprotectant
with the ability to penetrate the cell
instantaneously.
Hyperbaric cryopreservation
Hyperbaric cryopreservation is another method
to reduce the ionic concentration at subzero Cel-
sius temperatures [15]. It has some similarity to
the isochoric cryopreservation proposed in this
study because isochoric cryopreservation also
leads to elevated pressures, but in a different
124 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

way. Isochoric cryopreservation is a two phase vice technology needed is cumbersome and
thermodynamic equilibrium process. In hyperbaric expensive while in the former the device technol-
cryopreservation, the solution is maintained as a ogy is extremely simple and inexpensive.
single phase in a liquid form. By increasing the
pressure of the solution ice is completely avoided.
The absence of ice causes the solution in the bio- Materials and methods
logical material to remain at its original composi-
tion. Hyperbaric pressures are used more often in Experimental isochoric cryopreservation system
food preservation and in preservation of tissue
structure for cryomicroscopy as opposed to cryo- Isochoric cryopreservation systems are simple.
preservation [9,17]. There, elevated pressures, fol- This is a system in which ice and the solution exist
lowed by rapid freezing lead to preservation of in thermodynamic equilibrium at a constant tem-
the biological material structure and are used for perature and constant volume. They require only
this purpose. Several attempts have been also re- a constant volume chamber, capable of withstand-
ported on the use of hyperbaric pressures for or- ing the pressures that develop in the system. For
gan preservation at reduced temperatures, control, or analysis, there is the need to measure
without the deleterious aspects of freezing, e.g., the internal pressure and temperature. Fig. 1 illus-
kidney [5], liver [19] or cell preservation [18]. In trates the system which we have used and which
general, the elevated pressures were obtained by can withstand pressures up to 75.8 MPa. The
means of mechanical devices. In the kidney pres- chamber is made from stainless steel and has an in-
sures of 1000 atm were used and it was found that ner diameter of 1 in and outer diameter of 2.13 in
pressure reduces the concentration of chemical and an inner length of 3 in. The constant volume
additives needed for vitrification [5]. No survival chamber is sealed with a screw and metal seal
was reported. In the liver, pressures were obtained and is connected to an Omega electronic pressure
to about 70 MPa. It was found that livers can sur- transducer with a rupture disk of 60 MPa. To cool
vive preservation of about 35 MPa but succumb to the system, it was introduced in a controlled tem-
higher pressures. Survival was found to be a func- perature bath (Neslab RT-140). This arrangement
tion of the rate in increase in pressure, as well as allows controlling the temperature of the system
the magnitude of the pressures. In cells, where
pressure was examined independent of tempera-
ture, it was found that they can survive to
200 MPa for brief periods of time and that the
ATP is depleted [18]. There are certain mecha-
nisms which may have led to damage in the hyper-
baric cryopreservation protocols described in
those studies. Thermodynamic analysis points to
these mechanism and they will be discussed in fu-
ture papers. The most obvious is that the elevated
pressure is detrimental to cells. However, in tradi-
tional hyperbaric cryopreservation there is no at-
tempt to minimize the pressure for a certain
cryopreservation temperature. In isochoric cryo-
preservation, the system naturally adjusts itself to
the minimal pressure for the particular cryopreser-
vation temperature and thereby minimizes the tox- Fig. 1. Isochoric cryopreservation chamber. It consists of a
constant volume chamber that is hermetically sealed and in
icity of the pressure. Another compelling argument which the pressure is monitored with a pressure gage. The
in favor of isochoric cryopreservation over hyper- chamber is filled with fluid and is cooled by immersion in a
baric cryopreservation is that in the later the de- constant temperature bath.
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138 125

