3

1
Institute of Biomedical Technology, State University of New York at Binghamton, Binghamton, New York.
2
Department of Biological Sciences, Binghamton University, Binghamton, New York.
3
Cell Preservation Services, Inc., Owego, New York.
Changing Paradigms in Biopreservation
John M. Baust,
13
Kristi K. Snyder,
13
Robert G. VanBuskirk,
13
and John G. Baust
1,2
The feld of cryopreservation has a long and successful history of in-depth study and progress. Advances in our
knowledge base and our ability to cryopreserve cells have been consequential and have led to its widespread
integration into academic, clinical, and agricultural settings. While many cell systems are successfully cryopre-
served today, there remains signifcant cell loss associated with cryopreservation. Moreover, even today some
cell systems remain uncryopreservable from a practical perspective. This is due to the diversity of post-freeze
responses of individual cells to the various stressors experienced during the freeze-thaw process. In 1998, sev-
eral independent groups reported on the direct involvement of apoptotic and necrotic cell death following cryo-
preservation (Baust, et al., 1998 and Borderie, et al., 1998). In addition to those reports, a substantial literature base
describing the modulation of cell death through the use of various protease inhibitors, free radical scavengers,
media formulations, and other novel compounds exist. These studies have identifed diverse molecular-based,
cellular responses to cryopreservation and have further demonstrated the signifcant improvements in cell sur-
vival through the modulation of molecular events. Numerous studies have reported on the molecular-based
phenomena of cryopreservation-induced delayed onset cell death, yet our understanding of the pathway activa-
tion, progression, control, and the downstream effect on cell function remains in its infancy. To this end, mod-
ulation studies, such as targeted apoptotic control (TAC), have shown promise in furthering our understanding
of the activation pathways and are proving to be a critical next step in the evolution of the cryopreservation
sciences. This review provides an overview of the current literature on the mechanisms of cell death associated
with cryopreservation failure.
Introduction
T
he field of biopreservation is experiencing rapid expan-
sion
1
due in part to the growing interest in personalized
medicine, and drug discovery. The recent successes in cell
therapy reported by Geron coupled with the lift in restric-
tion on stem-cell research, interest should continue to grow.
As such, increasing demands have been placed on the pres-
ervation sciences to improve the viability and function of
complex and sensitive cells including stem cells and engi-
neered cells and tissues. These demands have now stretched
traditional preservation sciences to a limit.
2
As a result,
cryobiology has morphed its focus into the disciplines of
cell and molecular biology to drive continued scientifc
advancement.
3
Underlying this shift is the discovery of the activation of
apoptosis during and following preservation.
4
In 1998, Baust
et al.
5
reported the involvement of apoptosis contributing to
cryopreservation failure. Since that time, numerous studies
into the molecular-based cell death following cryopreserva-
tion have been reported.
4,626
Emphasis over the past 10 years
to adopt the evolving cellular and molecular approaches to
further the understanding of cryopreservation failure has
resulted in a series of studies, many of which are reviewed
and expanded upon in this article.
1,15,18,19,2731
Understanding biopreservation
Interdisciplinary efforts to advance the effective-
ness of cell, tissue, and organ preservation have led to the
development of the scientifc specialtybiopreservation.
Biopreservation, an interdisciplinary approach, incorporates
the felds of cryobiology, engineering, cellular and molecu-
lar biology, including cell signaling, genomics, proteom-
ics, metabolomics, systems biology, and computer sciences.
BIOPRESERVATION AND BIOBANKING
Volume 7, Number 1, 2009
Mary Ann Liebert, Inc.
DOI: 10.1089/bio.2009.0701.jmb
REVIEW
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BAUST ET AL. 4
availability and biological reducing power in a cell, to name
a few.
53
When considering the full context of the oxidative
stressors presented to a cell in the cold, in conjunction with
the generation of free radicals, it is clear that low-tempera-
ture exposure provides multiple routes for the initiation of a
molecular-based stress response.
Cryopreservation
Cryopreservation represents the storage of biological
material at ultra subfreezing temperatures (80C) for
extended periods (weeks to years). Cryopreservation proto-
cols begin with hypothermic exposures, extend through the
hypothermic continuum, and reach equilibrium in the glassy
state (vitrifed). This journey is reversed during the thawing
process. It is essential to recognize that despite the presence
of extracellular ice, cells that are structurally preserved (avoid
intracellular ice formation) remain in a state of deepening
hypothermia until reaching the vitrifcation state (Tg) of
the preservation medium. During this period, solute levels
continue to elevate due to freeze concentration.
54
Cell func-
tion, while suppressed and uncoupled, does not cease until
vitrifcation has been achieved.
55
In order to reduce the prob-
ability of intracellular ice formation during freezing, cryo-
protective agents (CPAs) are added during the initial cooling
phase. CPAs include a diversity of penetrating (membrane
permeable) and nonpenetrating agents, such as DMSO, glyc-
erol, dextrans, sugars, and so on, often contained within a
buffered electrolyte media.
15,32,33
With the frst reports of glycerol serving as a protective
solute and its application to freezing of avian spermatozoa
56

and human erythrocytes (RBC),
57
mammalian cryopreser-
vation research began a decade of advancements that cul-
minated with the addition of DMSO to the preservation
cocktail mix.
58
By focusing on two highly differentiated
cellular products (RBC and spermatozoa) with fxed life
spans, the full spectrum of the impacts of preservation stress
on the complex biology of normal functioning cells was ob-
scured. In effect, these model systems provided a cloak
that obscured the spectrum of events associated with post-
thaw, cryopreservation-induced delayed onset cell death. As
methodological developments proceeded with nontermi-
nally differentiated mammalian systems, many cell types
proved refractory to cryopreservation. Even those cells that
are successfully preserved often demonstrate signifcant
post-thaw death (3070%) within 2448 h.
8
Following addition in the cryoprotective cocktail, cooling
continues at a given rate (1C/min is typical). A seeding
step (ice nucleation) is included in the 2 to 6C range to
prevent excessive undercooling (supercooling) of the cell
and the cryopreservation cocktail. If cooling rates are too
rapid, inadequate cellular dehydration occurs and the proba-
bility of lethal intracellular ice formation increases, resulting
in cell rupture and early-stage necrosis upon thawing.
8,14,23,25

If cooling rates are too slow, it is believed that the extended
exposure to the freeze-concentrated solutes (now multimo-
lar levels) will result in toxic solution effects.
5963
As temperature is lowered below the freezing (melting)
point of the preservation medium, controlled slow cooling
is again utilized to reduce sample temperature to 40 to
80C followed by transfer to ultra low-temperature storage
(ie, liquid nitrogen immersion, liquid nitrogen vapor, or less
than 135C mechanical storage). This temperature range is
Through this integration, biopreservation represents the
simultaneous management of numerous, lethal conditions
(physical and biochemical), with the expectation of normal
recovery. Efforts to sustain living biologics in a dormant state
supportive of reanimation have included either hypother-
mic (refrigerated) or frozen storage.
27
Hypothermic storage
involves maintenance at temperatures in the range of 0C
to ~32C, typically between 2C and 10C. Cryopreservation
is defned as the long-term maintenance of biologics at tem-
peratures below 80C and typically below 140C (below
the reported range of the nominal glass transition tempera-
tures of pure water).
What is striking about the developments within the dis-
tinct subdiscipline of biopreservation is the relative isolation
of cryopreservation studies from organ-based hypothermic
storage research. Studies within the hypothermic stor-
age area have focused primarily on improving tissue and
organ preservation in support of transplantation, target-
ing ion balance, buffering capacity, free radical scavenging,
oncotic support, and the provision of nutrients.
2,5,24,28,29,32,33

