5.

11 Cryopreservation and Desiccation Preservation of Cells

Siyuan Chen*, Jie Ren*, and Rongjun Chen, Department of Chemical Engineering, Imperial College London, South Kensington
Campus, London, United Kingdom
© 2019 Elsevier B.V. All rights reserved.

5.11.1 Introduction 157

5.11.2 Key Mechanisms of Cell Damage in Preservation 158
5.11.2.1 Cryopreservation Damage 158
5.11.2.1.1 Cell Damage During Freezing 158
5.11.2.1.2 Cell Damage During Cryostorage and Thawing 158
5.11.2.2 Desiccation Damage 159
5.11.2.2.1 Mechanical Strains and Structural Damage 159
5.11.2.2.2 Metabolically Derived Damage 159
5.11.3 Cell Preservation Technologies 159
5.11.3.1 Cryopreservation 159
5.11.3.1.1 Ice Crystal Formation and Vitrification 159
5.11.3.1.2 Cryoprotectants 160
5.11.3.1.3 Challenges in Cryopreservation 160
5.11.3.2 Dry Storage of Cells 160
5.11.3.2.1 Drying Methods 161
5.11.3.2.2 Desiccation Protectants 161
5.11.3.2.3 Challenges in Cell Desiccation 161
5.11.4 Current Thrusts in Cell Preservation 162
5.11.4.1 Cryopreservation 162
5.11.4.2 Desiccation 162
5.11.5 Conclusions 163
References 163
Further Reading 164

Glossary
Anhydrobiosis A state of suspended metabolic activity induced by extreme removal of cell water.
Cryopreservation Preservation of cells at an ultralow temperature.
Cryoprotectants Compounds used in cryopreservation to protect biological samples from freezing damage.
Desiccation A process of drying an object to an extreme level or to the state of extreme dryness.
Glass transition temperature (Tg) The critical temperature at which a liquid-to-glass transition occurs.
Lyophilization A process that involves the removal of most unbound water from biological materials through controlled
freezing followed by sublimation of ice under vacuum.
Vitrification A process where the aqueous solution within cells forms an amorphous, glassy solid instead of a crystalline, icy
solid (ice).

5.11.1 Introduction

Cell preservation is an integral part of biopreservation and biobanking. It is crucial for the delivery of emerging cell-based therapies
such as blood transfusion, immunotherapy, reparative and regenerative medicine, and fertility preservation. Long-term preservation
of red blood cells (RBCs) plays a key role in both blood transfusion and serologic testing, especially in the case of rare RBCs where
blood samples need to be collected in advance to deal with any emergency. Several cell-based immunotherapies have already been
approved by the US Food and Drug Administration (FDA), such as Sipuleucel-T (Provenge) for prostate cancer treatment (Den-
dreon Corporation, 2010), and two CAR T cell therapies, Kymriah (Novartis, 2017) and Yescarta (Gilead Sciences Inc., 2017),
for large B cell lymphoma treatment. Stem cell–based therapies have been used for the treatment of various diseases, including

*
Equal contribution to this chapter.

Comprehensive Biotechnology, 3rd edition, Volume 5 https://doi.org/10.1016/B978-0-444-64046-8.00451-1 157