and measuring the pressure during isochoric freez-
ing in a constant volume two phase system.
The principle of operating the isochoric cham-
ber is illustrated in Fig. 2. In a typical experiment
the solution is introduced in the chamber. Biolog-
ical samples, such a suspension of cells or organs
are also introduced in the chamber. The open
chamber is filled to the rim and immersed in a con-
stant temperature bath. The temperature of the
bath is lowered to the value of the phase transition
temperature of the solution in the chamber. When
the temperature of the chamber has reached the
phase transition temperature, a 3 · 3 mm ice crys-
tal is introduced in the chamber to serve as a seed
and to induce nucleation at a predetermined loca-
tion—usually far away from the cells or organ.
The chamber is then sealed in the constant temper-
ature bath. The temperature of the bath is gradu-
ally reduced and the temperature of the chamber
follows that of the bath. The isochoric chamber
internal pressure is continuously monitored to
determine that the chamber performs properly.
In experiments, to verify the fundamental thermo-
dynamic correlations of isochoric cryopreserva-
tion, we continuously measured pressure and
correlated the pressure to the bath temperature.
In the experimental part of this study, we per-
formed a variety of experiments with solutions of
potential interest to cryopreservation and com-
pared the experimentally determined tempera-
ture–pressure correlations to the correlations
obtained from thermodynamic analysis.
Thermodynamic analysis
The goal of the thermodynamic analysis is to
develop mathematical tools that facilitate the
understanding and design of isochoric cryopreser-
vation systems. In addition, we will use the analy-
sis to illustrate theoretically the advantages of
isochoric cryopreservation over isobaric and
hyperbaric cryopreservation. The analysis of iso-
choric cryopreservation processes follow certain
fundamental principles, which will be listed here:

Fig. 2. Typical freezing process in the isochoric chamber. The open chamber with the solution and the biological material is first
brought to the phase transition temperature of the solution. An ice crystal is introduced to serve as a nucleation seed to avoid
supercooling and random nucleation. The chamber is tightly sealed and the temperature of the chamber uniformly reduced. The
process of freezing starts from the nucleation site. The constant volume chamber contains a mixture of liquid and ice, which is a unique
function of temperature.
126 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
(a) The isochoric cryopreservation process
occurs in a two phase thermodynamic sys-
tem, in which the ice and liquid co-exist
and are in thermodynamic equilibrium.
(b) In a two phase system in thermodynamic
equilibrium, the temperature and pressure
are interrelated and one specifies the other.
(c) In an isochoric system the volume is constant.
(d) In a two phase isochoric system, at a pre-
scribed temperature or pressure, the only
possible variable that adjusts itself to fulfill
the requirement of thermodynamic equilib-
rium and constant volume is the ‘‘quality’’
of the system, i.e., the relative percentage of
ice and liquid in the system.
(e) Either temperature or pressure and the qual-
ity at each temperature or pressure com-
pletely specify the process of freezing in
isochoric cryopreservation.
We will now describe how to fully specify an
isochoric two phase system using first the freezing
of pure water as an example.
Isochoric freezing of pure water
The phase change temperature of a simple ther-
modynamic system comprised of a pure substance
in a gravity field is a unique function of pressure.
In other words, for any pressure there is a specific
and unique temperature at which change of phase
takes place, and vice versa. The pressure tempera-
ture phase diagram for pure water is well known,
e.g., [8]. Particularly relevant to the isochoric cryo-
preservation process is the interface between ice I
and water on the phase diagram (Fig. 3, after
[8]). Table 1 [8], gives the thermodynamic proper-
ties upon phase transition between pure water
and ice I and ice III. To facilitate analysis, we have
developed a regression curve for the relation be-
tween pressure and temperature during phase tran-
sition between pure water and ice I, to the corner
of liquid water, ice I and ice III. This is given in
Fig. 4 and in Eq. (1).
P ¼
0:1461T 2
12:58T þ 0:1013: ð1Þ
In this correlation, the temperature is expressed in
C and pressure in MPa. The corner between li-
Fig. 3. Phase diagram for water in the region of ice I (after [8]).
quid water, ice I and ice III is
21.985 C at
209.9 MPa.
Table 1 illustrates an important property that
critically affects isochoric cryopreservation. It is
seen that ice I, unlike ice III, has a higher specific
volume than the water in thermodynamic equilib-
rium with it. Therefore, when water freezes to ice
I in an isochoric system the pressure of the system
will increase—because the specific volume of the
composite will increase, in a constant volume. At
the pressures analyzed here, water and ice cannot
be considered incompressible—they will compress.
The temperature and pressure of a two phase iso-
choric system are dependent. Therefore, if for in-
stance the temperature of the isochoric system is
fixed and there is freezing, the percentage of ice
that will form will self-adjust to generate in the
constant volume the pressure required for thermo-
dynamic equilibrium between ice and water at that
pressure. The percentage of ice or water in an iso-
choric two phase system can be therefore calcu-
lated for each temperature and the system
completely specified.
The calculations shown in Appendix A, lead to
an expression for the quality Z, which gives the
percentage weight of water relative to the total
weight of the mixture in a two phase mixture of
ice I and water in an isochoric system at equilib-
rium at a particular temperature and pressure.
This expression takes the form
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138 127