Methodological developments falling under the cryopres-
ervation rubric link principles relating survival of cells in
solution to cooling. In other words, cryopreservation has
focused primarily on the physical parameters associated
with freezing events during the preservation process
2,14,34
at
the expense of understanding that a chill-freeze continuum
exists (hypothermic continuum) that impacts survival.
20
This
disconnect has contributed, in part, to the limitations of
obtaining complete survival of normally functioning cells
from cryogenic storage (ie, cell in = cell out).
The hypothermic continuum
Nearly all biopreservation procedures begin with a re-
duction in temperature from 37C to most typically the
0 to 10C range. A maintenance target of 4C is common.
Cooling represents a change in the energy state of a system.
In effect, kinetic energy necessary to support the chemical
reactions that defne the metabolome is reduced resulting in
the uncoupling and shunting of biochemical reactions.
35,36

These biochemical imbalances cause the depletion of ade-
nylates (ATP), and disrupt membrane-mediated transport.
With the progressive drop in temperature, cells experience
rapid gains in calcium,
16,37,38
the loss of potassium,
17,38,39
and
intracellular acidosis (pH levels approaching 4). In addition,
changes in cell and organelle membrane characteristics have
been reported as phase changes in the lipid domains
29,4042

from a liquid-crystalline to the solid-gel state occur.
29,41,42
As
a result, membranes become leaky, thereby contributing
to transmembrane ionic imbalances.
These events occur with minor changes in the kinetic
energy levels. One measure of the advantage total change
in metabolism is Q
10
, which in mammalian systems calculates
to an ~50% decrease in oxygen consumption (metabolism)
for each 10C decrease in temperature.
29,4245
Accordingly,
the oxygen consumption of a cell at 5C is ~6% of that at
37C.
4650
Q
10
represents a simplifcation, as it does not refect
individual reactions but an average of regulatory and non-
regulatory enzymatic processes and hence the net of uncou-
pling/recoupling and shifts in metabolic pathways.
29,40,41,51
Q
10

has been observed to increase dramatically with the onset of
freezing.
52
Accordingly, hypothermia impacts energy status,
macromolecular reactivity and stability, adenylate levels,
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UPDATE ON THE MODERN STATE OF CRYOPRESERVATION 5
infuence ice formation/growth within a cell continue to aid
our understanding of the cryopreservation process. The pro-
cess, which depends on CPAs, has provided for the effective
control of intracellular ice formation.
54,5658,69,7685
Necrosis
Necrotic cell death has also been investigated and reported
in numerous cases of cryopreservation failure.
4,70,71,86,87

Traditionally, necrosis, or pathological cell death, is used
to describe cellular murder.
73,85,88
Necrosis is an energy-
independent form of cell death characterized by cell and
organelle swelling, loss of membrane integrity, lysosomal
rupture, and random DNA fragmentation, ultimately result-
ing in cell lysis (Fig. 1B).
78,79,85,89,90
As a result, cytokines are
released causing the activation of immune and infamma-
tory responses in vivo.
73,79,85,88,90
The initiation and progres-
sion of necrosis often occurs rapidly, in a response to severe
cellular stress resulting in the activation of detrimental in-
tracellular signaling cascades. Necrosis has been shown to
be activated in response to ischemia, osmotic shock, severe
thermal stress, ionic dysregulation, toxic agents, and so on.
Many of these necrotic activating stressors are linked to
cryopreservation.
Apoptosis
Apoptosis plays an integral role in the homeostatic main-
tenance of cell number and tissue size in complex organ-
isms.
91
Apoptotic processes are also a critical line of defense
controlling the daily deletion of damaged cells. Kerr et al.
89
coined the term apoptosis in 1972 referring to cells under -
going a form of cell death described as shrinking necrosis.
Following this report, a distinct feld of investigation describ-
ing, characterizing, and unraveling the associated processes
(genes, proteins, cascades, time course, and morphology)
ideal due in part to it falling below the reported glass tran-
sition temperatures (T
g
) of pure water.
64
The glass transition
temperatures for cryoprotective mixtures vary substantially
and have been reported to be in the 115 to 90C range.
Below T
g
, system viscosity increases exponentially yielding
cessation of all measurable molecular translational motion.
Hence, the presumption is that molecular interactions (ie,
metabolism) halt during the sub-T
g
storage interval.
65
Prior
to reaching the T
g
, chemical reactivity continues at reduced
rates yielding the potential for sustained free radical damage.
It is for this reason that long-term storage at 80C (>612
months) is ill-advised, even for biologics such as serum or
macromolecules. With the transition through T
g
, the hypo-
thermic continuum effectively ends. Structural preservation
is afforded to these cells, but a clear inability to manage the
preservation-induced stresses is apparent. When one con-
siders the stress factors associated with cryopreservation, it
creates a relatively clear picture of the critical involvement
played by the cells biology in responding to freezing.
Accordingly, a focal shift in investigations in cryopreser-
vation has occurred centering around cell stress response
biology.
Understanding Cell Death
A generic listing of cell-based stress factors serves as a
template to guide the design of improved preservation meth-
ods, assuming adequate structural preservation. There are
well-noted differences in the sensitivity of various cell types
to preservation processes.
66
Van Buskirk et al.
20
reported on
these variations in three cell types indicating a possible need
for cell-matched preservation media and protocols. This
study suggested that distinct cell types manage their stress
response through differing molecular pathways. Given this,
the new challenge facing biopreservation is the integration
of a molecular-based logic to develop an in-depth under-
standing of a cells round-trip excursion through the hypo-
thermic continuum.
Modes of cell death
It is now understood that multiple paths of cell death are
associated with cryopreservation failure occurring hours to
days post-thaw (Fig. 1).
15,16,25
Descriptions of the preservation
process have been previously discussed with an emphasis
on the importance of the effect of a cells response to low-
temperature exposure.
1,27,67
In general, increases in cellular
stress results in the activation of apoptotic and necrotic
cascades leading to increased cell death and as such man-
agement of this stress response plays a critical role in pres-
ervation outcome.
Physical events related cell death
Ice-related cell rupture is most commonly associated
with cryopreservation (Fig. 1A). Thousands of studies have
been dedicated to increasing the understanding of the con-
trol and prevention of ice-related cell rupture since Polge et
al.
56
published on the use of glycerol as a CPA in success-
ful cryopreservation. As a result, a plethora of studies have
been devoted to understanding and preventing intracellu-
lar ice formation to facilitate successful cryopreservation
outcome.
8,58,60,63,65,6877
Numerous studies on compounds that
Intracellular
Ice Formation
A
B
C
D
Cellular
Stress
Apoptotic Cascade
ATP Loss
2 Necrosis
Apoptotic Cell
Disassembly
Cell Lysis
Necrosis
FIG. 1. Cell death pathways associated with cryopreser-
vation failure. Diagrammatic representation of the various
paths of death that a cell may undergo as a result of cryo-
preservation stresses: (A) physical ice rupture, (B) necrotic
cell death, (C) apoptotic cell death, or (D) secondary necrosis.
(Adapted from Baust et al., 2002,
14
Baust JM, 2007.
67
)
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BAUST ET AL. 6
also been noted as a consequence of genomic and proteomic
alterations in cells.
114,117120
Gene mutations, either expression
alterations or deletion, often result in the inability of a cell
to progress properly through classical apoptotic cascades,
thereby switching to necrosis. The transitional nature of cell
death pathways in response to similar stressors creates an
extremely complex environment to characterize.
Apoptosis in Cryopreservation
Apoptosis following cryopreservation has now been
documented in a wide variety of cellular systems. Studies
identifying post-thaw apoptosis have appeared in a myriad
of systems including renal cells, fbroblasts, hepatocytes, pe-
ripheral blood mononuclear cells (PBMC), cord blood, sper-
matozoa, oocytes, ovarian tissue, vascular tissue, and so
on.
6,8,12,15,17,121123
Molecular-based cell death
Recently, it has been determined that cell death follow-
ing cryopreservation is linked with apoptotic and secondary
necrotic mechanisms.
3
Many stress phenomena associated
with cryopreservation and a sampling of known apoptotic
initiating stressors. A simple comparison reveals the pleth-
ora of commonalities between cryopreservation stress and
apoptotic activation. A retrospective review of the stresses
associated with cryopreservation intuitively suggests the
involvement of apoptotic processes. In 1995, Jurisicova et
al.
118
reported observations of apoptosis in preimplanted
human embryos and identifed programmed cell death
(PCD) as a contributing factor to post-cryopreservation
embryo demise. That same year, a number of other studies
also described the reduction of cell stress in both cryopres-
ervation and hypothermic storage resulting in improved cell
survival.
69,124130
These studies described the utilization of
compounds including vitamin E, EDTA, protease inhibitors,
and free radical scavengers, all known inhibitors of apop-
tosis, in preservation media (both hypothermic and cryo-
preservation) to positively infuence cell survival. Reports
detailing discrepancies in cell survival following frozen
storage of human keratinocytes observed apoptotic cells
infuencing post-thaw viability assessments.
69
The presence
of apoptotic cells in cryopreserved allograft heart valves fol-
lowing transplantation has also been reported.
131
Although
apoptosis was reported in association with these systems,
it was not until 1998 that studies directly linked apoptosis
to cryopreservation failure.
5
Since then, there have been
many studies looking to identify apoptotic involvement in
cryopreservation failure.
4,7,8,10,14,17,19,23,26,116119,121,131135
In 2000,
Fowke et al.
86
reported on apoptosis following cryopres-
ervation in PBMC. The following year, Fu et al.
6
and Yagi
et al.
7
reported on the involvement of apoptosis following
cryopreservation in mouse and porcine hepatocytes, respec-
tively. Additionally, Schuurhuis et al.
123
and Lund et al.
136

