Fisher John P. e.a. (ed.) Tissue Engineering
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12-10 Tissue Engineering
interaction between the two phases present simultaneously: the frozen liquid phase, which rejects solids
and the supercooled liquid phase, which cannot freeze due to the increased concentration of the solutes
rejected by the ice. Presence of solutes depresses the freezing temperature of the water, as a function of
their mole fraction. The tug-of-war between these two phases introduces compartmentalization yielding
to very high osmotic and electrical gradients within the cytosol. This may explain the low survival rates
recorded at relatively high subzero temperatures.
To this date numerous post-thaw viability and function experiments have been performed with virtually
every kind of cell using cryoprotectants at different concentrations, freezing/thawing rates, and storage
conditions. Interested readers are directed to reviews by Mazur [48] and McGrath [36]. Interestingly,
the mechanism of cryoprotection offered by the most widely used chemicals, for example DMSO and
glycerol, is not known [49,50] beyond the colligative action (that they replace the water molecules in the
cytosol) and their “strong interaction potential with water” at low temperatures. The colligative action
of cryopreservatives in the absence of water can be divided into the space-filling effect, which prevents
structural collapse and the osmotic effect, which presents the cryoprotectant as an alternative solvent
reducing solute concentration. DMSO has a very high hydrogen bonding affinity toward water creating
a nonideal mixture behavior (e.g., even though the freezing point of water and DMSO are 0 and 18.6
◦
C
respectively, the freezing point of a 1 : 3 molar ratio DMSO–water solution is −70
◦
C).
Given that, DMSO is toxic at high temperatures [51] and has been shown to cause gene activation
(for a review, see Ashwood-Smith [52]) and have mutagenic potential [53] at high concentrations, it is
surprising that it works so well for preservation at low temperatures in spite of its biological inadequacy: an
indication that the preservation phenomena is directly related to the thermodynamical properties of most
cryoprotectants (freezing point depression), their effects on the structure of water (such as eliminating
ice crystallization), and to the degree of molecular mobility reduction they offer (e.g., DMSO increases
cytoplasmic viscosity [50]).
A mechanism offered to explain the protective potential of membrane permeable cryoprotectants and
certain solutes introduced artificially into the cell (such as carbohydrates) is that they have the ability to
retardice formation by replacing water and imposing an ordered water structure resistant to crystallization.
Raman Scattering, NMR, dynamic molecular modeling, and Quasi Elastic NeutronScattering data indicate
that especially trehalose, even at low concentrations breaks the structure of adjacent water molecules and
slows down their mobility [54,55] and therefore makes them more resistant to crystallization at low
temperatures [56]. Similarly, DMSO can alter the structure and the rotational and translational mobilities
of water (e.g., 10% DMSO reduces the self-diffusion coefficient of water by half [50]) as a function of its
concentration and environmental temperature [57]. Analysis of osmotic stress injury to frozen cells [58]
in the presence of DMSO, glycerol, and ethylene glycol [59] also point to different reasons to explain the
protective mechanism offered by these cryoprotectants beyond their colligative action.
Even though the chemical reaction rates are expected to slow at low temperatures following Arrhenius
kinetics, due to freeze concentration, which decreases the distance between reactants and changes the pH
of the medium in addition to the possible catalytic effects of ice crystals [60] (and given that proton mobil-
ity is higher in ice than in water due to the organized crystal structure), some enzyme-catalyzed chemical
reaction rates do increase by orders of magnitude in the frozen state as compared to supercooled state
[61]. Combined with the detrimental effects of IIF, the factors stated above formed the scientific reasoning
behind development of an alternative preservation method: preservation of cells in an undercooled state
[62]. It is known that with the addition of each mole of solute to one liter of water suppresses the freezing
temperature by approximately 2
◦
C by increasing the entropy of the liquid phase. It was shown that if
structural stability at subzero temperatures could be ensured so that the probability of intra/extracellular
ice formation is at a minimum, short-term storage could be feasible for certain cells. This can be
achieved for example, by adding ice nucleation retarding solutes, applying high pressures, or using smaller
water volumes with minimum peripheral contact (such as utilization of small water droplets suspended
in oil).