158 Cryopreservation and Desiccation Preservation of Cells

hematological cancers, bone marrow failure, corneal injury, and burns. The first stem cell–based regenerative medicinal product,
Holoclar, was approved by the European Medicines Agency (EMA) in 2015 for corneal and skin regeneration. One major hurdle
for cell-based therapies to become a routine clinical reality is cell preservation. Currently there is only an approximately 5-day
time window from production of cells to patient delivery, which may cause problems since safety and efficacy testing could take
more than 2 weeks. In addition, cell manufacture may take place at some distance from the point of final use, so preservation during
transport and shelf life of cellular products have become important limiting issues. Therefore, in order to create reproducible and
cost-effective cell-based therapies, it is of critical importance to preserve validated therapeutic cells for a sufficient period of time
without losing their viability and functionality.
A better cell preservation method allows the better decoupling of cell collection/expansion from final patient delivery. Transport
of blood from donors to clients is a time-critical operation due to its limited shelf life using the present hypothermic preservation
method. With a long-term preservation technique, the abundant on-site storage of blood products can be achieved. It is also critical
for banking of therapeutic cells such as stem cells and immune cells, thus allowing flexible quality and safety testing before research
or clinical use, and transportation among sites of cell collection, processing, and clinical administration. These would set the foun-
dation to develop a manufacturing paradigm for cell-based therapies. It can also significantly reduce the costs of human labor and
clinical cell production facilities.
Cell-based therapies could win regulatory approval, but lack of suitable technology for effective long-term preservation of valu-
able therapeutic cells could result in serious manufacturing and supply chain issues, enormously affecting the business. For
example, Sipuleucel-T (Provenge),1 the first therapeutic cancer vaccine approved by FDA in 2010, is made up of peripheral blood
mononuclear cells collected from patients 3 days before treatment and should be used within 18 h under storage at 2–8  C. The
high manufacturing costs and transportation constraints were among major reasons that Dendreon Corporation went into bank-
ruptcy in 2014. Another issue is blood wastage due to the limited shelf life of RBCs; millions of dollars are wasted by discarding the
expired blood units every year.2
In addition, cell preservation has been applied in other fields, such as preservation of anammox bacteria for waste treatment3
and preservation of fecal microbiota for Clostridium difficile infection treatment.4

5.11.2 Key Mechanisms of Cell Damage in Preservation

Cell preservation involves a series of complex processes, during which cell damage could easily occur. Hence, it is required to under-
stand key mechanisms of cell damage in order to develop an optimal preservation technology.

5.11.2.1 Cryopreservation Damage

5.11.2.1.1 Cell Damage During Freezing
Cryopreservation, also called freeze-thawing, is a widely used method for long-term storage of cells or tissues at an extremely low
temperature such as 80  C or even lower. When cells are frozen in a solution, ice forms first in the extracellular space and rejects
solutes, which leads to the dramatically increased solute concentration at the interface between ice and water. This accumulation of
solutes causes ice to grow in the form of finger-like structures at microscale, a phenomenon known as “constitutional supercool-
ing.”5 Cells are entrapped between the finger-like ice structures, and as a result, the solution surrounding the cells becomes
more hypertonic. The chemical potential difference between the supercooled intracellular solution and the hypertonic extracellular
solution provides the driving force to efflux water from cells, which leads to cell shrinkage, dehydration, or even rupture. The
damage to cells during freezing increases with extending exposure time and increasing extracellular concentration. Cooling rate,
which relates to the process of water transport across the membrane, is an important thermal parameter of cryopreservation. The
water transport across the membrane and the cell shrinkage are dependent on incubation time, membrane permeability, and extra-
cellular solution osmolality. The chemical mode of damage during freezing could be eliminated if cells are rapidly frozen to cryo-
genic temperatures. Therefore, the cell survival rate improves with increasing the cooling rate. However, when the cooling rate
exceeds a certain optimal value, the cell survival rate decreases, probably due to the sudden formation of intracellular ice. When
cells are cooled too rapidly, the intracellular solution is thermodynamically supercooled in order to equilibrate osmotically with
the extracellular solution, which increases the possibility for intracellular ice nucleation. This “inverse U-shaped survival curve”
may vary from cell type to cell type.5
There are also other modes of cell damage. The “unfrozen fraction” hypothesis has been proposed based on an observation that
the survival rate of human erythrocytes during slow freezing was dependent predominantly on the concentration of extracellular
solutes in the unfrozen fraction.6 The mechanical interaction of ice crystals with cells entrapped between them has also been
demonstrated to cause cell destruction.7 In addition, it has been reported that membrane lipids may undergo topological changes
and lateral phase separation may occur at low temperature, leading to further damage of cell membranes.5