documented apoptosis in PBMCs following thawing. The
presence and contribution of apoptosis has also now been
reported in renal cells,
4,5
fbroblasts,
8
blood cells,
137139
cor-
nea,
140
stem cells,
9,141
cord blood,
10
lymphocytes, sperm,
142

ovarian tissue,
143
and oocytes.
121,144
These reports as well as
others continue to solidify the foundation of molecular-
based cell death following cryopreservation as a universal
phenomena infuencing outcome.
24,26,119,133,145
emerged.
83,8698
These studies have led to the characteriza-
tion of apoptosis as a highly conserved set of cellular pro-
cesses among complex organisms ranging from nematodes
to primates.
93,94,99101
Apoptotic cell death is defned by three stages: initiation,
execution, and termination (Fig. 1C). During each stage, a
series of specifc events is activated as part of a complex cas-
cade leading to cell death. Progression through each stage
requires energy input (ATP) throughout the process without
which, cells may shunt to a necrotic cell death pathway.
102

This shunting has been termed secondary necrosis
15,20,103
and
is discussed in the transitional cell death section further
(Fig. 1D). Apoptosis has been shown to initiate as a result
of stresses including radiation, cytotoxic agents, nutrient
deprivation, excess or diminished gene products, anoxia,
growth factor withdrawal, and temperature.
4,8,11,20,90,96100,104108

Following induction at any number of organelles, apoptosis
proceeds through a cascade of events including caspase ac-
tivation, mitochondrial release of cytochrome C, cell cycle
arrest, externalization of membranous phosphatidylserine,
or alterations in gene expression.
89,91,96,101,102,104,105,109112
These
events lead to the termination stage where DNA is cleaved
into ordered fragments, the membrane blebs and apop-
totic bodies form, and the complete disassembly of the cell
occurs.
Transitional cell death
Molecular-based cell death has been perceived as a
black or white process, proceeding through apoptosis or
necrosis. At the intracellular signaling level, apoptosis is
viewed as true organized molecular response while ne-
crosis involves random molecular events. With that said,
the cell death landscape has evolved substantially over the
past 10 years suggesting that apoptosis and necrosis repre-
sent extremes on each end of the molecular-based cell death
continuum. Bras et al.
113
have suggested that three types of
apoptosis occur. Type I: the conventional apoptosis does not
involve lysosomes but relies on caspase activation; Type II
is characterized by lysosomal-linked autophagocytosis,
whereas Type III is lysosomal-independent, necrosis-like
apoptosis marked by swelling of intracellular organelles. In
fact, many of the caspases now appear to play roles in both
apoptosis and necrosis.
114
It is now thought that when a cell
commits to death, an apoptotic response is activated. This
proceeds through cellular execution (classical apoptosis) or to
the point where the initiation stress becomes too great or en-
ergy levels too low for continuation. At this point, cell death
shunts from apoptosis to necrosis for completion (secondary
necrosis)
20,111,112,115,116
(Fig. 1D). The vacillating nature of apop-
totic and necrotic cell death was demonstrated in Jurkat cells
by Leist et al.
102
as the apoptotic-induced population could be
shifted to necrotic characteristics with the removal of energy
substrates. Conversely, the replenishment of energy returned
the system to the apoptotic program, up to a nonreversible
point. This transitional cell death has been demonstrated in
a number of studies and provides a basis for the cell death
continuum in cryopreservation. Common stressors such as
nutrient deprivation, DNA damage, cytokine exposure, cy-
totoxic agents, oxygen deprivation, and ionic imbalance
may result in both apoptosis and necrosis. The relative de-
gree of the stress experienced by the cell determines the
mode of death. Observations of transitional cell death have
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UPDATE ON THE MODERN STATE OF CRYOPRESERVATION 7
activation of caspase in human spermatozoa and Yagi et al.
7

in a porcine hepatocytes model. Expanding on these fnd-
ings, Vogel et al.
146
reported numerous alterations in human
fbroblast protein levels following cryopreservation. In this
study, the authors further described the utilization of cel-
lular proteomic fngerprinting in a diagnostic manner to
assess the quality of biologics following preservation.
Initiation of cryopreservation-induced
molecular death
While much research has been focused on identifying
and quantifying apoptosis following cryopreservation,
few detailed investigations into the initiating stresses exist.
Inherent in the process is the exposure of cells to numer-
ous stressors, many of which can initiate a molecular death
response. Many of these factors include metabolic uncou-
pling, production of free radicals, alternations in cell mem-
brane structure and fuidity, dysregulation of cellular ionic
balances, release of calcium, osmotic fuxes, and CPA expo-
sure.
14,15,24
This list of stresses associated with cryopreserva-
tion is by no means complete, but illustrates stress response
complexity and multiplicity of potential initiation points. It
is believed that the accumulation of sublethal stressors dur-
ing the preservation process results in activation of apopto-
sis followed by a shift to secondary necrosis. In an effort to
provide insight into the effect of the various stressors associ-
ated with cryopreservation, studies have begun to focus on
the various cellular initiation sites of apoptosis. These stud-
ies remain in their infancy, but have begun insight into the
pathways associated with cryopreservation-induced molec-
ular cell death, including the cell membrane, nucleus, and
mitochondria.
Control of Cryopreservation-Induced
Molecular Response
With the discovery of molecular responses in cells to the
preservation process, there have been a number of attempts
to control these events in an effort to improve preservation
outcome. These approaches vary and include alteration in
solution design (cryoprotectant carrier media), and addition
of protective agents for targeted apoptotic control (TAC).
Cryopreservation solution design
One shift in the approach to improving cryopreservation
outcome in recent years is that of carrier media formulation.
Traditional cryopreservation media consists of a basal cul-
ture media with serum protein and DMSO supplementa-
tion. While providing for physical protection through the
DMSO and protein components, the basal solutions do not
provide adequate control or maintenance of an appropriate
physiological environment for cells during the cryopres-
ervation process. These traditional solutions fall short in
addressing changes in solution pH, free radical production,
energy deprivation, and so on. Further, culture media-based
solutions designed for use at normothermic conditions do
not provide the appropriate ionic environment necessary for
cell maintenance during preservation,
24
as these media are
considered extracellular-like with regards to ionic concen-
trations (high Na
+
, low K
+
). Accordingly, the solution prop-
erties of these historical preservation media do not provide
Cryopreservation-induced delayed onset cell death
Reviewing the literature, it can be concluded that molec-
ular-based cell death (apoptosis) plays a critical role in cryo-
preservation outcome in many systems. One critical aspect
is the temporal component of post-cryopreservation cell
death.
3
To this point, evaluation of cell populations within
a few hours post-thaw does not allow for the identifcation
of the full extent of apoptosis or necrosis (Fig. 2). Molecular-
based cell death may take many hours to days to manifest
following thawing due to the chronological nature of the cell
death machinery. It is this temporal component that contin-
ues to elude many investigators attempting to characterize
molecular cell death following preservation. In 2001, a re-
port detailed the timing of cell death following cryopres-
ervation, termed the phenomena cryopreservation-induced
delayed onset cell death (CIDOCD).
8
This study documented
a delayed peak in necrosis (6 h) and a subsequent peak in
apoptosis (12-h) post-thaw. Due to the ordered temporal pro-
gression of the cell death cascades, the nadir in cell survival
was not observed until 24-h post-thaw. Subsequently, a series
of investigations into the path of molecular cell death pro-
gression ensued. In 2002, Baust et al.
14
reported on a genomic
response following thawing in the form of up-regulation of
transcriptional activity of key apoptotic enzymes (caspase-3,
-8, -9) in a delayed manner (1218 h post-thaw). Vogel et al.
146