The intracellular ice formation (IIF) has been thought to be the main source of viability loss for
preserved biological systems. It is postulated that the ice crystals in the cytosol mechanically disrupt the
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Roles of Thermodynamic State and Molecular Mobility 12-11
cytoskeleton and the macromolecules in addition to causing very high localized solute concentrations due
to solute rejection from the frozen phase. It is also suggested that IIF can not form by itself and is usually
induced by Extracellular Ice Formation (EIF) breaching the cell membrane through membrane pores,
enlarging them and causing the membrane to rupture.
It was therefore suggested that if IIF and EIF can be eliminated altogether (in spite of the disadvantages
of having to load very high concentrations of cryoprotectants into the cells and keeping them at liquid
nitrogen temperatures) then the preservation of cells, organs, and even whole human bodies would
be possible in a vitrified state [63]. With high cryoprotectant concentrations (as high as 9 M [63])
and low temperature storage, ice formation within the cells can be completely eliminated and also an
intracellular amorphous (or glassy?) state may be reached. This line of work however, is not supported by
detailed theoretical, experimental, and numerical analysis of molecular motion confirming the presence
of intracellular glass. Given that the glass transition temperatures for DMSO, glycerol, and ethylene
glycol (at 100% w/w) are −122.2, −187, and −115
◦
C, respectively [64] when compared to that of
pure water (−135
◦
C) even if their primary mechanism of action in preservation at high concentration
is by vitrification, then their only role is relaxing the high cooling rate (10
6
K/sec) requirement for
the vitrification of water. Actually, for a 5 M solution of DMSO in water, the critical cooling rate for
vitrification (the cooling rate at which crystal formation is minimum) is approximately 10 K/sec. This is
achievable, however the critical warming rate is still unattainable (10
8
K/sec) [65].
12.2.3 Vitrification
The discovery of the presence of a vitrified state in the cytoplasms of desiccated plant seeds [66], Artemia
cysts, and fungal spores started extensive research for desiccation preservation of mammalian cells
and tissues. For plants and certain animals, it was shown that the transition to the glassy state was
enabled through the accumulation of certain carbohydrates (glucose, sucrose, raffinose, and stachyose in
plants, and trehalose in microorganisms and animals making up about 15 to 25% of their dry weight).
Having the same chemical groups (–OH) as water, carbohydrates are hypothesized to replace the hydrogen
bonds formed between water molecules and the macromolecules in the cytoplasm and the lipid mem-
branes stabilizing their conformations in the absence of water [5]. This has formed the basis of the “water
replacement hypothesis” [67] that was offered as an explanation for the protective capacity of certain
carbohydrates against freezing and desiccation damage. Another theory proposed is based on the “prefer-
ential exclusion” of carbohydrates from macromolecule surfaces [68,69]. This theory predicts that when
water is scarce in the cytoplasm (as would be the case during desiccation or freezing), water molecules
in the hydration shell of the macromolecules remain undisturbed while the void left by the removed free
water is filled by the carbohydrates eliminating structural collapse. It is not known to this date which
one of these hypotheses explains the protection mechanism responsible for reducing macromolecule and
membrane denaturation during desiccation.
Additionally, a recently introduced hypothesis based on molecular mobility measurements suggests that
the main protective effect of carbohydrates is by breaking the structure of the surrounding water molecules
(especially at low water concentrations) and creating more structured water clusters, therefore enabling
encapsulation of biological macromolecules in a rigid matrix [70]. This hypothesis is in agreement with
the additional claim made by the first two hypotheses that upon removal of the free water, a cytosolic
glass with reduced molecular mobility is formed, virtually stopping all biochemical processes. In addition
to carbohydrates, certain solutes abundant in the cytosol (such as proteins, amino acids, ions, and salts)
are also thought to be participating in the formation of the cytoplasmic glass [71] (for a recent review,
see Buitink et al. [72]). It should be noted that the glass transition temperature of an ideal mixture is
determined by the glass transition temperatures of its constituents. Since it has a very low glass transition
temperature (T
g
=−135
◦
C), water significantly reduces the glass transition temperature of carbohydrates
as a function of its concentration.