Table 1
Thermodynamic properties with phase transition from liquid to ice I and ice III, after [9]
Phase transition Temperature (C) Pressure (MPa) Volume change (cm3/g) Enthalpy change (kJ/kg)
Liquid to ice I
20 193.3 +0.1313
241
Liquid to ice I
15 156 +0.1218
262
Liquid to ice I
10 110.9 +0.1122
285
Liquid to ice I
5 59.8 +0.1016
308
Liquid to ice I 0 0.1 +0.09
334
Liquid to ice III
22 207.5
0.0466
213
Liquid to ice III
20 246.2
0.0371
226
Liquid to ice III
17 346.3
0.0241
257

aT 1 ðP 0 ; T Þ ¼ A1 þ A2 T þ A3 T 2 þ A4 T 3 ; ð4Þ

b0T 1 b1
bT 1 ðP ; T Þ ¼ ; b0T 1 ¼ ; ð5Þ
1 þ m1 b0T 1 P 1
b2 T
where A1 is the 1.5756 · 10
4, A2 is the
5.556 · 10
7, A3 is the 2.655 · 10
8, A4 is the
7.11 · 10
10, b1 is the 1.827 · 10
5, b2 is
the 1.418 · 10
3, m1 is the 5. The units in Eqs.
(3)–(5) are degrees Celsius for temperature and
bars for pressure.
The correlations and constants for ice are from
[3].
For water
" Z Z #
P Tk
0 0 0 0
v2 ¼ v10 exp
bT 2 ðP ;T ÞdP þ aT 2 ðP 0 ; T ÞdT :
Fig. 4. Regression correlation between pressure and tempera- P0 T k0
ture for phase transition between water and ice I.
ð6Þ

v0
v1 The compressibility and thermal expansion coeffi-

Z¼ ; ð2Þ cients are
v2
v1
!
where the subscript 0, 1, and 2 represent initial X
4
bT 2 ðP ; T Þ ¼ bi P  10
4 ;
i
ð7Þ
conditions at the onset of freezing (1 atm in our
i¼0
calculations), ice and water, respectively.
The specific volumes for ice and water as a func-  
B
tion of temperature and pressure will be given in aT 2 ¼ Aþ  10
4 : ð8Þ
CþC
the following set of equations.
For ice In Eq. (8) A, B, C, and C are functions of temper-

Z P Z T  ature and pressure expressed as follows.
v1 ¼ v10 exp
bT 1 ðP 0 ;T ÞdP 0 þ aT 1 ðP 0 ; T 0 ÞdT 0 :
P0 T0 A ¼ a1 þ a2 T K þ a3 T 2K
ð3Þ B ¼ a4 þ a5 T K þ a6 T 2K þ a7 T K C þ a8 C
ð9Þ
Here, b and a are the compressibility coefficient C ¼ a8 þ a9 T K þ a10 T 2K þ a11 T 3K
and the coefficient of thermal expansion, respec-
C ¼ P þ a13 P 2 þ a14 P 3 ;
tively [17]. The subscript 0 represents the proper-
ties at the freezing point of pure water at 1 atm. where
128 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