also reported on the post-thaw activation of caspase-3 in
a renal model and demonstrated substantial alterations in
proteolytic activity throughout the initial 24-h recovery pe-
riod. Schmidt-Mende et al.
132
has reported post-thaw pro-
tease activation in a bone marrow cell model as well. This
study found a high level of intrinsic proteolytic activity
following preservation leading to the cleavage of various
apoptotic proteins. Further implicating caspase involvement
following cryopreservation, Paasch et al.
134
reported on the
Post-Thaw Recovery Interval (h)
True
Survival
Onset and Progression of Delayed Cell Death
Population Regrowth Interval /
Functional Utilization Interval
Apparent
Survival
0
0
10
20
30
40
50
S
a
m
p
l
e

V
i
a
b
i
l
i
t
y

60
70
80
90
100
4 8 12 16 20 24 48
FIG. 2. Timing and progression of cell death following
cryopreservation. Representation of the progression of the
temporal sample survival status during recovery from cryo-
preservation. Cell viability is typically seen as elevated
immediately post-thaw and then progressively decreases
during the initial 24 to 48 h of recovery as apoptotic and
necrotic events manifest, yielding a true survival that is
much lower than initially observed. (Adapted from Baust
JM, 2005.
122
)
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BAUST ET AL. 8
during cold storage of rat liver prevented apoptotic induc-
tion in endothelial cells as a result of cold ischemia.
More recently, focus in TAC-based preservation has
shifted to understanding and inhibiting the activity of Rho
kinases as a means of improving cell survival.
66,157159
In
2008, Martin Ibaez et al.
159
reported on the benefts of Rho-
associated kinase (ROCK) inhibition for the cryopreserva-
tion of embryonic stem cells. In this study, ROCK inhibitor
was added to both the freeze and recovery medium. ROCK
inhibition resulted in an increase in stem-cell survival, and
reduced the level of stress-induced spontaneous differentia-
tion while maintaining differentiation capacity of the cells.
Li et al.
158
expanded investigations into the action of ROCK
inhibition and has suggested that ROCK inhibition might not
directly effect cold-induced apoptosis, but rather reduces the
negative effects of the cell dissociation and handling process
associated with preservation protocols, thereby reducing the
overall cell loss. While dissecting two interlinked portions
of the preservation process (preparation of sample for freez-
ing and freezing), this study demonstrated the infuence of
Rho kinase activity on stem-cell cryopreservation outcome.
Further, this study again illustrates the infuence of the entire
process on cell state and ultimate outcome of the cell preser-
vation process. Most recently, Heng and colleagues have con-
tinued this line of TAC study, incorporating ROCK inhibitors
into preservation media for bone marrow mesenchymal stem
cells (MSC), and reported a marked increase in MSC sur-
vival.
66,157
Interestingly, as discussed earlier, Heng reported
that the beneft of ROCK inhibition was not seen immediately
post-thaw, but manifested by 24-h post-thaw. These fndings
are consistent with previous reports in other cell systems such
as renal cells, fbroblasts, PBMCs, and liver cells among oth-
ers.
4,8,86,118,147,151
Taken together, studies focused on TAC have
shown tremendous promise for cell- and application-specifc
development of improved cryopreservation processes.
Summary
Many cell-based applications in regenerative and repar-
ative medicine, biobanking, tissue engineering, and so on
require normal, predictable, and timely return of cells follow-
ing cryopreservation. This is often not the case with todays
technologies and approaches. Additionally, there exists a
growing body of evidence suggesting that CPAs may also
have a potentially negative impact on the proteome, genome,
and fragmentome. Accordingly, it has become imperative
that new lines of investigation into cellular response to cryo-
preservation commence. As our understanding continues to
grow, advancements will continue to push the present-day
limits of successful preservation. Molecular-based study
has once again helped to propel cryopreservation forward.
A union between the optimized structural protection and
cellmolecular-based modulation is most likely to provide
the next level of improvements in post-preservation out-
come. The frst generation of cryopreservation developments
focused on structural preservation of cells through the
inclusion of cryoprotectants and ice management. Second-
generation strategies, focusing on preservation media com-
position, have integrated with frst-generation strategies and
improved preservation outcome.
15
Current studies are now
focused on linking the management of gene-regulated
stress-dependent effects on a cell (TAC) with that of frst-
and second-generation approaches.
for protection at the cellular level, thereby in many cases ex-
acerbating cell death.
15
In contrast to extracellular-like media, the development of
intracellular-like media has shown beneft in increasing cell
survival. Reports on media such as ViaSpan (University of
Wisconsin Solution), CryoStor, Unisol, Adesta, and Celsior,
to name a few, have detailed varying levels of improved
survival when combined with CPAs for cryopreservation.
Successes with this approach have been reported in cellular
systems including hepatocytes,
147,148
cord blood,
149
stem cells,
PBMCs,
122
fbroblasts,
8
keratinocytes,
14
blood vessels,
145
and
engineered tissues.
25
In the majority of these studies, eval-
uation of the cryopreservation media was conducted and
correlated with improvements in cell survival, function,
and growth. Interestingly, in most of these studies, the im-
provement in viability and sample quality was not noted
immediately post-thaw; it was not until the molecular-based
events fully manifested that the improvement was observed.
Continuation studies have suggested that the improvement
in cell survival and function was due to a reduction of both
apoptosis and necrosis during post-thaw recovery although
the mechanism of which is unknown.
9,15,24
Application of targeted apoptotic control
As previously discussed, the processes and pathways
associated with the induction of apoptosis and necrosis
are complex. The current state of knowledge relating to the
extent and activation sites of these molecular-based events
continues to grow. While new cryopreservation media have
provided for an improvement in cryopreservation outcome,
the involvement of apoptosis in cryopreservation failure has
led to many studies investigating the feasibility of TAC.
15