5.11.2.1.2 Cell Damage During Cryostorage and Thawing

In general, cells can be potentially stored for decades in liquid nitrogen. However, chemical reactions continuously take place at
temperature above absolute zero. Hence, damaging chemical reactions may still occur during cryostorage, although at a slow
rate. Thawing is another source of cell injury. Recrystallization upon thawing is the most likely mechanism of cell damage.8 Frozen
 Cryopreservation and Desiccation Preservation of Cells 159

cells are filled with tiny ice crystals acting as nucleation sites because there is no sufficient time for crystal growth during rapid
freezing. Once thawed, those small ice crystals may grow into larger ones through the annealing of ice grain boundaries, causing
damage to organelles or other cellular structures. In addition, the frozen domains contain different solute compositions with
various melting temperatures. When ice melts, cells are likely exposed to a less concentrated solution. The resulting osmotic imbal-
ance will lead to the influx of water into cells and consequent rupture of the cell membrane.

5.11.2.2 Desiccation Damage

Water plays a crucial role in all living organisms. Removal of water from cells, especially mammalian cells which lack natural mech-
anisms to resist desiccation injuries, is a severe or often lethal process.7 Dehydration and desiccation have tremendous physiological
effects toward cells, including mechanical strains, structural damage, and metabolically derived damage.

5.11.2.2.1 Mechanical Strains and Structural Damage

Extensive loss of water from cells can cause morphological changes, and cell shrinkage is the most common consequence. With the
loss of water during drying, the concentration of solutes increases dramatically. Exposure of cells to highly concentrated solutes not
only leads to cell shrinkage but also induces pH shifts and some toxicities. Furthermore, due to lack of physiochemical support
provided by water solvency, cell membranes may experience severe stress, leading to the reduction of cell volume. Loss of large
amount of membrane surface area may result in cell membrane leakage or breakage during desiccation, or burst upon rehydration.
Membrane properties can also be impaired in response to desiccation stress. Removing water from cells induces intermolecular
association of polar lipids. The tighter packing of membrane lipids increases the rigidity of the fatty acid domain and causes lateral
compression in the acyl domain, which would eventually result in a lamellar fluid-to-gel transition. This transition may exclude
membrane proteins from the lipid bilayer or even trigger membrane leakage due to the imperfect lipid packing in different domains.
Moreover, the lipid bilayer conformation may be changed to a hexagonal II phase, a conformation that leads to membrane fusion.9
In addition, drying may induce protein and DNA damage. Denaturation of proteins can occur when their hydration shield and
their hydrogen bonding with water are removed by drying, resulting in an irreversible conformational change and aggregation of
proteins. This damage can be reduced by increasing the drying rate. However, the response of proteins to desiccation stress varies
from protein to protein. For example, porcine pancreatic elastase and bovine pancreatic ribonuclease A have been reported to main-
tain their functional activity after freeze-drying without excipients, while lactate dehydrogenase lost its functionality under similar
conditions.9,10 Although DNA structures are more stable in the absence of water compared to proteins, cells are susceptible to desic-
cation injury during germination when DNA is decondensed. Dehydration of cell nucleus may result in chromosomal aberrations.

5.11.2.2.2 Metabolically Derived Damage

Loss of water can slow down the metabolism and affect the metabolic pathways of cells. In the cryptobiosis state of desiccation-
tolerant species, downregulation of protein synthesis in response to dehydration is applied as a cost-effective strategy to conserve
energy. Desiccation may also be accompanied by a reduction in intracellular pH for reduced proteolysis and an increase in sugar
polyols for improved biostabilization.9
While some other metabolic processes such as lipid and carbohydrate metabolic processes are shut off, respiration continues at
a much slower level in cells upon desiccation. This results in accumulation of high-energy intermediates that may leak out of mito-
chondria, forming reactive oxygen species (ROS) and free radicals. Dehydration can lead to an oxidative stress as a result of ROS
production and/or inactivation of antioxidant enzymes. When ROS production exceeds antioxidant defenses, chemical injuries
will occur and eventually lead to lipid peroxidation, protein denaturation, and nucleic acid damage, thus severely impairing overall
cellular metabolism. Oxidative stress has been suggested by researchers as one of the major causes of desiccation damage.11 Even
though normal metabolism may be resumed once returned to hydration state, the damage caused by metabolic imbalance and
oxidation may be permanent.