(3)–(9) and the insertion of the values of the spe-

a1 = 4.78506 · 101
cific volume at that temperature and pressure in
a2 =
8.12847 · 10
2
Eq. (2).
a3 = 8.49849 · 10
5
a4 = 5.56047 · 105
Isochoric freezing of a solution
a5 =
3.76355 · 103
a6 = 5.56395
Biological materials are made of aqueous solu-
a7 = 5.59682 · 10
3
tions. During cryopreservation, cryoprotectants
a8 =
2.76522 · 101
are often added to reduce the chemical damage
a9 =
4.28067 · 103
during freezing, thereby further increasing the
a10 =
3.39150 · 101
complexity of the solution. Therefore, while iso-
a11 = 3.65873 · 10
1
choric freezing of pure water is of fundamental
a12 =
5.89617 · 10
4
interest, during isochoric cryopreservation there
a13 = 3.28892 · 10
4
is freezing of a solution. To the best of our knowl-
a14 = 2.65933 · 10
8
edge, there is no thermodynamic information on
the process of freezing of aqueous solutions in an
and isochoric system. From fundamental concepts of
thermodynamics it is possible to state that the
b0 = 4.41753 · 10
1
phase change temperature of a solution in a simple
b1 =
1.09205 · 10
4
gravity field is a function of pressure and solution
b2 = 1.99785 · 10
8
composition. In this study, we propose that as a
b3 =
2.08128 · 10
12
first-order approximation the freezing temperature
b4 = 8.86050 · 10
17
of an aqueous solution in an isochoric system can
be expressed as
The specific volume of water can be calculated T ph ðP ; cÞ ¼ T 0 þ DT ðP Þ þ DT ðcÞ; ð11Þ
numerically using the constants given above. The
units are degrees Kelvin for temperature and bars where Tph is the phase change temperature for iso-
for pressure. The expressions and the constants for choric freezing of a solution, T0 is the freezing tem-
water are from [13]. perature of pure water at the reference pressure of
From Eqs. (1) and (3)–(9) it is possible to ob- 1 atm, DT (P) is the temperature depression due to
tain, through numerical integration the specific an increase in pressure, and DT (c) is the tempera-
volumes of ice and water in a two phase, equilib- ture depression as function of solute concentration,
rium, ice–water system, at each temperature and c. In this first-order approximation we assume that
its corresponding pressure. From knowledge of the effect of pressure and solute concentration in
the original specific volume of the water, and the water freezing temperature are independent of each
specific volumes of water and of ice at the new other. We anticipate that this assumption is correct
thermodynamic equilibrium conditions, we can at lower pressures and concentrations and that at
obtain from Eq. (2) the percentage weight of water higher pressures and concentrations the effects
relative to the percentage weight of the mixture in may not be linearly additive. Nonlinear correlations
an isochoric system, at each temperature and pres- will probably be obtained later, when experimental
sure. The percent of ice in the mixture is given by data becomes available.
Ice % ¼ ð1
Z Þ  100: ð10Þ Pressure
In summary, the set of Eqs. (1)–(11) completely
specify an isochoric two phase system of pure The correlation for DT (P) is given bellow.
water–ice I, in thermodynamic equilibrium. For
each temperature the pressure is given by Eq. (1) DT ðP Þ ¼
4E
10P 3
3E
7P 2
and the quality by the numerical solution of Eqs.
0:00809P þ 0:00819547826: ð12Þ
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
This equation is a best fit correlation obtained
from Fig. 4 in a similar way to Eq. (1). Here, the
temperature change is in degrees Centigrade or
Kelvin and the pressure in MPa.
Concentration
In this paper, we will develop the methodology
for thermodynamic analysis of isochoric freezing
of solutions using for illustration two aqueous solu-
tions of interest to cryopreservation: sodium chlo-
ride and a solution of physiological concentration
of sodium chloride with glycerol. In the derivation
of the methodology we will further assume, as a
first-order approximation, that when the solution
contains several solutes the effect of each compound
is to depress the freezing temperature independent
of the other and therefore the effect of different com-
pounds is also linearly additive. Obviously this
assumption is rigorously correct only in a dilute
solution. For instance in a composite solution of
glycerol and sodium chloride the freezing point
depression due to the solutes is taken to be
DT ðcÞ ¼ DT NaCl ðcNaCl Þ þ DT glycerol ðcglycerol Þ:
Data on the freezing point depression of NaCl and
other additives of interest to cryopreservation can
be found in reference [2]. Fig. 5 shows the freezing
point depression of NaCl as a function of temper-
Fig. 5. Freezing point depression as a function of concentration of NaCl.
129
ature. An analytical correlation can be obtained
from Fig. 5, of the form
DT NaCl ðcÞ ¼
294:15c3
80:193c2
54:402c:
ð14Þ
The freezing point depression, due to ethylene gly-
col and due to glycerol in water, are given in Figs.
6A and B, respectively [2]. The correlation for
freezing point depression of ethylene glycol as a
function of temperature is given by
DT e
glycol ðcÞ ¼ 231:48c4
354:94c3 þ 76:389c2

49:559c þ 0:0132: ð15aÞ
In the case of glycerol the relation between freez-
ing temperature and concentration was so irregu-
lar that different correlations where produced for
segments of the temperature in different ranges of
concentration.
0 6 c < 0:2895
DT glycerol ðcÞ ¼ 92:743c3
75:17c2
16:408c
0:2895 6 c < 0:3378
ð13Þ DT glycerol ðcÞ ¼
1006:1c3 þ 561:08c2
128:97c þ 5:9249
0:3378 6 c < 0:4825
DT glycerol ðcÞ ¼
9811:1c3 þ 12223c2
5085:7c þ 689:01
0:4825 6 c < 0:6755
DT glycerol ðcÞ ¼ 394:85c2
573:06c þ 165:43: ð15bÞ
130 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

Fig. 6. Freezing point depression as a function of concentration of (A) ethylene glycol and (B) glycerol.