Chemical reaction rates have been shown to decrease in the presence of high concentrations of carbo-
hydrates, facilitating protein dynamics studies [73,74], slowing down oxygen diffusion therefore increasing
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12-12 Tissue Engineering
the stability of fluorescent molecules [73], reducing degradation of enzymes [75,76] and proteins [74,77],
and enabling lyophilization of bacteria [78] and viruses [79] supporting the cytosolic vitrification hypo-
thesis by experimental evidence. However, contradicting reports also exist, where researchers did not find
any difference between the state diagrams (glass transition temperature as a function of water content)
of desiccation tolerant and intolerant plants [80]. This has been offered as the basis of why cytosolic
vitrification alone can not be responsible for preservation in the desiccated state.
It was shown that for the carbohydrates to protect against desiccation damage, they should be present
on both sides of the membrane [81]. Almost all of the glass-forming carbohydrates and polymers are
membrane impermeable and should be introduced artificially with the exception of glucose. Currently
applied reversible membrane breaching methods are microinjection [82], osmotic shock [83], electropor-
ation [84,85], thermal shock [86], acoustical exposure [87,88], endocytosis [89], and transport through
switchable membrane pores [90,91]. For details of these methods, see Acker et al. [81]. An alternative
method utilized is the genetic modification of mammalian cells to express genes for coding carbohydrates
intracellularly [92]. It is still perceived as a challenge to upload mammalian cells with high concentrations
(0.2 to 0.3 M) of carbohydrates required for preservation.
A glass is a metastable liquid with very low molecular mobility of its main structural ingredient, which
forms an amorphous matrix rather than an energetically more favorable crystal. Whether achieved by
rapid cooling, desiccation, addition of cryoprotectants or carbohydrates, amorphous to glassy phase
transition is characterized by a sharp discontinuity in the temperature–density curve. As an indication of
very compact packing in the molecular arrangement in the glassy phase, density is a very weak function
of temperature. At the glass transition temperature, specific heat also decreases significantly since a
higher percentage of the thermal energy transferred to the system causes a temperature change without
being dissipated by the exceedingly confined molecular motions of the matrix molecules. Even though
vitrification is characterized by a very significant change in viscosity (O(7–9)), and the stopping of α-
relaxation processes, depending on the relative sizes of the matrix molecules and the solvent remaining in
the system, molecular motion does not completely stop [42]. During drying of thin films or small droplets
of high molecular weight polymer and carbohydrate solutions for example, evaporation continues even
though early on a glassy film is formed on the surface. The decoupling of matrix mobility from that of the
solvent at glass transition may present a challenge that the diffusion of the solvent and the small solutes
can not be prevented during storage of biological materials. The glass transition temperature increases
with increasing molecular weight. Therefore, with increasing molecular weight of the matrix, the mobility
of the solvent at matrix glass transition also increases [93,94]. This has led some researchers [95] to
conclude that a glassy matrix (actin embedded in a glass formed by dextran, a very high molecular weight
carbohydrate) can not protect against desiccation damage. Apparently, in this particular case even though
the matrix was glassy, decoupling of matrix and solvent (and small solute) mobilities were very significant
resulting in denaturation of actin.
It would be wrong to conclude however, that the diffusion of small solutes and the solvent in a glassy mat-
rix would not be affected by the presence of the glass. With increasing solute concentration, the mobility of
the solvent is increasingly limited and therefore the solvent switches from the free, unrestricted diffusion
to jump-diffusion (Figure 12.3) between the cages formed by the glassy matrix [96]. There is evidence
that the transition in the diffusion mode of water coincides with the point, where the carbohydrates have
been shown to form a three-dimensional hydrogen bonded network [97]. The diffusion constant shows
an Arrhenius type dependence on temperature above and below the glass transition temperature (with
different activation energies) indicating decoupling of the solvent and the carbohydrate matrix mobility
[98–100]. For example, during desiccation diffusivity of water in the Artemia cysts decreases with decreas-
ing water content, however below a critical water content corresponding to 0.15 g
water
/g
drymatter
[101], it
starts to increase.
