Fisher John P. e.a. (ed.) Tissue Engineering

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Roles of

Thermodynamic State

and Molecular

Mobility in

Biopreservation

Alptekin Aksan

University of Minnesota

Center for Engineering in

Medicine/Surgical Services

Harvard Medical School

Massachusetts General Hospital

Shriners Hospital for Children

Mehmet Toner

Center for Engineering in

Medicine/Surgical Services

Harvard Medical School

Massachusetts General Hospital

Shriners Hospital for Children

12.1 Water–Solute Interactions and Intracellular

Transport ................................................. 12-3

Intracellular Water and Molecular Mobility •

Transmembrane Water Transport Effects

12.2 Molecular Mobility in Preservation .................... 12-6

Molecular Mobility in Supercooling and Phase Change •

Cryopreservation • Vitriﬁcation • Vitriﬁcation by Ultrafast

Cooling • Vitriﬁcation by Desiccation • Lyophilization

12.3 Storage .................................................... 12-14

12.4 Summary ................................................. 12-15

Acknowledgments............................................... 12-16

References ....................................................... 12-16

In a very broad sense, preservation can be deﬁned as the process of reversibly arresting the biochemical

reactions and therefore the metabolism of an organism (in a state of suspended animation [1]) in order

to sustain function after a “prolonged” exposure to otherwise lethal conditions. The lethal conditions are

created by the inadequacy of the surrounding medium in supplying nutrients and removing by-products,

exposure to draught, or the extremes of temperature that would disturb the biochemical processes vital

to the organism.

The rates of biochemical reactions are dependent on the proximity and mobility of the reactants.

Mobility is determined by the mutual interactions of the solvent with the solutes. The state of water (the

solvent) determines the mobility of the solutes and in return, the solutes change the structural organ-

ization of nearby water molecules through hydrophilic and hydrophobic interactions. In the cytoplasm,

the thermodynamic state of the medium (and therefore the molecular mobility) determines the rate of

metabolic activity.

12-1

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12-2 Tissue Engineering

: Mass transfer timescale

: Heat transfer time

: Trans-membrane water flux

q ⬙: Heat flux

Ice

Glass

Cell

∆Π

q ⬙

⬙

q ⬙

⬙

q ⬙

⬙

q ⬙

⬙

3q0

rr∆T

T: Temperature

<<1

FIGURE 12.1 Effect of timescales on cell response.

In this chapter, the mechanisms enabling preservation of biological systems will be examined from

the perspective of “molecular mobility” exploring the effects of the timescales for cooling, freezing,

crystallization, vitriﬁcation, structural relaxation, and diffusion. Following example underlines the

importance of timescales in preservation.

The timescales of biochemical reactions and the preservation conditions applied to the organism play

crucial roles in determining the success of preservation. For example, the ratio of the timescale of water

diffusion, τ

, across the cell membrane (τ

= r/3L

,wherer, L

, and  are the cell radius,

membrane permeability, and osmotic pressure differential, respectively) to the timescale of cooling the

cell experiences, τ

(τ

= (2c

ρrT)/(3q



),wherec

, ρ, q



, and T are the speciﬁc heat, mass density,

heat ﬂux, and temperature differential, respectively) determines the fate of a cell during freezing such that

(Figure 12.1):

• τ

/τ

> 1 causes excessive dehydration of the cell.

• τ

/τ

∼ 1 establishes an intra/extracellular equilibrium such that the intracellular water trans-

ported across the membrane balances the extracellular osmotic increase induced by freezing

(the solute-concentration effect [2]) minimizing the amount of intracellular free water.

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Roles of Thermodynamic State and Molecular Mobility 12-3

• τ

/τ

< 1 results in rapid cooling (faster than the cell can reach equilibrium with its surroundings)

inducing Intracellular Ice Formation (IIF) known to be lethal to most cells(see Figure 12.2 Toner [3],

for the correlation between IIF and post-thaw viability of mammalian cells).