In 2000, our group reported on the attempt to control the
activation of apoptosis following cryopreservation through
direct caspase inhibition,
4
which markedly improved cell
survival.
8
Subsequent studies further demonstrated that
TAC could be used to modulate both the levels of post-thaw
apoptosis and necrosis.
8
Yagi et al.
7
reported on the beneft
of TAC in improving hepatocyte cryopreservation, a signif-
icant investigative milestone. These data provided a basis
for the hypothesis describing transitional cell death in cryo-
preservation failure. A number of additional reports have
emerged describing incorporation of caspase inhibitors into
cryopreservation media to improve cell survival.
15
In addition to these studies, there have been numerous
other reports on the infuence of TAC on cell survival in both
cryoprevention and hypothermic storage. In the mid- to late
1990s, several reports on the utilization of protease inhibi-
tors, free radical scavengers (vitamins), and ion chelation
emerged.
124130,150
While not specifcally targeting apoptosis
as the mechanism of death, these reports nonetheless clearly
demonstrated the beneft of this approach. With the specifc
identifcation of apoptosis contributing to cryopreservation
failures
5
along with other studies,
3,6,7,9
the benefts employ-
ing TAC to improve cryopreservation outcome became
obvious. Subsequent independent studies from 2001 to 2003
recognized this and reported the beneft of TAC using cas-
pase inhibitors.
3,68,26,151154
A report describes the benefts
of calpain inhibitors during cryopreservation to improve
cell survival.
155
The Robilotto et al.
155
study was, in part, an
extension of studies by Baust et al.
3,4,8
in combination with
studies by Sindram et al.
156
that showed calpain inhibition
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UPDATE ON THE MODERN STATE OF CRYOPRESERVATION 9
24. Taylor MJ, Bailes JE, Elrifai AM, et al. A new solution for life
without blood. Asanguineous low-fow perfusion of a whole-
body perfusate during 3 hours of cardiac arrest and profound
hypothermia. Circulation 1995;91(2):431444.
25. Baust JM, Van Buskirk R, Baust JG. Cryopreservation of an
engineered human skin equivalent: The apoptosis paradigm.
J Am Soc Mech Eng (Adv Heat and Mass Trans Biotech).
1999;363:7176.
26. Mathew AJ, Van Buskirk RG, Baust JG. Improved hypo-
thermic preservation of human renal cells through suppres-
sion of both apoptosis and necrosis. Cell Preserv Technol
2002;1(4):239253.
27. Gao D, Mazur P, Critser KK. Fundamentals of mammalian
spermatozoa. In Karow AM, Critser JK, eds. Reproductive Tissue
Banking: Scientifc Principles. San Diego, CA: Academic Press;
1997:263328.
28. Southard JH, van Gulik TM, Ametani MS, et al. Important
components of the UW solution. Transplantation
1990;49(2):251257.
29. Southard JH, Belzer FO. Organ preservation. Annu Rev Med
1995;46:235247.
30. Peng XR, Liu T, Zhang Y. Addition of alpha-tocopherol to cul-
ture medium improves the quality and cryosurvival of nuclear-
transferred ovine embryos. J Reprod Dev 2008;54(6):403407.
31. Wundrich K, Paasch U, Leicht M, et al. Activation of caspases
in human spermatozoa during cryopreservationan immu-
noblot study. Cell Tissue Bank 2006;7(2):8190.
32. Taylor MJ, Campbell LH, Rutledge RN, et al. Comparison of
Unisol with Euro-Collins solution as a vehicle solution for
cryoprotectants. Transplant Proc 2001;33(12):677679.
33. Baicu SC, Taylor MJ. Acid-base buffering in organ preser-
vation solutions as a function of temperature: new parameters
for comparing buffer capacity and effciency. Cryobiology
2002;45(1):3348.
34. Lovelock JE. The haemolysis of human red blood-cells by freez-
ing and thawing. Biochim Biophys Acta 1953;10(3):414426.
35. Huang JS, Downes GL, Childress GL, et al. Oxidation of
14C-labeled substrates by dog kidney cortex at 10 and 38C.
Cryobiology 1974;11(5):387394.
36. Fuller BJ. Gene expression in response to low temperatures
in mammalian cells: a review of current ideas. Cryo Letters
2003;24(2):95102.
37. Tani M, Neely JR. Role of intracellular Na+ in Ca2+ overload
and depressed recovery of ventricular function of reperfused
ischemic rat hearts. Possible involvement of H+-Na+ and Na+-
Ca2+ exchange. Circ Res 1989;65(4):10451056.
38. Renlund DG, Gerstenblith G, Lakatta EG, et al. Perfusate
sodium during ischemia modifes post-ischemic functional
and metabolic recovery in the rabbit heart. J Mol Cell Cardiol
1984;16(9):795801.
39. Neely JR, Grotyohann LW. Role of glycolytic products in
damage to ischemic myocardium. Dissociation of adenosine
triphosphate levels and recovery of function of reperfused is-
chemic hearts. Circ Res 1984;55(6):816824.
40. Wilson JM, McMurdo AC. Chilling injury in plants. In Morris
GJ, Clarke A, eds. Effects of Low Temperature on Biological
Membranes. London: Academic Press; 1973:4(1):285309.
41. Lyons JM, Raison JK. A temperature-induced transition in
mitochondrial oxidation: Contrast between cold and warm
blooded animals. Comp Biochem Physiol 1970;37:405411.
42. Raison JK. The infuence of temperature-induced phase
changes on the kinetics of respiratory and other membrane-
associated enzyme systems. J Bioenerg 1973;4(1):285309.
43. Fuhram GJ, Fuhram FA. Oxygen consumption of animals and
tissues as a function of temperature. J Gen Physiol 1959;42:215.
44. vant Hoff JH. tudes sur la Dynamique Chimique. Amsterdam:
F. Muller & Co; 1884.
45. Zimmermann KC, Green DR. How cells die: apoptosis path-
ways. J Allergy Clin Immunol 2001;108(4 Suppl):S99S103.
References
1. Baust JG. Concepts in biopreservation. In Baust JG, Baust JM, eds.
Advances in Biopreservation. Boca Raton: CRC Press; 2007:114.
2. Fuller BJ, Lane N, Benson EE. Life in the Frozen State. Boca
Raton: CRC Press; 2004.
3. Baust JM. Molecular mechanisms of cellular demise asso-
ciated with cryopreservation failure. Cell Preserv Technol.
2002;1(1):1731.
4. Baust JM, Van Buskirk, Baust JG. Cell viability improves fol-
lowing inhibition of cryopreservation-induced apoptosis. In
Vitro Cell Dev Biol Anim 2000;36(4):262270.
5. Baust JM, Van Buskirk R, Baust JG. Cryopreservation outcome
in enhanced by intracellular-type medium and inhibition of
apoptosis. Cryobiology 1998;37(4):410411.
6. Fu T, Guo D, Huang X, et al. Apoptosis occurs in isolated
and banked primary mouse hepatocytes. Cell Transplant
2001;10(1):5966.
7. Yagi T, Hardin JA, Valenzuela YM, et al. Caspase inhibition
reduces apoptotic death of cryopreserved porcine hepatocytes.
Hepatology 2001;33(6):14321440.
8. Baust JM, Vogel MJ, Van Buskirk R, et al. A molecular basis
of cryopreservation failure and its modulation to improve cell
survival. Cell Transplant 2001;10(7):561571.
9. de Boer F, Drager AM, Pinedo HM, et al. Extensive early apop-
tosis in frozen-thawed CD34-positive stem cells decreases
threshold doses for haematological recovery after autologous
peripheral blood progenitor cell transplantation. Bone Marrow
Transplant 2002;29(3):249255.
10. Abrahamsen JF, Bakken AM, Bruserud O, et al. Flow cytomet-
ric measurement of apoptosis and necrosis in cryopreserved
PBPC concentrates from patients with malignant diseases.
Bone Marrow Transplant 2002;29(2):165171.
11. Peter ME, Heufelder AE, Hengartner MO. Advances in apopto-
sis research. Proc Natl Acad Sci USA 1997;94(24):1273612737.
12. Xiao M, Dooley DC. Assessment of cell viability and apoptosis
in human umbilical cord blood following storage. J Hematother
Stem Cell Res 2003;12(1):115122.
13. Matsushita T, Yagi T, Hardin JA, et al. Apoptotic cell death and
function of cryopreserved porcine hepatocytes in a bioartif-
cial liver. Cell Transplant 2003;12(2):109121.
14. Baust JM, Van Buskirk RG, Baust JG. Gene activation of the
apoptotic caspase cascade following cryogenic storage. Cell
Preserv Technol 2002;1(1):6380.
15. Baust JM. Advances in media for cryopreservation and hypo-
thermic storage. Bioprocess Int 2005;3(Supp 3):4656.
16. Martin G, Sabido O, Durand P, et al. Cryopreservation induces
an apoptosis-like mechanism in bull sperm. Biol Reprod
2004;71(1):2837.
17. Paasch U, Grunewald S, Agarwal A, et al. Activation pat-
tern of caspases in human spermatozoa. Fertil Steril
2004;81(Suppl):18021809.
18. Snyder KK, Van Buskirk RG, Baust JM, et al. Biological pack-
aging for the global cell and tissue therapy markets. BioProcess
J 2004;3(3):3445.
19. Van Buskirk RG, Baust JM, Snyder KK, et al. Cryopreservation
Its not just about cell yield. BioProcess Int 2005;3(4):6474.
20. Van Buskirk RG, Snyder KK, Baust JM, et al. Hypothermic stor-
age and cryopreservation: The issues of successful short-term
and long term preservation of cells and tissues. BioProcess Int
2004;2(10):4249.
21. Mathew AJ, Baust JM, Van Buskirk RG, et al. Cell preservation in
reparative and regenerative medicine: evolution of individual-
ized solution composition. Tissue Eng 2004;10(1112):16621671.
22. Baust JG, Gage AA. The molecular basis of cryosurgery. BJU
Int 2005;95(9):11871191.
23. Anzar M, He L, Buhr MM, et al. Sperm apoptosis in fresh and
cryopreserved bull semen detected by fow cytometry and its
relationship with fertility. Biol Reprod 2002;66(2):354360.
B
i
o
p
r
e
s
e
r
v
a
t
i
o
n