As opposed to diffusion in a glassy matrix, where translational mobility is dictated by cooperative
diffusion, in cryopreservation, diffusion is dependent on the presence of defects in the crystalline structure
(and the presence of grain boundaries). In a perfect crystal (without any defects or grain boundaries),
mobility of the solvent and small solutes are exceedingly hindered (Figure 12.2).
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Roles of Thermodynamic State and Molecular Mobility 12-13
For glass-forming liquids, with increasing cooling rate, the probability of reaching the glassy state
increases (when compared to crystallization). However, since a glass is intrinsically a liquid with a higher
energy than its crystal, given enough time (depending on the characteristics of the carbohydrate and the
storage conditions this could be weeks, years, or even centuries) it will crystallize.
In the absence of chemical and electrical interactions, the size of a molecule determines its mobility
in a glassy matrix. In dextran solutions, for example with increasing molecular weight of the dextran,
water mobility at glass transition increases. For successful storage therefore, the size of the molecules to be
preserved should be larger than that of the molecules making up the glassy matrix and the free volume of
the matrix they form. Rigidity of the glassy matrix may also contribute to its storage potential by reducing
solvent mobility. For example, it was shown by Elastic Incoherent Neutron Scattering measurements that
the glassy matrix formed by trehalose (which is known to be superior in terms of its protective potential) is
more rigid than the glasses formed by maltose and sucrose [102]. If the preserved molecules are polar, their
mobility may further be reduced since they can form strong hydrogen bonds with the glassy matrix. Due to
the facts presented above, we may conclude that a high glass transition temperature alone is not sufficient
in determining the storage potential offered by a particular glass former. Even though dextran has a higher
glass transition temperature than trehalose and sucrose, it has been repeatedly shown to have inferior
protective capacity against desiccation and freezing damage. One reason for its low protective capacity
may be its large size making it less flexible and therefore its hydration sites less accessible. However, there
is reason to believe that the main factor is the high pore size of the glassy matrix it forms presenting lower
resistance to the diffusion of solutes and solvents. With the technological advances making it feasible to
measure molecular mobility in the glassy state, this hypothesis can be put to test.
12.2.4 Vitrification by Ultrafast Cooling
If the kinetic energy can be reduced faster than the rotational and translational diffusion timescales at any
temperature, then vitrification without crystallization can be achieved for an aqueous glass former at any
water content. If it can be cooled fast enough (10
6
to 10
7◦
C/sec), even water can be vitrified at a tem-
perature of −135
◦
C. For biological materials, these cooling rates can not be achieved using conventional
techniques such as liquid nitrogen immersion. However, methods utilizing cooling with simultaneous
heating are promising [103]. Ultrafast cooling, if achieved, does not expose cells to osmotic and dehyd-
ration stresses and therefore also eliminates compartmentalization (Figure 12.1). However, if storage
at noncryogenic temperatures is desired, methods to reduce mobility at high temperatures should be
developed.
12.2.5 Vitrification by Desiccation
During isothermal drying, the glass transition temperature of the solution increases. Evaporation of the
solvent causes a decrease in the free volume increasing the viscosity and the structural relaxation times.