The concentrations c, used in Eqs. (14) and (15)
are in terms of mass ratio, i.e., mass of substance
per volume of solution. The following equation
can be used to correlate the concentration (mass
ratio) with molarity.
c ¼ MW  M  v;
where MW is the molecular weight, M molarity,
and v is the solution specific volume.
The methodology for thermodynamic analysis
of isochoric cryopreservation employs the entire
set of Eqs. (1)–(16). The analysis also incorporates
the observation that ice I has a very tight crystal-
lographic structure and therefore does not incor-
porate solutes. Consequently, during freezing of
ð16Þ
solutions the solutes are rejected into the liquid
phase and their mass is conserved. Because the
data is not available, we will assume as a first-or-
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
der approximation that the compressibility of glyc-
erol equals that of the water. When available, ther-
modynamic data on the compressibility and
thermal expansion of cryoprotectants can be used
similarly to the way the compressibility and ther-
mal expansion of water are used in this study.
The goal of the analysis is to completely charac-
terize the thermodynamic process of isochoric freez-
ing. The thermodynamic state of the isochoric
freezing solution is fully characterized by the corre-
lation between temperature and pressure in thermo-
dynamic equilibrium and by the quality of the
solution. Therefore, the analysis will produce curves
that correlate for each initial composition of aque-
ous solution: (a) the pressure of the system and the
temperature in which the system is in two phase
equilibrium during isochoric freezing; and (b) the
quality and the temperature. The following algo-
rithm has been developed to characterize the pro-
cess of freezing in an isochoric cryopreservation
system.
Since the freezing temperature is a function of
pressure and concentration, and since the concen-
tration in the solution is dependent on the
amount of ice formed which in turn is function
of pressure and temperature, the freezing temper-
ature can not be calculate directly. The freezing
temperature has to be calculated iteratively. The
following iterative algorithm was used to deter-
mine for a certain initial composition of solution
the freezing temperature at each pressure and the
corresponding quality, during isochoric
cryopreservation.
1. Start with the pressure, P and the initial solu-
tion concentration, c0
2. Calculate DT (P) using Eq. (12).
3. Calculate DT (c) using the corresponding Eq.
13,14,15a,15b,16.
4. Calculate the freezing temperature using Eq.
(12).
5. Evaluate the quality, Z, with Eqs. (2)–(9) for
the pressure P and the temperature in step 4,
following procedure used for pure water.
6. Evaluate the new concentration due to the
formation of ice, from the assumption
that ice does not incorporate solutes and
conservation of mass using the following
equation:
131
c0
c1 ¼ ;
Z
where, c1 is the new calculated concentration,
c0 and Z is the initial concentration and Z
the quality.
7. Repeat steps 3 and 4.
8. Evaluate the percent difference of freezing
temperature with the temperature of the pre-
vious iteration.
 
 i 
T ph
T i
1
ph 
%diff ¼ ;
T iph
where T iph is the temperature obtained in the
i
1
present iteration and T ph is the temperature
of the previous iteration.
9. If the percent of difference is less than a spec-
ified tolerance terminate the loop and record
the freezing temperature and the quality at
the analyzed pressure.
10. If not repeat the loop.
Results and discussion
The process of isochoric freezing of pure
water is completely characterized by Eqs. (1)–
(10). Figs. 4 and 7 completely characterize the
thermodynamic state of pure water during iso-
choric freezing. Fig. 4 is the relation between
pressure and temperature during freezing of
pure water. This correlation is universal for
any ice–water solution in equilibrium and there-
fore also holds for isochoric freezing. Fig. 7 was
obtained from Eqs. (1)–(10) and shows the per-
centage mass of ice during the isochoric freezing
of pure water as a function of the temperature
of the system. Fig. 7 also shows an important
aspect of design of isochoric cryopreservation.
This aspect is also illustrated in Fig. 2. It is
important to notice that during isochoric freez-
ing, a substantial portion of the water has
transformed to ice. Therefore, if during cryo-
preservation it is desirable to avoid ice in the
biological media the nucleation site for ice
should be at a distance from the biological
material. Additionally, the volume of the system
should be designed in such a way as to avoid
132 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

Fig. 7. Percentage mass of ice as a function of the temperature of the system during isochoric freezing of pure water.