5.11.3 Cell Preservation Technologies

Cryopreservation at ultralow temperature and dry storage at ambient temperature are two promising cell preservation methods for
long-term storage. Other conventional preservation methods, such as hypothermic preservation which is currently used for blood
storage, have drawbacks such as limited shelf life, high maintenance cost, and limited transportation ability. In this article, we
mainly discuss cryopreservation and dry storage.

5.11.3.1 Cryopreservation
5.11.3.1.1 Ice Crystal Formation and Vitrification
The entire process of cell cryopreservation includes freezing and storage of a cell sample at an ultralow temperature, followed by
thawing of the cell with its function recovered. At the initial stage of freezing, extracellular temperature will be lower than the intra-
cellular one, which could lead to formation of extracellular ice crystals, while the intracellular water stays liquid. Another process
that may occur in cell cryopreservation is vitrification, which is a state where living cells become glasslike solids. It can restrict the
160 Cryopreservation and Desiccation Preservation of Cells

motion of intracellular biomolecules such as proteins and improve their structural stability. Factors controlling ice crystal formation
and vitrification, including freezing and thawing rates as well as the type and concentration of cryoprotectants, need to be consid-
ered when developing suitable cryopreservation approaches.
Meryman has concluded five common strategies for cryopreservation of living cells, such as ultrarapid freezing and thawing,
controlled-rate freezing, freezing with nonpenetrating polymers (e.g., polyvinyl pyrrolidone and polyethylene oxide), vitrification,
and equilibrium freezing.12

5.11.3.1.2 Cryoprotectants
Cryoprotectants are used to protect the cell membrane integrity and intracellular environment to reduce or avoid the damage to the
structures and properties of membrane lipids, proteins, and nucleic acids. They are divided into two categories depending on their
cell membrane permeability.
Nonpermeable cryoprotectants, such as sugars (e.g., trehalose and sucrose), starches (e.g., hydroxyethyl starch), polyvinyl pyr-
rolidone, and polyethylene oxide, cannot enter cells and thus stay extracellular during cryopreservation. They are normally applied
to protect cells during rapid cooling. After cooling starts, the extracellular solution will go through vitrification due to the extracel-
lular presence of cryoprotectants. As a result, the water flux between inside and outside of cells will be inhibited as the viscosity of
extracellular increases.12
When a slow freezing rate is applied, there is sufficient time for intracellular water to move out of cells under the osmotic pres-
sure, resulting in a reduction in cell volume. To address this problem, permeable cryoprotectants that can enter cells, including
dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol, can be used to induce the vitrification of intracellular
environment before ice crystal formation, thus preventing excessive loss of cell volume.5
Recently, more cryoprotectants have been developed from natural products. A novel soybean flour product made up of protein
(34.20 g/100 g), carbohydrate (36.00 g/100 g), and lipid (21.10 g/100 g) has been demonstrated to prevent Bacillus subtilis SB-MYP-
1 cell damage after freeze-drying stress. Rhodiola sachalinensis saccharide extracted from the rhizome of Herba Rhodiolae has also
been used as a new cryoprotectant for bovine spermatozoa. In addition, it has been reported that a natural zwitterionic molecule,
L-carnitine, could greatly improve the survival rate of ornamental cichlid fish after exposure to a cold shock. Antifreeze proteins
found in marine organisms have also been utilized in cryopreservation of cells, embryos, and organs.