Mode Coupling Theory [40] predicts a crossover temperature located at approximately 40 to 50 K above
the glass transition temperature of a glass-forming solution, where there is a transition from liquid-like
to solid-like molecular dynamics. The structural relaxation time, τ
α
, is related to the collective motion of
a group of molecules to loosen up the cage they form around a single molecule and enable it to make a
translational diffusive motion. The short timescale relaxation time, τ
β
, however involves the temperature
dependent vibrational motion of a single molecule, which also involves rotation. τ
α
reaches a value of
10
2
sec (τ
α
= 10
−7
sec at the crossover temperature) very close to glass transition (for an extensive review,
see Novikov, 2003 [104]). The O(9) decrease in the structural relaxation time with proximity to glassy state
(each evaporating water molecule making it more difficult for the next one in the solution to evaporate) is
thought to make vitrification improbable in the experimental timescales considered. However, vitrification
of carbohydrate solutions can be accelerated using a cocktail of carbohydrates [105] and salts [120].
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12-14 Tissue Engineering
12.2.6 Lyophilization
Cryopreservation (either in the frozen or the vitrified state) requires very low temperatures for storage
and transportation and therefore is not economical for many purposes. An alternative to cryopreservation
is lyophilization, where the reduction of molecular mobility reached at low temperatures by cryopreser-
vation can be achieved at relatively high temperatures by the removal of water (the universal plasticizer),
thus increasing the glass transition temperature [106]. The product to be stored is initially frozen in
the presence of cryoprotectants and carbohydrates. Then, by the application of vacuum, frozen water
is sublimated at progressively increasing temperatures. Removal of water increases the glass transition
temperature of the product and establishes stability at ambient temperatures. In the pharmaceutical and
food industries, lyophilization is widely used for the preservation of proteins, enzymes, bacteria, and
foodstuff.
The main advantage of lyophilization over desiccation is in minimizing the exposure to osmotic stresses.
During diffusive or convective drying, by the removal of water, the solutes concentrate and the product
to be preserved is exposed to increasing osmotic stresses over long periods of time. In lyophilization, the
product is first frozen and then the water is removed, minimizing osmotic stress buildup. However, the
removal of water from the frozen structure leaves pores within the protective matrix and the product.
These pores in time may collapse and the structural integrity of the product may be lost. In order to
prevent this during lyophilization, the primary and secondary drying temperatures are kept above the
collapse temperature of the matrix. Certain high molecular weight carbohydrates (such as dextran) are
also incorporated to increase the structural stability of the matrix.
12.3 Storage
During storage the preserved organism does not need a steady supply of nutrients (and removal of the
by-products). On the down side, it does not have the metabolic activity to repair the accumulating
damage. In the preserved state, the harmful environmental factors are, to a degree, physically isolated
from the organism by the surrounding matrix (the frozen liquid crystal structure in cryopreservation or
an amorphous matrix of carbohydrates during desiccation) reducing the amount of accumulated damage.
Regardless of the processing method, the final thermodynamic state of the product and the molecular
mobility at that state determine the storage stability as a function of storage parameters (primarily humid-
ity, temperature, and pressure). If at the storage condition, the product is not at equilibrium (internally
or externally with the environment), the chemical processes will continue even at slow rates toward the
minimum energy state. For example in vitrification, since the glass formed is inherently at a higher
energy state it will crystallize in time. Crystallization is accompanied by compartmentalization [105] and
heteregeneous devitrification, resulting in an increase in mobility. It also creates high mechanical stresses
that can damage the product. The structural mobility of the glass determines its crystallization lifetime,
while the mobilities of the solvents and small solutes within the glass determine the degradation of the
stored product. One way to eliminate crystallization is to use cocktails of carbohydrates and salts. For
example, raffinose and stachyose are very effective in inhibiting sucrose crystallization [107]. The pres-
ence of small solutes and proteins in the cytoplasm may also help reduce crystallization of sugars [108].
In cryopreservation, the grain boundaries between the frozen sections and the interfaces between the ice
crystals and the solutes rejected during freezing have higher free energy and therefore mobility. Molecular
mobility in these regions determine the stability of the stored product.