• τ

/τ

 1 theoretically, yields to ultrafast cooling without ice crystallization (if as an additional

constraint τ

/τ

 1 where, τ

is the timescale of structural relaxations) enabling vitriﬁcation of

the extracellular medium, and more importantly the cytosol.

12.1 Water–Solute Interactions and Intracellular Transport

Water is the most abundant substance in and around an organism, yet it is the least understood in terms

of its role in biological function and preservation. Water has unique physical and chemical properties

[4] (for a complete review, see Franks [5], for an extensive collection of the properties and the anom-

alies of water, see the excellent electronic source by Chaplin [6]). Hydrogen bonds (E

= 4 to 7 kJ/mol

[7]) with bond energies similar to the local thermal ﬂuctuations are continuously formed and broken

between neighboring water molecules organizing them into ﬂickering clusters of minimum free energy.

These loosely bonded hydrogen clusters have very short life spans (τ

= 10

−11

to 10

−12

sec) and are

quickly destroyed just to form new ones in a never-ending cycle. This behavior establishes the basis of

molecular mobility of water such that even in pure liquid form, a single water molecule is not inde-

pendent in its motion but, at any instant of time, moves in coordination with a cluster of molecules. It is

therefore widely believed that for water a cluster (rather than an individual water molecule) is the element-

ary structural unit and the interactions of clusters are responsible for its unique chemical and physical

properties [8].

There is a continuous tug-of-war between the hydrogen bonds trying to stabilize the network of

water molecules and the temperature dependent random motions breaking these bonds. With decreasing

temperature, the magnitude of local thermal ﬂuctuations decrease, increasing the lifetime of the hydrogen

1.E-14

1.E-12

1.E-10

1.E-08

1.E-06

1.E-04

1.E-02

200 250 300 350 400

Temperature (K)

water

(cm

/sec)

Supercooled water

Ice

Liquid water

Water in 75% sucrose

Water in 70% trehalose

Water in erythrocyte cytoplasm

Water in water

FIGURE 12.2 Self-diffusivity of water. Data of water diffusivity in 70% trehalose solution: NMR by Ekdawi-Sever

et al. [112], NMR by Rampp et al. [42] and DMS by Conrad and de Pablo [41]; water diffusivity in 75% sucrose

solution: Ekdawi-Sever et al. [112]; water diffusivity in the supercooled region: DMS by Paschek and Geiger [113] and

NMR by Price et al. [114]; water diffusivity in ice: Onsager and Runnels [115] and Petrenko and Whitworth [116];

water diffusivity in liquid phase by Mills [117], NMR by Harris and Newitt [118]; water diffusivity in 75% sucrose:

NMR by Moran et al. [119].

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12-4 Tissue Engineering

bonds among water molecules (i.e., the number of available neighboring hydrogen bonding sites per water

molecule at any given time decreases). Water mobility (and its self-diffusion coefﬁcient, D

, as shown in

Figure 12.2) therefore decreases [9,10] while the water clusters they participate in get more densely packed

and grow [7]. Water mobility is not only a function of temperature but also the thermodynamic state. For

example, D

of liquid water decreases only by an O(2) over a range of 150 K whereas it drops by an O(6)

upon freezing at 0

◦

C (Figure 12.2). In the frozen state, each water molecule makes hydrogen bonds with

only four neighboring molecules in a three-dimensional tetrahedron-like conﬁguration. The degree of

tetrahedricity (perfectness of the tetrahedral conﬁguration) increases with decreasing temperature [10].

The strong interations between water molecules also cause an unexpected decrease in D

when the density

is decreased by increasing hydrostatic pressure. In water, density decrease lowers the hydrogen bonding

possibility, therefore reduces mobility. In other liquids however, mobility is increased due to the increase

in the free volume.