a
n
d

B
i
o
b
a
n
k
i
n
g

2
0
0
9
.
7
:
3
-
1
2
.

d
o
w
n
l
o
a
d
e
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f
r
o
m

o
n
l
i
n
e
.
l
i
e
b
e
r
t
p
u
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.
c
o
m
b
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T
H
E

U
N
I
V
E
R
S
I
T
Y

O
F

M
A
N
C
H
E
S
T
E
R

o
n

0
3
/
2
6
/
1
3
.

F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
BAUST ET AL. 10
69. Borderie VM, Lopez M, Lombet A, et al. Cryopreservation and
culture of human corneal keratocytes. Invest Ophthalmol Vis
Sci 1998;39(8):15111519.
70. Donaldson C, Armitage WJ, Denning-Kendall PA, et al.
Optimal cryopreservation of human umbilical cord blood.
Bone Marrow Transplant 1996;18(4):725731.
71. Frim J, Snyder RA, McGann LE, et al. Growth kinetics of
cells following freezing in liquid nitrogen. Cryobiology
1978;15(5):502516.
72. Zambelli A, Poggi G, Da Prada G, et al. Clinical toxicity of cry-
opreserved circulating progenitor cells infusion. Anticancer
Res 1998;18(6B):47054708.
73. De Loecker P, Fuller BJ, Koptelov VA, et al. Cryopreser-
vation of isolated rat hepatocytes: effects of iron-mediated
oxidative stress of metabolic activity. Cryobiology 1997;34(2):
150156.
74. Mazur P, Cole KW. Infuence of cell concentration on the con-
tribution of unfrozen fraction and salt concentration to the
survival of slowly frozen human erythrocytes. Cryobiology
1985;22(6):509536.
75. Fahy GM. The relevance of cryoprotectant toxicity to cryobi-
ology. Cryobiology 1986;23(1):113.
76. Meryman HT. The Exceeding of a minimum tolerable cell
volume in hypertonic suspension as a cause of freezing in-
jury. In Wolstenholme GEW, OConnor M, eds. The Frozen Cell.
Churchill: Ciba Foundation Symposium; 1970:5164.
77. Paynter SJ, Fuller BL, McGrath JJ, et al. The effects of cryo-
protectant permeability on mouse oocytes. Cryo Letters.
1995;16:321324.
78. Mazur P. Freezing of living cells: mechanisms and implica-
tions. Am J Physiol 1984;247(3 Pt 1):C125C142.
79. Pegg DE, Diaper MP. On the mechanism of injury to slowly
frozen erythrocytes. Biophys J 1988;54(3):471488.
80. Coger R, Toner M. Preservation techniques for biomaterials.
In Brunzion JD, ed. The Biomedical Engineering Handbook. Boca
Raton: CRC Press; 1995:15671579.
81. Morris CB. Cryopreservation of animal and human cell lines.
Methods Mol Biol 1995;38:179187.
82. Eroglu A, Russo MJ, Bieganski R, et al. Intracellular trehalose
improves the survival of cryopreserved mammalian cells. Nat
Biotechnol 2000;18(2):163167.
83. Loretz LJ, Li AP, Flye MW, et al. Optimization of cryopreser-
vation procedures for rat and human hepatocytes. Xenobiotica
1989;19(5):489498.
84. Fuller BJ. Cryoprotectants: the essential antifreezes to protect
life in the frozen state. Cryo Letters 2004;25(6):375388.
85. Walker NI, Harmon BV, Gove GC, et al. Patterns of cell death.
Methods Achiev Exp Pathol. 1988; 13:1854.
86. Fowke KR, Behnke J, Hanson C, et al. Apoptosis: a method
for evaluating the cryopreservation of whole blood and pe-
ripheral blood mononuclear cells. J Immunol Methods
2000;244(12):139144.
87. Martin H, Bournique B, Sarsat JP, et al. Cryopreserved rat
liver slices: a critical evaluation of cell viability, histolog-
ical integrity, and drug-metabolizing enzymes. Cryobiology
2000;41(2):135144.
88. Searle J, Kerr JF, Bishop CJ. Necrosis and apoptosis: distinct
modes of cell death with fundamentally different signifcance.
Pathol Annu 1982;17(Pt 2):229259.
89. Kerr JF. Shrinkage necrosis of adrenal cortical cells. J Pathol
1972;107(3):217219.
90. Columbano A. Cell death: current diffculties in discrimi-
nating apoptosis from necrosis in the context of pathological
processes in vivo. J Cell Biochem 1995;58(2):181190.
91. Habibovic S, Hrgovic Z, Bukvic I, et al. [Molecular mechanisms
in apoptosis]. Med Arh 2000;54(1):3340.
92. Wyllie AH, Kerr JF, Currie AR. Cell death: the signifcance of
apoptosis. Int Rev Cytol 1980;68:251306.
46. Lanir A, Clouse ME, Lee RG. Liver preservation for trans-
plant. Evaluation of hepatic energy metabolism by 31P NMR.
Transplantation 1987;43(6):786790.
47. Fuller BJ, Gower JD, Green CJ. Free radical damage and organ
preservation: fact or fction? A review of the interrelationship
between oxidative stress and physiological ion disbalance.
Cryobiology 1988;25(5):377393.
48. Stubenitsky BM, Ametani M, Danielewicz R, et al. Regeneration
of ATP in kidney slices after warm ischemia and hypothermic
preservation. Transpl Int 1995;8(4):293297.
49. White BC, Wiegenstein JG, Winegar CD. Brain ischemic an-
oxia. Mechanisms of injury. JAMA 1984;251(12):15861590.
50. Jennings RB, Ganote CE. Mitochondrial structure and function
in acute myocardial ischemic injury. Circ Res 1976;38(5 Suppl
1):I80I91.
51. Southard JH, Van der Laan NC, Lutz M, et al. Comparison
of the effect of temperature on kidney cortex mitochondria
from rabbit, dog, pig, and human: Arrhenius plots of ADP-
stimulated respiration. Cryobiology 1983;20(4):395400.
52. Scholander PP, Flaff W, Waters V, et al. Climatic adaptations in
arctic and tropical poikilothermas. Physiol Zool 1953;26:6792.
53. Storey KB, Storey JM. Metabolic rate depression and biochem-
ical adaptation in anaerobiosis, hibernation and estivation. Q
Rev Biol 1990;65(2):145174.
54. Mazur P. Principles of cryobiology. In Fuller BJ, Lane N,
Benson EE, eds. Life in the Frozen State. Boca Raton: Academic
Press; 2004;365.
55. Taylor MJ, Song YC, Brockbank KGM. Vitrifcation in tissue
preservation. In Fuller BJ, Lane N, Benson EE, eds. Life in the
Frozen State. Boca Raton: Academic Press; 2004:603692.
56. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after
vitrifcation and dehydration at low temperatures. Nature
1949;164(4172):666.
57. Smith AU. Prevention of haemolysis during freezing and thaw-
ing of red blood-cells. Lancet 1950;2(6644):910911.
58. Lovelock JE, Bishop MW. Prevention of freezing damage to
living cells by dimethyl sulphoxide. Nature 1959;183(4672):
13941395.
59. Mazur P. The role of intracellular freezing in the death of cells
cooled at supraoptimal rates. Cryobiology 1977;14(3):251272.
60. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freez-
ing injury. Evidence from Chinese hamster tissue-culture
cells. Exp Cell Res 1972;71(2):345355.
61. Rhoads LS, Zlobinsky Y, Van Buskirk RG, et al. Patterns of
latent heat liberation during controlled rate cooling: absence
of effects on the survival of cryopreserved cells. Cryo Letters
1991;12:329338.
62. Brown RT, Baust JG. Time course of peripheral heterothermy
in a homeotherm. Am J Physiol 1980;239(1):R126R129.
63. Williams RJ. The mechanisms of cryoprotection in the inter-
tidal mollusk. Cryobiology 1969;42:5055.
64. Franks F. The properties of aqueous solutions at sub-zero
temperature. In Franks F, Mathias S, eds. Biophysics of Water.
Hoboken, NJ: John Wiley & Sons; 1982:279294.
65. Rall WF, Mazur P, McGrath JJ. Depression of the ice-nucleation
temperature of rapidly cooled mouse embryos by glycerol and
dimethyl sulfoxide. Biophys J 1983;41(1):112.
66. Heng BC. Effect of Rho-associated kinase (ROCK) inhibitor
Y-27632 on the post-thaw viability of cryopreserved human
bone marrow-derived mesenchymal stem cells. Tissue Cell
2009.[Epub ahead of print]
67. Baust JM. Properties of cells and tissues infuencing preser-
vation outcome: molecular basis of preservation-induced cell
death. In Baust JG, Baust JM, eds. Advances in Biopreservation.
Boca Raton: CRC Press; 2007:6387.
68. Eberl T, Amberger A, Herold M, et al. Expression of stress proteins,
adhesion molecules, and interleukin-8 in endothelial cells after
preservation and reoxygenation. Cryobiology 1999;38(2):106118.
B
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s
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y