5.11.3.1.3 Challenges in Cryopreservation

DMSO is currently the most widely used cryoprotectant. However, the toxicity and complex operation of DMSO as a cryoprotectant
have limited its medical use. Even a small amount of DMSO remaining in the preservation media can cause adverse side effects
toward central nervous, cardiovascular, and respiratory systems after transfusion or transplantation. Methods have been tried to
reduce the amount of DMSO yet with limited success. Therefore, significant interest exists in the development of alternative
cryoprotectants.
Active research seeks to apply trehalose, which is a hydrophilic disaccharide of glucose accumulated in a variety of freezing
and desiccation-tolerant organisms.13 Trehalose needs to be present on both sides of the cell membrane to achieve optimal
cryoprotection. However, mammalian cells are not permeable to trehalose nor can they synthesize it. This makes the intracel-
lular delivery of trehalose a vital step to realize its application in cell preservation. A few attempts have been made so far to
load trehalose into cells. However, methods such as liposomal delivery,14 osmotic shock,15 and trehalose modifications16 have
difficulties to reach target concentrations of intracellular trehalose ranging from 100 to 200 mM,17 which are thought to be
required for bioprotection of mammalian cells. Other methods yielding sufficient intracellular trehalose for biopreservation
may have safety concerns, such as mutant bacterial toxins,18 cell-penetrating peptides,19 and electroporation,20 or lack scalabil-
ity, such as microinjection.21 A safe and scalable method based on the use of pH-responsive, fusogenic peptide–mimicking,
amino acid–based biopolymers, such as PP-5022,23 and PLP-NDA,24,25 has been demonstrated to achieve sufficient intracellular
trehalose through transient cell membrane permeabilization without causing significant cell damage. This biomimetic
polymer-based method has been successfully applied in mammalian cell cryopreservation, although its clinical viability
remains to be further validated.

5.11.3.2 Dry Storage of Cells

Cryopreservation is the most widely used method for cell preservation so far. However, it has limitations, including requirements
for specialized equipment to maintain ultralow temperature, high cost for long-term storage, and difficulties in sample ship-
ping.26 Given the increasing demand for cell banking, there is a need to develop alternative storage options. The existence of
desiccation-tolerant anhydrobiotic creatures in nature, such as tardigrades (also called water bears), suggests the possibility for
storage of cells in dry form. Dry preservation of cells initially started in the 1950s, with attempts made to lyophilize spermatozoa,
red blood cells, and platelets. Storage of cells in dry state would overcome the limitations of refrigeration. It allows easier trans-
portation and storage and is also energy-saving and environment-friendly. In spite of tremendous challenges, developing a method
that enables long-term storage of cells at ambient temperatures with high viability and functionality remains a highly attractive
goal for researchers.
 Cryopreservation and Desiccation Preservation of Cells 161

5.11.3.2.1 Drying Methods

5.11.3.2.1.1 Lyophilization
Lyophilization, also referred as freeze-drying, plays an important role in dry preservation of cells. It is a process that removes water
from samples through controlled freezing, followed by primary and secondary drying under vacuum. Cryoprotectants need to be
added during the freezing stage to minimize the damage associated with freezing. Majority of water in biological samples is
removed by sublimation during primary drying. Careful control of the heating rate is required to avoid excessive vapor generation
that may cause cellular structural breakdown or alteration. Due to the importance to retain a slow sublimation, primary drying is
typically the longest step in lyophilization.27 After primary drying, there is still residual unfrozen water that forms hydrogen bonds
with biological molecules, such as membrane lipids or proteins. A secondary drying step is then applied to reduce the residual
bound water to an optimal level to ensure the biostabilization in dry state. Secondary drying usually takes place at an increased
temperature and a decreased pressure, thus less water exists in a glassy state, consequently allowing the removal of remaining water.