Any surface (hydrophilic or hydrophobic) or solute (charged or uncharged) disrupts the bonding

patterns of the water molecules in its near vicinity causing local polarization and altering the life cycles of

the surrounding water clusters [6,11]. This results in variations in water mobility, which can be detected

by methods such as Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared Spectroscopy

(FTIR). Close to a hydrophilic surface exerting a higher attraction force, water mobility decreases (the

water molecules make stronger bonds with the surface and they are less available to join in a cluster).

This causes depression of the freezing temperature and is the origin of the “unfreezable water” concept

frequently used by the cryobiologists. Similarly, in close proximity to a hydrophobic surface or a solute, in

this case entirely due to geometrical factors limiting hydrogen bonding possibility (that the water molecules

can not make bonds with the hydrophobic surface), in the direction perpendicular to the surface, water

mobility and therefore diffusion decreases. Parallel to the hydrophobic surface however, water diffusivity

is not different from that of free water [12]. The coexistence of hydrophobic and hydrophilic surfaces on

most proteins therefore creates large spatial gradients of water mobility, which may be closely related to

protein function (e.g., the alternating regions of high and low water mobility within the hydration shells of

actin ﬁlaments are thought to be contributing to the movement of myosin along these ﬁlaments [13]). Ions

also affect nearby water molecules and alter their mobility [14]. For example, structure-breaking solutes

such as urea [15] and large ions such as I

−

and Cs

[14] increase the mobility of the water molecules in

their immediate vicinity. Small ions such as Mg

and F

−

, on the other hand, have the opposite effect on

their hydration layer. Interactions with nearby surfaces and solutes change the lifetime and the stability of

each vicinal water cluster and change their physical properties (e.g., low mobility vicinal water has lower

mass density, lower freezing point, and higher speciﬁc heat than bulk water).

The interaction of water with solids and surfaces is mutual. Water is not only a solvent but is also a react-

ant itself. It is a substance functioning in cooperation with the solutes [16] altering their charge, conforma-

tion, and reactivity. The range of water–solute interactions (the distance a water molecule should be from a

surface or a solute to be fully isolated from its effects) is one of the most controversialtopics in the literature,

however it is widely accepted that vicinal water layers do not extend beyond 1 to 10 water molecules.

12.1.1 Intracellular Water and Molecular Mobility

In isotonic conditions, approximately 70% of the cell’s volume is water. However, it would be wrong to

think that the intracellular solutes and macromolecules bathe in a dilute solution. It has long been known

that most, if not all, of the intracellular water exhibits physical properties unlike those in the bulk [17]

(see the D

in erythrocytes in Figure 12.2). This is attributed to the presence of high concentrations

of proteins (200 to 300 g/l) [18], ions, amino acids, fatty acids, sugars, and other small solutes in the

cytoplasm enmeshed in a network of cytoskeletal macromolecules (actin ﬁlaments, microtubules, and

intermediate ﬁlaments). In individual organelles (such as mitochondria) the protein concentration may

be even higher [19]. Within the cytoplasm, at any given time, water molecules are either a part of a

tight cluster (bulk water) or in the close vicinity (vicinal water) of a surface (cell or organelle membrane)

or a solute (a macromolecule, ion, or amino acid). There is not a consensus in the literature on the relative

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Roles of Thermodynamic State and Molecular Mobility 12-5

populations of vicinal and bulk water within the cytosol. The estimates vary in a range of 0 to 100% of the

total intracellular water (for details, see Clegg [17] and the references therein). Similarly, the names given

to the various subpopulations of water molecules in the close proximity of surfaces/solutes also vary from

one source to another (hydration, bound, vicinal, essential, structural, ordered, unfreezable, osmotically

inactive, etc.).