T
H
E

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E
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O
F

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N
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H
E
S
T
E
R

o
n

0
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/
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/
1
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.

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o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
UPDATE ON THE MODERN STATE OF CRYOPRESERVATION 11
116. Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis
in hepatic ischemia/reperfusion injury. Gastroenterology
2003;125(4):12461257.
117. Chavez-Reyes A, Parant JM, Amelse LL, et al. Switching mech-
anisms of cell death in mdm2- and mdm4-null mice by deletion
of p53 downstream targets. Cancer Res 2003;63(24):86648669.
118. Jurisicova A, Varmuza S, Casper RF. Involvement of pro-
grammed cell death in preimplantation embryo demise. Hum
Reprod Update 1995;1(6):558566.
119. Borderie VM, Laroche L. Ultrastructure of cultured and
cryopreserved human corneal keratocytes. Cornea 1999;18(5):
589594.
120. Thornberry NA, Lazebnik Y. Caspases: enemies within.
Science 1998;281(5381):13121316.
121. Men H, Monson RL, Parrish JJ, et al. Degeneration of cryopre-
served bovine oocytes via apoptosis during subsequent cul-
ture. Cryobiology 2003;47(1):7381.
122. Baust JM, Cosentino M, Meeks E, et al. Apoptotic cell death
contributes signifcantly to peripheral blood mononuclear
cells cryopreservation failure. Cryobiology 2005;51(4):354355.
123. Schuurhuis GJ, Muijen MM, Oberink JW, et al. Large popu-
lations of non-clonogenic early apoptotic CD34-positive cells
are present in frozen-thawed peripheral blood stem cell trans-
plants. Bone Marrow Transplant 2001;27(5):487498.
124. Van Buskirk RG, Taylor MJ, Shen R, et al. Optimization of
HypoThermosol using cultured cardiomyocytes and the fuo-
rescent multiple endpoint assay. Cryobiology 1995;32:590.
125. Mathew A, Baust JG, Van Buskirk RG. Optimization of
HypoThermosol for the hypothermic storage of cardiomyo-
cytesAddition of EDTA. In Vitro Toxicol 1997;10(4):407415.
126. OFlaherty C, Beconi M, Beorlegui N. Effect of natural anti-
oxidants, superoxide dismutase and hydrogen peroxide on
capacitation of frozen-thawed bull spermatozoa. Andrologia
1997;29(5):269275.
127. Hadj-Aissa A, Ramella-Virieux SG, Steghens JP, et al. Calcium
antagonists improve kidney function in the rat after cold stor-
age in high-Na UW but not in high-K UW solution. Transplant
Proc 1997;29(5):24392441.
128. Nagasaki H, Nakano H, Boudjema K, et al. Effcacy of precon-
ditioning with N-acetylcysteine against reperfusion injury
after prolonged cold ischaemia in rats liver in which gluta-
thione had been reduced by buthionine sulphoximine. Eur J
Surg 1998;164(2):139146.
129. Roberts RF, Nishanian GP, Carey JN, et al. Addition of aproti-
nin to organ preservation solutions decreases lung reperfusion
injury. Ann Thorac Surg 1998;66(1):225230.
130. Mathew AJ, Hollister WR, Addona T, et al. Vitamin E and
EDTA Improve the Effcacy of Hypothermosol-Implication of
Apoptosis. In Vitr Mol Toxicol 1999;12(3):163172.
131. Hilbert SL, Luna RE, Zhang J, et al. Allograft heart valves: the
role of apoptosis-mediated cell loss. J Thorac Cardiovasc Surg
1999;117(3):454462.
132. Schmidt-Mende J, Hellstrom-Lindberg E, Joseph B, et al.
Freezing induces artifcial cleavage of apoptosis-related
proteins in human bone marrow cells. J Immunol Methods
2000;245(12):9194.
133. Villalba R, Pena J, Luque E, et al. Keratocyte injury in human
corneas cryopreserved under standard conditions. Cell Tissue
Bank 2004;5(4):201204.
134. Paasch U, Sharma RK, Gupta AK, et al. Cryopreservation and
thawing is associated with varying extent of activation of
apoptotic machinery in subsets of ejaculated human sperma-
tozoa. Biol Reprod 2004;71(6):18281837.
135. Sarkar S, Kalia V, Montelaro RC. Caspase-mediated apoptosis
and cell death of rhesus macaque CD4+ T-cells due to cryo-
preservation of peripheral blood mononuclear cells can be
rescued by cytokine treatment after thawing. Cryobiology
2003;47(1):4458.
93. Alnemri ES. Mammalian cell death proteases: a family of
highly conserved aspartate specifc cysteine proteases. J Cell
Biochem 1997;64(1):3342.
94. Cohen GM. Caspases: the executioners of apoptosis. Biochem
J 1997;326(Pt 1)116.
95. Kanzler S, Galle PR. Apoptosis and the liver. Semin Cancer
Biol 2000;10(3):173184.
96. Sheikh MS, Fornace AJ, Jr. Death and decoy receptors and p53-
mediated apoptosis. Leukemia 2000;14(8):15091513.
97. Gewirtz DA. Growth arrest and cell death in the breast tumor
cell in response to ionizing radiation and chemotherapeutic
agents which induce DNA damage. Breast Cancer Res Treat
2000;62(3):223235.
98. Nicotera P, Leist M, Fava E, et al. Energy requirement for
caspase activation and neuronal cell death. Brain Pathol
2000;10(2):276282.
99. Xue D, Shaham S, Horvitz HR. The Caenorhabditis elegans
cell-death protein CED-3 is a cysteine protease with substrate
specifcities similar to those of the human CPP32 protease.
Genes Dev 1996;10(9):10731083.
100. Yin XM. Signal transduction mediated by Bid, a pro-death
Bcl-2 family proteins, connects the death receptor and mito-
chondria apoptosis pathways. Cell Res 2000;10(3):161167.
101. Hengartner MO, Horvitz HRC. elegans cell survival gene
ced-9 encodes a functional homolog of the mammalian proto-
oncogene bcl-2. Cell 1994;76(4):665676.
102. Leist M, Single B, Castoldi AF, et al. Intracellular adenosine tri-
phosphate (ATP) concentration: a switch in the decision between
apoptosis and necrosis. J Exp Med 1997;185(8):14811486.
103. Duru NK, Morshedi M, Schuffner A, et al. Semen treatment
with progesterone and/or acetyl-l-carnitine does not improve
sperm motility or membrane damage after cryopreservation-
thawing. Fertil Steril 2000;74(4):715720.
104. Fink KB, Andrews LJ, Butler WE, et al. Reduction of post-trau-
matic brain injury and free radical production by inhibition of
the caspase-1 cascade. Neuroscience 1999;94(4):12131218.
105. Emery E, Aldana P, Bunge MB, et al. Apoptosis after traumatic