5.11.3.2.1.2 Drying at Ambient Temperatures

In nature, anhydrobiots do not freeze before drying, which indicates the possibility to remove the freezing step in dry preservation of
cells. Traditional air-drying or natural evaporation is not suitable for dehydration of cells due to inconsistent drying rates, long pro-
cessing time, and difficulties to control the final moisture content. Those issues could be addressed by vacuum drying, during which
uniform and rapid drying of liquid samples can be achieved at reduced air pressure. Using arbutin (4-hydroxyphenyl-b-glucopyr-
anoside) and trehalose as protectants, more than 40% of human mesenchymal stem cells (hMSCs) were reported to survive after
vacuum drying to below 0.3 g H2O/g dry weight, with their metabolic activity and osteogenic differentiation ability retained after
rehydration.28 Spray-drying is another method for cell drying at ambient temperatures. Spray-drying atomizes liquid samples, fol-
lowed by rapid evaporation of water by heated air during sample falling by gravity. This process is potentially destructive to cells
because it usually takes place at high temperatures (e.g., 150–200  C).26 Spray-freeze-drying technique, which is a combination of
both spray-drying and freeze-drying, has been developed to overcome this problem.9

5.11.3.2.2 Desiccation Protectants

Protectants are needed to protect cells from damage during desiccation. For lyophilization, protectants should prevent cell damage
during both freezing and drying. Cryopreservation of cells using membrane-permeable protectants, such as the most widely used
DMSO, has been carried out with some successes, but little work has been reported on their successful application in desiccation of
cells.27 In addition, membrane-permeable protectants such as DMSO are toxic at high concentrations. Extensive washing of cellular
products is thus required before clinical use. However, cell washing is not only time-consuming but can also cause significant cell
damage and loss. Therefore, there is significant interest in the development of alternative desiccation protectants inspired by anhy-
drobiots in nature.

5.11.3.2.2.1 Sugars
Many anhydrobiotic organisms are able to synthesize trehalose in response to environmental stress in order to survive freezing and
desiccation. Nonreducing sugars such as trehalose can protect cells against desiccation damage probably by (1) formation of
hydrogen bonds with lipids and proteins to inhibit membrane fusion, membrane phase separation, and damage of the structures
and properties of biological molecules (water replacement hypothesis) and (2) formation of a glassy matrix that limits possible
chemical and/or physical interactions between biological molecules and prevents cellular collapse (vitrification hypothesis). In
addition, these sugars are believed to act as antioxidants to protect cellular constituents from oxidation. Reducing sugars such as
maltose are also effective lyoprotectants, but their high reactivity makes them less favorable for dry storage of cells compared to
nonreducing sugars.
Trehalose is considered to be superior to other nonreducing sugars for desiccation application. It can interact more strongly with
polar residues in cellular components than other nonreducing sugars, thus creating a physical state that mimics a fully hydrated
condition. In addition, trehalose has a higher glass transition temperature, Tg, and is believed to form a stronger glass compared
to other sugars. The Tg for trehalose and sucrose is 115 and 65  C, respectively, suggesting that the sample dried in the presence
of trehalose should be more resistant to structural changes than the one with sucrose.29

5.11.3.2.2.2 Proteins
Some proteins, such as small heat-shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins, are capable of
improving the desiccation tolerance of cells. HSPs are expressed in anhydrobiotic creatures facing desiccation stress. It has been re-
ported that HSPs were engaged in the inhibition of apoptosis and oxidative damage.11 LEA proteins, expressed in seeds and animals,
can protect target proteins from deactivation and aggregation due to desiccation, interact with cell membranes to offer protection,
and stabilize vitrified sugar glasses by increasing Tg.26 Although sugars are more widely used as desiccation protectants, those
proteins also play an important role in desiccation preservation since they are more stable above their Tg than sugars.29,30

5.11.3.2.3 Challenges in Cell Desiccation

It remains a major hurdle to retain cell viability and functionality during desiccation. Use of suitable protectants can limit or prevent
desiccation damage to cells. Trehalose is a widely used desiccation protectant. It is required to be abundantly present both inside
and outside cells to provide maximum desiccation protection. Unfortunately, trehalose is membrane impermeable, and
162 Cryopreservation and Desiccation Preservation of Cells