Overall cytosolic mobility is directly related to the metabolism and function of a cell [20,21]. However,

the mobility of water in the cytosol is not spatially homogeneous [22,23] as evidenced by the presence of

compartmentalization inside the cytoplasm (regions of solute aggregation and variable water mobility)

using Fluorescence Recovery After Photobleaching (FRAP) [24] and Raman Scattering Microscopy (RSM)

[25]. It is postulated that the intracellular mobility gradients determine the active and resting states of

cells [26,27] and are altered in response to osmotic stress [28] and in the presence of carcinogens [26].

As opposed to dilute solutions, where the chemical reactions are transition-state-limited [29], most

of the biochemical reactions in crowded environments are diffusion-limited. However, the diffusion

mechanism in the cytoplasm is different from that in a dilute solution and is altered by the increased

frequency of close-range interactions such as binding of and collisions between solutes and surfaces.

In order to determine the hydrodynamic properties of the cytosol (translational, rotational diffusion

coefﬁcients and viscosity) various techniques have been utilized (NMR, FRAP, Electron Spin Resonance

(ESR), etc. See Table 12.1 for details). The values reported in the literature lie in a very broad range

(e.g., cytosolic viscosity values vary from 0.5 to 5 times that of water) and contradict each other (see

reviews byLuby-Phelps et al. [24] and Arrio-Dupont et al. [30] for cytosolic diffusivity measurements using

different methods and tracer molecules). The main reason for the discrepancy among the reported values

is believed to be originating from the differences in the methodologies applied (such as the measurement of

the translational diffusivity of a very large number of tracer molecules over a large volume [∼1/10 to 1/20

of the volume of an attached cell] with FRAP or the shortcoming of NMR in distinguishing the signals

from the intermolecular and intramolecular bonds and the requirement for relatively long acquisition

times [31]), the characteristics of the tracer used (e.g., its size [24]), and inability of most of these

methods to distinguish among different molecular interactions (free diffusion, binding, or collision) in this

crowded environment [32]. The differences observed between the cytoplasmic viscosity values measured

by rotational vs. translational diffusion of tracers indicate that physical interactions (such as binding and

TABLE 12.1 Most Common Methods for Measurement of Molecular Mobility

Method Quantity measured Range/limitations

Nuclear magnetic

resonance (NMR)

Relaxation times T

, T

of proton

(

H) and carbon (

C) nuclei of

water–carbohydrate samples

Cannot distinguish between the intermolecular and

intramolecular bond signals. Measurement times are

higher than the measured relaxation times

Dielectric spectroscopy Complex dielectric permittivity Water dipole moment relaxations in the kHz–GHz

range

Differential scanning

calorimetry (DSC)

Speciﬁc heat change C

T=T

May be used in the 100–1500 K range. Measures the

glass transition temperature of the bulk sample

Fluorescence recovery after

photobleaching (FRAP)

Translational diffusivity of the

tracer molecule

Measures mobility of very large number of molecules

in a large area (∼1 µm

). Measurements in a glass

are not feasible due to photobleaching

Electron spin resonance

(ESR)

Spin relaxation of molecular

probes (such as tempol)

Rotational mobility range [110]:

t = 10

−12

–10

−8

sec (continuous-wave EPR),

t = 10

−7

–10

sec (saturation tranfser EPR). Probe

properties change with hydration level [111]

Fourier Transform Infrared

Spectroscopy (FTIR)

Molecular bond vibration Strong absorption of IR light by water

Circular dichroism

Quasielastic neutron

scattering (QNS)

Measurement time ∼10

−12

sec,

Measurement distance ∼1A [17]

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12-6 Tissue Engineering

collisions) present a higher obstacle to diffusion when compared to ﬂuid phase viscosity (see e.g., Figure 1

in Mastro and Keith [33]). Crowding and solute concentration affect larger macromolecules more than

the small solutes and ions, and it is therefore not feasible to assign a single parameter for mobility. Even

though the viscosity of the cytosol is not signiﬁcantly higher than water, some large macromolecules

do not diffuse at all in the timescale of hours [34]. This would limit the reaction rates of some of the

intracellular biochemical processes, if they depended on diffusion only. Nature overcomes this problem

by crowding certain reactants in small regions (compartmentalization) of the cytoplasm [35], which also

explains the spatial heterogeneity of water mobility observed intracellularly [22,23].