human spinal cord injury. J Neurosurg 1998;89(6):911920.
106. Elsasser A, Suzuki K, Schaper J. Unresolved issues regarding
the role of apoptosis in the pathogenesis of ischemic injury
and heart failure. J Mol Cell Cardiol 2000;32(5):711724.
107. Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in
apoptosis. Eur J Pharmacol 1998;351(3):261272.
108. Saikumar P, Dong Z, Weinberg JM, et al. Mechanisms of
cell death in hypoxia/reoxygenation injury. Oncogene
1998;17(25):33413349.
109. Reutelingsperger CP, van Heerde WL. Annexin V, the regu-
lator of phosphatidylserine-catalyzed infammation and coag-
ulation during apoptosis. Cell Mol Life Sci 1997;53(6):527532.
110. Condo I, Testi R. Intracellular mediators of programmed cell
death initiated at the cell surface receptor Fas. Transpl Int
2000;13(Suppl 1):S3S6.
111. Blankenberg FG, Katsikis PD, Tait JF, et al. In vivo detection and
imaging of phosphatidylserine expression during programmed
cell death. Proc Natl Acad Sci USA 1998;95(11):63496354.
112. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-
dependent formation of Apaf-1/caspase-9 complex initiates an
apoptotic protease cascade. Cell 1997;91(4):479489.
113. Bras M, Queenan B, Susin SA. Programmed cell death via mi-
tochondria: different modes of dying. Biochemistry (Mosc).
2005;70(2):231239.
114. Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome
c-initiated caspase cascade: hierarchical activation of caspas-
es-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J
Cell Biol 1999;144(2):281292.
115. Liu CY, Liu YH, Lin SM, et al. Apoptotic neutrophils under-
going secondary necrosis induce human lung epithelial cell
detachment. J Biomed Sci 2003;10(6 Pt 2):746756.
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y
.
BAUST ET AL. 12
a new target for therapeutic intervention? Ann Thorac Surg
2001;72(5):14571464.
151. Cosentino LM, Corwin W, Baust JM, et al. Preliminary report:
evaluation of storage conditions and cryococktails during pe-
ripheral blood mononuclear cell cryopreservation. Cell Preserv
Technol 2007;5(4):189204.
152. Peter AT, Linde-Forsberg C. Effcacy of the anticaspase agent
zVAD-fmk on post-thaw viability of canine spermatozoa.
Theriogenology 2003;59(7):15251532.
153. Heng BC, Clement MV, Cao T. Caspase inhibitor Z-VAD-FMK
enhances the freeze-thaw survival rate of human embryonic
stem cells. Biosci Rep 2007;27(45):257264.
154. Fujita R, Hui T, Chelly M, et al. The effect of antioxidants and
a caspase inhibitor on cryopreserved rat hepatocytes. Cell
Transplant 2005;14(6):391396.
155. Robilotto AT, Baust JM, Van Buskirk R, et al. Calpain activation
infuences cryopreservation outcome. Cell Preserv Technol
2006;41(1):1730.
156. Sindram D, Kohli V, Madden JF, et al. Calpain inhibition pre-
vents sinusoidal endothelial cell apoptosis in the cold ischemic
rat liver. Transplantation 1999;68(1):136140.
157. Heng BC, Richards M, Cao T. Are stem cells inherently more
prone to cryopreservation-induced apoptosis compared to or-
dinary somatic cells? Hum Reprod 2009;24(2):492; author reply
492493.
158. Li X, Krawetz R, Liu S, et al. ROCK inhibitor improves survival
of cryopreserved serum/feeder-free single human embryonic
stem cells. Hum Reprod 2009;24(3):580589.
159. Martin-Ibanez R, Unger C, Stromberg A, et al. Novel cryo-
preservation method for dissociated human embryonic
stem cells in the presence of a ROCK inhibitor. Hum Reprod
2008;23(12):27442754.
Address reprint requests to:
Dr. John G. Baust
Lead Professor and UNESCO Chair of Cryobiology
Institute of Biomedical Technology
Science 3 Suite 144
State University of New York
Binghamton, NY 13902
E-mail: JBaust@Binghamton.edu
Received 22 February, 2009/Accepted 16 March, 2009
136. Lund PK, Westvik AB, Joo GB, et al. Flow cytometric evalua-
tion of apoptosis, necrosis and recovery when culturing mono-
cytes. J Immunol Methods 2001;252(12):4555.
137. Bontadini A, Tazzari PL, Manfroi S, et al. Apoptosis in leucode-
pleted packed red blood cells. Vox Sang 2002;83(1):3541.
138. Greco NJ, Seetharaman S, Kurtz J, et al. Evaluation of the reac-
tivity of apoptosis markers before and after cryopreservation
in cord blood CD34(+) cells. Stem Cells Dev 2006;15(1):124135.
139. Bakken AM. Cryopreserving human peripheral blood progen-
itor cells. Curr Stem Cell Res Ther 2006;1(1):4754.
140. Ohno K, Nelson LR, Mitooka K, et al. Transplantation of cryo-
preserved human corneas in a xenograft model. Cryobiology
2002;44(2):142149.
141. Heng BC, Ye CP, Liu H, et al. Loss of viability during freeze-
thaw of intact and adherent human embryonic stem cells
with conventional slow-cooling protocols is predominantly
due to apoptosis rather than cellular necrosis. J Biomed Sci
2006;13(3):433445.
142. Moran JM, Madejon L, Ortega Ferrusola C, et al. Nitric oxide
induces caspase activity in boar spermatozoa. Theriogenology
2008;70(1):9196.
143. Hussein MR, Bedaiwy MA, Falcone T. Analysis of apoptotic
cell death, Bcl-2, and p53 protein expression in freshly fxed
and cryopreserved ovarian tissue after exposure to warm is-
chemia. Fertil Steril 2006;85(Suppl 1):10821092.
144. Parks JE. Hypothermia and mammalian gametes. In Karow
AM, Critser JK, eds. Reproductive Tissue Banking: Scientifc
Principles. San Diego: Academic Press; 1997:229262.
145. Snyder KK, Baust JM, Van Buskirk RG, et al. Improved cryopres-
ervation of vascular tissue. Cryopreservation 2005;51:357358.
146. Vogel MJ, Baust JM, Van Buskirk RG, et al. Apoptotic cascades
are activated following cryopreservation. Cryobiology 2000;
41:390.
147. Sosef MN, Baust JM, Sugimachi K, et al. Cryopreservation of
isolated primary rat hepatocytes: enhanced survival and long-
term hepatospecifc function. Ann Surg 2005;241(1):125133.
148. Sugimachi K, Sosef MN, Baust JM, et al. Long-term function
of cryopreserved rat hepatocytes in a coculture system. Cell
Transplant 2004;13(2):187195.
149. Stylianou J, Vowels M, Hadfeld K. CryoStor signifcantly
improves cryopreservation of Haematopoetic Stem Cells
(HSC). Cryotherapy 2005;7(Suppl 1):117.
150. Hagl C, Tatton NA, Khaladj N, et al. Involvement of apoptosis
in neurological injury after hypothermic circulatory arrest:
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