Vitrified products can be stored at higher temperatures, however their storage conditions still need
to be regulated carefully since molecular mobility increases significantly with increasing water con-
tent. For a trehalose glass the glass transition temperature decreases by 50 K by a very slight change
in the water content (0.05 g
water
/g
drymatter
). If the water activity in the environment is higher than
that in glass, the product absorbs water and the molecular mobility within increases. On the opposite
end, if the environmental water activity is lower, the stored product may desiccate further, losing its
hydration water. For most lyophilized bacteria, it has been shown that irrespective of the preservation
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Roles of Thermodynamic State and Molecular Mobility 12-15
agent used, for successful revival, the water content within the product should not drop below a certain
limit.
The other important factors that need to be considered for successful storage in vitrified state are
oxygen and light. Free oxygen radicals have very high diffusivity and are extremely reactant. Even at
very low mobility environments, they may jump-start enzymatic reactions. Some carbohydrates, such as
trehalose are known to prevent oxygen radical damage [109] as well.
12.4 Summary
The “molecular mobility” hypothesis analyzed here suggests that, if all of the reactants in an organism
can be reversibly immobilized in their native configuration, the biochemical processes can be stopped
and preservation can be achieved (Figure 12.4). The condition set forth by the hypothesis is sufficient to
halt the diffusion-limited biochemical reactions, which dominate in a crowded, confined environment
such as in the cytoplasm [29]. However, not all of the chemical reactions in an organism is governed
by diffusion. In order to respond to sudden changes in its environment (under certain conditions, the
metabolic rate has been known to increase up to 35 times), the mammalian cell has devised strategies to
accelerate certain reactions without being limited by diffusion timescales, for example by accumulating
reactants in aggregates (compartmentalization), reducing the degree-of-freedom of the reactants (such
as the case with membrane proteins) and advection (bulk mixing due to cell motion). The effects of the
currently applied and proposed biopreservation methods on this kind of chemical reactions remain to be
researched.
The important points that are highlighted in this chapter are:
(1) Glass transition is a collaborative phenomena involving the solvent and the solute. With increasing
difference in the molecular sizes of the solvent and the solute mobility of the solute molecules making
up the glassy matrix and that of the solvent (or other small solutes suspended in the solvent) decouple
strongly at glass transition. Mobility of the solvent may be significantly higher than that of the matrix,
even below the glass transition temperature of the system, enabling certain chemical reactions to proceed.
High molecular weight carbohydrates increase the glass transition temperature of the system, however
they do not necessarily decrease the solvent (or small solute) mobility in the same degree.
(2) If noncryogenic temperature storage is desired, the mobility within and surrounding the preserved
product should be lowered. This may be achieved by loading high amounts of protectants (cryoprotectant
solvents such as glycerol, DMSO, etc. or carbohydrates, proteins, etc.) into the product to displace water
or change the properties of the intracellular water. It is unlikely that any organism can be successfully
preserved by vitrification at conditions requiring complete removal of all of its water. The “essential
water” in the product upon removal causes irreversible damage. This quantity is not easily measurable.
The water affinities of the macromolecules and membranes in the preserved product and that of the added
protectant are different and also change with the decrease in the availability of water. Decreasing the water
content of the overall system comprised of the product to be preserved and the surrounding protectant
matrix (frozen, amorphous, or glassy) does not necessarily mean that the water contents of the matrix
and the product are equally reduced.
(3) The importance of diffusion-limited reactions for the metabolic activity of the product should
be established. Reduction of mobility within the medium is most likely to reduce the diffusion of any
size molecule, however there is increasingly more evidence collected showing that the majority of the
biochemical reactions do not depend on diffusion alone. For preservation to be successful, mobility of all
molecules in all size scales should be stopped without irreversibly disrupting their native configurations.
(4) The state of intracellular water is directly related to the metabolism and functioning of an organism.
All of the chemical agents known to have protective capacity against freezing and desiccation damage
modify the structure of the intracellular water. More research is required to establish the role of water
in organisms in order to develop successful preservation methods and to increase the efficiency of the
currently applied ones.
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12-16 Tissue Engineering
Acknowledgments
The authors thank Dr. James Clegg, Dr. John Bischof, and Dr. Xiang-Hong Liu for careful reading of the
manuscript and their constructive feedback. This work was supported by the National Institutes of Health
grants.
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