12.1.2 Transmembrane Water Transport Effects

The cell membrane shows very low resistance to water transport. However, it is the biggest obstacle to

the transport of solutes. Membrane permeability to solutes depends on the size, charge, and the hydrogen

bonding characteristics of the solute (for a review of membrane transport phenomena, see McGrath [36]).

Transport across the cell membrane in response to osmotic gradients is at the cornerstone of biopreser-

vation studies since it is directly related to administration of preservation agents and to the amount and

mobility of the intracellular water. Water is transported into the cell by three different methods (a) dif-

fusive transport across the membrane, (L

∼ 2–50 × 10

cm/sec), (b) facilitated transport through

membrane channels (L

∼ 200 × 10

cm/sec), and (c) cotransport through glucose transporters and

ion channels (L

∼ 4 ×10

cm/sec) [37]. Methods for quantifying membrane transport are reviewed by

Verkman [38].

Both desiccation and freezing (as well as their complementary processes; rehydration and thawing)

induce very high osmotic gradients across the cell membrane. Cells are capable of responding to mild

osmotic gradients by adjusting their volume, mainly by water transport. Applying an osmotic gradient

almost all of the free water (called the osmotically active water) in a cell can be removed temporarily without

any permanent damage. The water of hydration (participating in the osmotically inactive volume) on the

other hand, is tightly associated with the solutes and surfaces and upon removal causes polarization of

surfaces, aggregation and denaturation of the macromolecules [20,21].

12.2 Molecular Mobility in Preservation

In a dilute, nonreacting, binary solution diffusivities of the solvent and the solute depend on their relative

molecular sizes [39] as well as their concentrations and temperature. For this system, Stokes–Einstein

relationship correlates the hydrodynamical properties of the solution as,

translational

nπrη

, (12.1)

where, D, k, T , r, and η are the diffusivity, Boltzmann’s constant, absolute temperature, hydrodynamic

radius of the diffusing particle (van der Waals radius), and the viscosity, respectively. The constant n, takes

the value of 6 for a “stick (hydrophilic) boundary” condition and the value of 4, for a “slip (hydrophobic)

boundary” condition. With increased solute concentration, diffusion becomes more restricted and dif-

ferent interactions such as collisions with other solutes and binding between molecules start to dominate

and deviations from the Stokes–Einstein relationship is observed.

For a supersaturated solution, crystallization is the energetically most favorable path. However, if the

concentration increases very rapidly (or the temperature drops very fast) a meta-stable “glassy” form can

be reached. For a glass-forming system, the transition from a dilute to a concentrated solution diffusion

mechanism is determined by the concentration corresponding to the crossover temperature, T

, predicted

by the Mode Coupling Theory [40]. At the crossover temperature there is a transition from liquid-like

to solid-like dynamics. Note that T

∼ (1.14–1.6)T

for most glass-forming solutions, where T

is the

glass transition temperature. Diffusion in very high concentration solutions (close to glass transition

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Roles of Thermodynamic State and Molecular Mobility 12-7

Free diffusion:

Unrestricted diffusion down the osmotic gradient. With increased concentration,

limiting factors (such as chemical interactions between solutes or interparticle

collisions) start to dominate

Cooperative diffusion:

Appears with the transition from liquid- to solid-like behavior at the critical

temperature, T

, during rapid cooling (or at the critical concentration during

isothermal desiccation) requiring the collaboration of all of the molecules in a

non-crystalline cluster to loosen their cage to give enough space to a single

molecule to diffuse. At this regime a and b-relaxation times start to decouple

Decoupled diffusion:

Decoupling of the diffusion of the

matrix molecules making up the

glass from that of the solvent and

small solutes. Starts with the

stopping of the a-relaxation

processes of the glass-forming

matrix at the glass transition

Jump diffusion:

In a crystal, diffusion is directly

correlated to the presence of defects

(vacancies or additions). Solvent or

small solute molecules jump from

one vacancy in the crystal matrix to

another

FIGURE 12.3 Mechanisms of diffusion.

temperature) is governed by the frequency of jumping between the cages surrounding the tagged molecule

(either the solvent or a small solute) and is comparable to the time the molecule spends entrapped in the

cage rattling (β-relaxation) [41]. This is similar to the mechanism of diffusion in crystalline systems, where

the diffusing molecule jumps between the crystal defects (vacancies). Frequency of jumping is inversely

related to the structural relaxation (α-relaxation) time, τ

of the matrix. Temperature dependence of

distinguishes between the “fragile” and “strong” glasses, where the variation in τ

with temperature is

steeper in the former case. In Figure 12.3 changes in the mechanism of diffusion with the thermodynamic

state of the system is summarized.

In a concentrated and crowded environment such as in the cytosol, the motion of a small solute can

be divided into two main components (Figure 12.4a) (1) the translational diffusive motion (governed

by the α-relaxation timescale of the system), which results in a net displacement of the molecule down

its osmotic gradient and (2) the random motion, which does not result in a net displacement. The

random motion is governed by the physical and chemical interactions with the solvent and the sur-

rounding solutes and is characterized by the β-relaxation timescale of the system, which includes rotation

and Brownian motion. When the solvent is frozen, as a function of the storage temperature and the

perfectness of the crystal structure formed, α-relaxation timescale increases. Depending on the relat-

ive magnitudes of the solvent and the solute molecules (and the size of the pores formed) β-relaxation

may still continue (Figure 12.4b). Note the unfrozen bound water molecules in close proximity to the

protein surface with lower mobility. If the system is desiccated (to a point where some of the water

molecules in the hydration layer is also removed), both α and β-relaxations of the system may be stopped

completely, however, due to removal of the hydration layer, the protein may denature and its active

site may not be available for the binding of the ligand (Figure 12.4c). If denaturation of the protein is

irreversible, even after rehydration (when molecular mobility is restored) the ligand can still not bind

to the protein. Carbohydrates may be administered in order to prevent the denaturation of the pro-

tein while water is removed from the system lowering the mobility within the medium forming a glass

(Figure 12.4d,e).

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12-8 Tissue Engineering

hydration

water

bulk

water

|>0, |x

|>>0

|~0, |x

|>0

|~0, |x

|~0

|~0, |x

|=0

Molecular mobility

x=x

: net translational diffusion, x

: random

Freezing

Desiccation

Vitrification

Crystal

Glass

Ligand

Water

Protein

Carbohydrate

(b)

(a)

(c)

Preferential binding

Preferential exclusion

(d)

(e)

FIGURE 12.4 Molecular mobility in biopreservation.

For high solute concentrations in the absence of crystallization, Vogel–Tammann–Fulcher (VTF)

equation predicts the changes in the timescales of molecular motion as:

τ = τ

−(BT

)/(T−T

)

, (12.2)

where τ is the timescale of molecular motion, T

is the Kauzmann temperature corresponding to the zero

mobility state, τ

is the timescale of motion at the Kauzmann temperature (usually taken to be in the

order of 10

sec), and B is a constant related to the energy of activation of the relaxation process. The

values of B, for different carbohydrate solutions can be found in Rampp et al. [42].

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Roles of Thermodynamic State and Molecular Mobility 12-9

12.2.1 Molecular Mobility in Supercooling and Phase Change

At temperatures below the freezing temperature (0

◦

C, 1 atm) water may exist as a supercooled liquid or

ice. The theoretical limit for the presence of free water in the liquid form is −40

◦

C, where homogeneous

crystallization is initiated. For freezing to occur at any given temperature, certain number of water clusters

should form at the same time and reach a critical size (known as the formation of a nucleation embryo).