the possibility that the ice will extend into the
space occupied by the material.
Figs. 8 and 9 were obtained using the iterative
algorithm for thermodynamic analysis of isochoric
freezing in a solution. These figures completely
characterize the thermodynamic state of the ice-so-
lution mixture during freezing of a NaCl solution
which at 1 atm is at a physiological concentration
of 0.9% w/w. Fig. 8 shows the pressure–tempera-
ture correlation at thermodynamic equilibrium
during isochoric freezing. The solid line is the re-
sult of the calculations. The data points on the fig-

Fig. 8. The change of phase temperature–pressure correlation during the isochoric freezing of an aqueous solution of NaCl with an
initial concentration of 0.9% w/w at 1 atm.
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138 133

and methods section. Three experiments are shown

Fig. 9. Mass percentage of ice versus temperature for the
isochoric freezing of a solution of NaCl with an initial
concentration of 0.9% w/w at 1 atm.
ure were obtained by measuring the pressure as a
function of the temperature of the system with
the isochoric chamber described in the Materials
for each temperature. The experimental results are
within the range that can be obtained with our
chamber. It is evident that the experimental data
correlates well with the mathematical predictions
in this range. This suggests that in this range our
assumption in Eq. (11), on the linearly additive ef-
fects of pressure and concentration, is valid. Fig. 9
shows that in isochoric cryopreservation a sub-
stantial part of the volume of the chamber will
be occupied with ice. This reinforces our earlier
statement that in designing isochoric preservation
protocols which attempt to avoids ice in the bio-
logical material it is important to physically sepa-
rate between the biological material and the ice
and design the chamber with the ability to produce
this separation.
Fig. 10 is perhaps the most interesting figure
in this paper. It compares the concentration of
NaCl as a function of temperature during iso-
choric and isobaric (at 1 atm) freezing of a NaCl

Fig. 10. Comparison of concentration of NaCl as a function of temperature during isochoric and isobaric (at 1 atm) freezing of a NaCl
solution, that at a pressure of 1 atm had a physiological concentration of 0.9% w/w.
134 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

solution, that at a pressure of 1 atm had a phys-
iological concentration of 0.9% w/w. The curve
for isochoric freezing was obtained from step 6
in the iterative analysis and that for isobaric
freezing from the phase diagram for NaCl. This
figure shows that the effect of isochoric freezing
is to reduce the concentration of solutes at each
temperature by almost a factor of 10 relative to
the concentration during isobaric freezing. In ef-
fect the pressure acts as a very effective cryopro-
tectant. Because pressure instantaneously affects
the entire volume, it can be viewed as a cryopro-
tectant with an ability to enter the interior of the
cell, infinitely fast. We believe that in this prop-
erty of pressure is the yet untapped potential of
isochoric cryopreservation.

Fig. 11. Temperature–pressure correlation during isochoric freezing of solutions that at 1 atm have a concentration of 1 and 2 mol
ethylene glycol (A) and glycerol (B) in physiological concentration of NaCl.
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138 135

Fig. 12. Mass percentage of ice versus temperature during isochoric freezing of a solution that at 1 atm. had a physiological
concentration of NaCl and 1 and 2 mol concentration of ethylene glycol (A) and glycerol (B).

Figs. 11A and B and 12A and B were obtained
using the iterative algorithm for thermodynamic
analysis of isochoric freezing in a solution. They
also show results of experiments. These figures
completely characterize the thermodynamic state
of the ice-solution mixture during freezing of a
NaCl solution which at 1 atm is at a physiological
concentration of 0.9% w/w with 1 and 2 mol ethyl-
ene glycol (panels A) and glycerol (panels B). Fig.
11 shows the pressure–temperature correlation at
thermodynamic equilibrium during isochoric
freezing. The solid line is the result of the calcula-
tions. The data points on the figure were obtained
by measuring the pressure as a function of the tem-
perature of the system with the isochoric chamber
described in the Materials and methods section.
Three experiments were made for each data point.
The data points for glycerol essentially superim-
pose and only one is shown for clarity. The exper-
imental results are within the range that can be
obtained with our chamber. It is evident that the
experimental data correlates well with the mathe-
matical predictions in this range. The general ten-
dency of the mathematical analysis is to
underpredict the freezing point depression. While
in this range of measurements the assumptions in
Eq. (11) still hold, relatively well, we anticipate
that for higher pressures and concentrations there
136 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138

will be a synergistic effect between the concentra-
tion and pressure and our curve will serve as the
lower limit in the freezing point depression that
can be obtained with isochoric cryopreservation
at each pressure. At this stage in our research we
are in the process of completing the curves to the
triple point of ice I–Ice III and liquid, a new iso-
choric freezing chamber. Figs. 12 also show that
in isochoric cryopreservation a substantial part
of the volume of the chamber will be occupied with
ice. This further reinforces our earlier statement
that in designing isochoric preservation protocols,
which attempt to avoid ice in the biological mate-
rial it is important to physically separate between
the biological material and the ice, thus design
the chamber with the ability to produce this
separation.
Figs. 13A and B expand on the same interesting
observation seen in Fig. 10. They compare the
osmolality of the solution as a function of the