mammalian cells are incapable of synthesizing it. Therefore, it is of critical importance to deliver sufficient amount of trehalose into
cells without causing significant cell damage using a scalable method. Section 5.11.3.1.3 compares currently available technologies
for intracellular trehalose loading.
Rehydration may also cause cell injuries. Due to the liquid-to-gel membrane transition in the drying process, membrane leakage
and loss of intracellular homeostasis may occur during the rehydration process. In addition, the osmotically induced flow of water
through the cell membrane is another source of damage. Prehydration before direct rehydration is usually required to avoid imbi-
bitional damage, particularly for freeze-dried cells.27 Rehydration rate, medium, and temperature need careful optimization in order
to achieve a maximum cell survival.
Lyophilization has the risk of cross-contamination. Furthermore, variations of sample volume in a freeze dryer could remarkably
affect the appearance and quality of the final product. Thus, the parameters for lyophilization need to be modified once applied for
large-scale production.

5.11.4 Current Thrusts in Cell Preservation

5.11.4.1 Cryopreservation
A class of novel, pH-responsive, amino acid–based polymers, which are designed to mimic influenza viral peptides, have been
synthesized to facilitate intracellular delivery of trehalose.22,23,31 The amphiphilicity and structure of the biomimetic pseudopep-
tides were controlled by grafting hydrophobic natural amino acids, such as L-valine, L-leucine, and L-phenylalanine, to pendant
carboxylic acid groups of the parent polyamide, poly(L-lysine isophthalamide) (PLP).32,33 The polymers displayed a coil-to-globule
conformational transition in response to pH reduction, leading to transient membrane permeabilization for efficient intracellular
delivery.22,33,34 PP-50, with 50% L-phenylalanine relative to pendant carboxylic acid groups on the parent backbone, has been re-
ported to facilitate efficient intracellular loading of simply missed trehalose upon slight decrease of extracellular pH, reaching 123 
16 mM inside sheep RBCs and 251  6 mM inside human RBCs, respectively, after 9 h of incubation.22,23 Recently, a class of new
comblike biopolymers, such as PLP-NDA, have been developed by Chen et al.24,25 With relatively long hydrophobic alkyl side
chains such as decylamine (NDA), which act as membrane anchors, the PLP-NDA polymers can interact strongly with the cell
membrane and allow hydrophilic, membrane-impermeable trehalose to pass through the hydrophobic inner membrane layer
more rapidly and more efficiently than PP-50.24 It has been demonstrated that after only 15 min of treatment of erythrocytes
with the membrane-anchoring biopolymer, the intracellular trehalose already reached the targeted concentration of 100–
200 mM required for effective bioprotection of living cells.22 The erythrocytes loaded with trehalose by PP-50 or PLP-NDA were
suspended in extracellular trehalose solution, and it has been demonstrated that the presence of both intracellular and extracellular
trehalose during cryostorage in liquid nitrogen has yielded an improvement of the cryosurvival rate by approximately 20%
compared to the erythrocytes with extracellular trehalose alone.23,24 This work has been extended to cryopreservation of various
nucleated human mammalian cells.31,35 It has been reported that, after PP-50 mediated intracellular trehalose loading, cryostorage,
and 24-h post-thaw, the number of metabolically active Saos-2 cells normalized to that before freezing was between 91  5% and
103  4%, comparable to the cells frozen using DMSO. It is interesting to note that the doubling time of Saos-2 cells treated using
the PP-50/trehalose method was not statistically different to the nonfrozen control; however, the doubling time of cells frozen using
DMSO was significantly reduced.35