With decreasing temperature, the critical number of water molecules required to form a nucleation

embryo for the initiation of freezing decreases (from approximately 16,000 at −10

◦

C to 120 at −40

◦

[5]) and at −40

◦

C, it becomes statistically impossible for free water to remain in the liquid phase. In

biological systems, due to the presence of small hydrophobic solutes with low surface energy (such as

ice nucleating proteins in certain plants and bacteria that survive freeze injury), ice nucleation in the

supercooled state is initiated well before the theoretical limit is reached. This is believed to help protect

against the freeze-induced damage by minimizing compartmentalization and creating a more uniform ice

structure.

With decreasing temperature, the diffusivity of liquid water decreases (approximately O(2) 370 to 240 K,

see Figure 12.2) due to change in the mechanism of diffusion from unrestricted to cooperative

(Figure 12.3). Upon freezing, the drop in water diffusivity becomes even more signiﬁcant (approximately

O(6) as shown in Figure 12.2). The reduction in water mobility with supercooling and liquid-to-solid

phase change in addition to the decrease in most chemical reaction rates at low temperatures, makes

cryopreservation feasible.

12.2.2 Cryopreservation

Certain organisms are known to synthesize carbohydrates upon exposure to cold and desiccation (such as

trehalose synthesis by Escherichia coli [43], yeast [44], and nematodes [45]), which is crucial for their

survival [46]. It was discovered (by accident) that glycerol also protects against freeze injury. These ﬁndings

have fueled researchers to explore ways to use these chemical agents (cryoprotectants) for the preservation

of biological organisms, which are normally not freeze or desiccation resistant. Over the years, this has

led to the discovery of other cryoprotectants such as dimethylsulfoxide (DMSO) and ethylene glycol.

Cryoprotectants traditionally are divided into two main groups as membrane permeable and imper-

meable. Most effective and widely used cryoprotectants, DMSO [47], ethylene glycol, and glycerol are

highly membrane permeable whereas most of the carbohydrates (trehalose, hydroxyethyl starch, dextran,

etc.), proteins, and polymers are normally not. Exposure to membrane impermeable (or low permeability

when compared to that of water) cryoprotectants creates an osmotic gradient across the membrane, to

which a cell responds by shrinking. If a membrane permeable cryoprotectant is present on the other hand,

after initial shrinkage, with prolonged exposure and penetration of the chemical, the cell recovers to its

original volume. Similarly after thawing, to remove intracellular cryoprotectants, the cells are exposed to

hypotonic solutions. This results in swelling of the cell followed by return to its isotonic volume. It is widely

accepted that a signiﬁcant part of freeze damage is related to the uncontrolled swelling response during

thawing, that the membrane stretches beyond its mechanical limit and ruptures. The volume response of

the cell to cryoprotectants creates changes in the cytoplasmic molecular mobility due to the changes in

(a) the amount of cytoplasmic free water present at any time, (b) the intracellular solute concentration,

and (c) the changes in the electrical potential gradients due to proximity of macromolecular surfaces.

Additionally, during freezing, depending on the freezing-rate-dependent solute concentration (as presen-

ted previously in the ﬁrst part of this chapter) volume of the cell changes responding to osmotic gradients

(Figure 12.1). Brieﬂy, damage to cells during cryopreservation is attributed to various factors directly or

indirectly correlated to the presence of intra/extracellular ice (such as solute concentration, membrane

potential change, mechanical damage by ice crystals, steep electrical potential, and osmotic gradients,

etc.), however the exact mechanism of freeze injury is not known.

Cryopreservation is a process, which inherently disrupts intra/extracellular continuum and introduces

heterogeneity within the cytoplasm. During freezing of a complex solution, there always is a mutual