Fig. 13. Comparison of osmolality as a function of temperature during isochoric and isobaric (at 1 atm) freezing of a NaCl solution,
that at a pressure of 1 atm had a physiological concentration of 0.9% w/w with 1 and 2 mol ethylene glycol (A) glycerol (B).
 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
freezing temperature during isochoric and isobaric
(at 1 atm) freezing of a NaCl and ethylene glycol
(panel A) and glycerol (panel B) solution, that at
a pressure of 1 atm had a physiological concentra-
tion of 0.9% w/w NaCl and 1 or 2 mol ethylene
glycol or glycerol. The curves extend to the triple
point between ice I, ice III, and the liquid. These
figures also show that the effect of isochoric freez-
ing is to reduce the concentration of solutes at each
temperature by almost a factor of 10, relative to
the concentration during isobaric freezing. In effect
the pressure acts as a very effective cryoprotectant
that is additive to conventional cryoprotectants
and with an ability to enter the interior of the cell,
infinitely fast. This property of pressure should
have value in every aspect of isochoric cryopreser-
vation, which can be: (a) preserving biological
materials unfrozen at reduced temperatures; (b)
freezing or (c) partial freezing followed by rapid
freezing and perhaps vitrification.
Summary
We have developed thermodynamic principles
for isochoric (constant volume) cryopreservation
for low temperature preservation of biological
materials. Experiments with a simple isochoric
cryopreservation chamber have verified some of
our predictions. The equations, which were devel-
oped here could serve as guidelines in designing
isochoric cryopreservation protocols. The theoret-
ical studies in this paper show that in isochoric
cryopreservation, the increase in solution concen-
tration during freezing is lower at each tempera-
ture by almost an order of magnitude from that
in isobaric cryopreservation. This is obviously
the effect of pressure. In effect the pressure acts
as a very effective cryoprotectant that is additive
to conventional cryoprotectants and has the ability
to enter the interior of the cell, infinitely fast. The
technology for isochoric cryopreservation is very
simple; freezing in a constant volume chamber.
In contrast to hyperbaric freezing, here the process
is of thermodynamic equilibrium and equilibrium
thermodynamics can be used to design predictable
cryopreservation protocols. Isochoric cryopreser-
vation could be used as a means to maintain a bio-
137
logical organ, unfrozen to the temperature that
develops at the corner of ice I and ice III. There
are applications in which this could suffice. Fur-
ther slow cooling of the system will induce the
freezing of the biological material. However, this
will occur at lower temperatures at which the
chemical damage is reduced. Another possibility
is to rapidly cool the system from the corner of
ice I and ice III and induce either rapid freezing
or even vitrification from a lower temperature
and of a much smaller volume than that possible
at isobaric cryopreservation.
Appendix A. Expression for quality in a general
isochoric two phase system
Following is a general derivation for the qual-
ity, Z, which is the ratio between the mass of the
liquid solution to the total mass of the mixture
of liquid and ice in an isochoric, constant volume
system. In the derivation given below, V, m, v,
are volume, mass, and specific volume respectively.
The subscripts, 0, 1, 2, are for the original condi-
tions, ice and liquid, respectively. The original con-
ditions in our isochoric analysis are a pressure of
1 atm, the change of phase temperature of the li-
quid solution at that pressure and the assumption
that the original specific volume is that of the pure
liquid solution at that pressure and temperature.