5.11.4.2 Desiccation
In nature, there exist several different mechanisms employed by anhydrobiotic animals to survive in a dehydrated state. This has
inspired researchers to combine multiple protectants to enhance the desiccation protection of cells. The combination of sugars
(e.g., trehalose and sucrose) with HSPs or LEA proteins has demonstrated the synergistic effect on the cell recovery after desiccation
and rehydration. Human embryonic kidney cells 293H, which were transfected with the gene for Hsp26 protein (a small HSP) and
loaded with trehalose, displayed a significantly increased survival rate after desiccation compared to the cells treated with trehalose
alone.11 LEA protein has been reported to show synergistic interactions with sucrose in the formation of glass matrix, which is
important in the dehydration stage.30 In addition, as apoptosis is a damaging process in cell drying, the combination of trehalose
with an apoptosis inhibitor has also been utilized to enhance cell viability. CANARY cells treated with both trehalose and a pan-
caspase inhibitor, OPH-109, showed the improved post-rehydration viability and considerably lower the dehydration-induced
apoptosis.36
Efforts have been made to find other molecules that could increase cell viability after desiccation. Arbutin is an amphiphilic
glycoside found in high concentration in the resurrection plant M. flabellifolia. It can induce the expression of HSPs in hMSCs
and act as a strong antioxidant to protect cells against ROS damage under dehydrating conditions.28 The hMSCs that were dried
to 0.3 g H2O/g dry weight using a mixture of arbutin and trehalose showed the dramatically enhanced recovery and osteogenic
differentiation after rehydration compared with the cells dried with trehalose only.9 Arbutin is also believed to contribute to
membrane stabilization by inhibiting the enzymatic activity of phospholipase A2 in mostly dehydrated systems.30 In addition,
sorbitol has been used as a substitute of trehalose for bovine sperm desiccation. Although the mechanism is not fully understood,
sorbitol was found to improve the motility of sperms after drying and rehydration.9 Other molecules, such as proline, antioxidant
flavonoid rutin, glycine-betaine, deferoxamine, and polyols, have also been reported to improve cell recovery after desiccation.29,30
 Cryopreservation and Desiccation Preservation of Cells 163

Researchers have also tried to improve drying processes to enhance cell recovery. A new freezing method, which is superior to
conventional equiaxial freezing, has been developed by passing cell samples through a defined thermal gradient. Thanks to this,
more than 88% of mononuclear cells derived from human umbilical cord blood survived and retained the ability to differentiate
into different blood cells after freeze-drying and rehydration.26 The multithermal gradient method has also been applied to improve
the recovery of RBCs after lyophilization.6 In addition, minimizing cell exposure to hypertonic conditions at an optimal drying rate
could improve cell recovery. For example, a spin-drying method has been optimized through numerical simulation and employed
for Chinese hamster ovary (CHO) cell desiccation with trehalose as a desiccant protectant. With spin-drying for only 50 s, the water
content was rapidly decreased to 0.16 g H2O/g dry weight, but more than 95% of the cells maintained their membrane integrity.36
Moreover, intracellular loading of sufficient amount of trehalose is another strategy to improve cell protection during desiccation.
Recently, a class of novel, pH-responsive, fusogenic peptide–mimicking pseudopeptides, such as PP-5022,23,31 and PLP-NDA,24,25
have been reported to efficiently deliver trehalose into cells without causing significant damage. An increase in the membrane integ-
rity by a factor of 9  1 and reduced extent of hemoglobin oxidization from 66  1% to 23  3% have been observed in vacuum-
dried human erythrocytes loaded with 251  6 mM intracellular trehalose.23

5.11.5 Conclusions

The development of technologies that would permit stable, long-term preservation of cells without compromising their viability or
functionality is crucial for cell therapy and biobank. Cryopreservation is currently widely used for long-term cell storage. Desiccation
preservation of cells has also attracted much attention, since it overcomes the limitation of refrigeration, saves energy, and facilitates
easy transportation. However, preservation of cells, especially mammalian cells, faces formidable challenges due to the cell damage
during freezing and drying. A good understanding of the fundamental damage mechanisms would enable the development of effec-
tive preservation strategies for improved bioprotection of cells. So far, inspired by a variety of freezing and desiccation-tolerant
organisms in nature, researchers have made tremendous breakthroughs to retain the viability and functionality of cells after bio-
preservation, although there still exists ample room for further improvements. Future advances in this multidisciplinary area
will require concerted efforts of engineers, biologists, pharmaceutical scientists, and clinicians. The success in cell biopreservation
would spread widely the applications of cell therapy and revolutionize cell and even tissue banking.

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