The expression for Z, is obtained from conserva-
tion of mass in a constant volume.
At all times,
m0 ¼ m1 þ m2 ; ðA:1Þ
V 0 ¼ V 1 þ V 2: ðA:2Þ
The quality Z is,
m2 m2
Z¼ ¼ : ðA:3Þ
m0 m1 þ m2
Conservation of mass in a constant volume and
from the definition of specific volume yields,
V 0 V 1 þ V 2 m1 V 1 m2 V 2
v0 ¼ ¼ ¼ þ
m0 m0 m0 m1 m0 m2
ðm0
m2 Þ
¼ v1 þ Zv2 ¼ ð1
Z Þv1 þ Zv2 : ðA:4Þ
m0
138 B. Rubinsky et al. / Cryobiology 50 (2005) 121–138
Reorganization of the terms in Eq. (A.4), produces
a general expression for the quality of the mixture
during isochoric freezing in terms of the specific
volumes of the ice and unfrozen solution at each
state of thermodynamic equilibrium
v0
v1
Z¼ : ðA:5Þ
v2
v1
References
[1] F.O. Belzer, J.H. Southard, Principles of solid organ
preservation by cold storage, Transplantation 45 (1988)
673–676.
[2] R.E. Bolz, G.L. Tuve (Eds.), CRC Handbook of Tables
for Applied Engineering Science, The Chemical Rubber,
Cleveland, Ohio, 1920.
[3] V.E. Chizhov, O.V. Nagornov, Thermodynamic Properties
of Ice, Water and Their Mixture Under High Pressure,
Glaciers–Ocean–Atmosphere Interactions, Proceedings of
the International Symposium, St. Petersburg (1990).
[4] K.R. Diller, E.G. Cravalho, A cryomicroscope for the
study of freezing and thawing processes in biological cells,
Cryobiology 7 (1970) 191–199.
[5] G.M. Fahy, D.R. MacFarlane, C.A. Angel, Vitrification
as an approach to cryopreservation, Cryobiology 21 (1984)
407–427.
[6] F. Franks (Ed.), Water: A Comprehensive Treatise,
Plenum Press, New York, 1982.
[7] F. Franks, S. Mathias, P. Galfoe, S. Webster, D. Brown,
Ice nucleation and freezing in undercooled cells, Cryobi-
ology 20 (1983) 44–63.
[8] P.V. Hobbs, Ice Physics, Clarendon Press, Oxford, 1974.
[9] M.T. Kalichevsky, D. Knorr, P.J. Lilford, Potential food
applications of high-pressure effects on ice–water transi-
tions, Trends Food Sci. Technol. 6 (1995) 253–259.
[10] J.E. Lovelock, The haemolysis of human red blood cells by
freezing and thawing, Biochem. Biophys. Acta 10 (1953)
412–426.
[11] P. Mazur, Cryobiology: The freezing of biological systems,
Science 68 (1970) 939–949.
[12] H.T. Merryman, Cryobiology, Academic Press, New York,
1966.
[13] L.T. Minassian, P. Pruzan, A. Soulard, Thermodynamic
properties of water under pressure up to 5 kb, and between
28 and 120 C. Estimations in the supercooled region down
to
40 C, J. Chem. Phys. 75 (6) (1981) 3064–3072.
[14] S.A. Polge, A.V. Smith, A. Parkes, Revival of spermatozoa
after vitrification and dehydration at low temperature,
Nature 164 (1948) 666.
[15] R.W. Prehoda, Suspended Animation, Chilton Book,
Philadelphia, NY, London, 1969.
[16] B. Rubinsky, D.E. Pegg, A mathematical model for the
freezing process in biological tissue, Proc. Royal Soc. 234
(1988) 343–358.
[17] P.D. Sanz, L. Otero, C. de Elvira, J.A. Carasco, Freezing
processes in high pressure domains, Int. J. Refrig. 20 (5)
(1997) 301–307.
[18] G.J. Suppes, S. Egan, A.J. Casillan, W.J. Chan, B. Sekar,
Impact of high pressure freezing on DH5a Eschericia coli
and red blood cells, Cryobiology 47 (2003) 93–101.
[19] T. Takahashi, K. Kakita, Y. Takahashi, I. Sakamoto, K.
Yokoyama, T. Fujiu, S. Yamashina, T. Tamaki, Y.
Takazawa, R. Muratsubaki, Functional integrity of the
rat liver after subzero preservation under high pressure,
High Press. Transplant. Proc. 32 (2000) 1634–1636.
[20] K. Tatsutani, B. Rubinsky, A method to study intracellular
ice nucleation, J. Biomech. Eng. ASME Trans. 120 (1)
(1998) 27–31.
[21] K. Tatsutani, B. Rubinsky, G. Onik, R. Dahiya, The effect
of thermal variables on frozen human prostatic adenocar-
cinoma cells, Urology 48 (3) (1996) 441–447.
[22] M. Toner, E.G. Cravalho, M. Karel, Thermodynamics
and kinetics of intracellular ice formation during freezing
of biological cells, J. Appl. Phys. 69 (1990) 1582–1593.