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org/journal/abseba Review

Trehalose in Biomedical Cryopreservation−Properties, Mechanisms,

Delivery Methods, Applications, Benefits, and Problems
Yuying Hu, Xiangjian Liu, Fenglin Liu, Jingxian Xie, Qubo Zhu, and Songwen Tan*

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ABSTRACT: Cells and tissues are the foundation of translational medicine. At present, one of the main technological obstacles is
their preservation for long-term usage while maintaining adequate viability and function. Optimized storage techniques must be
developed to make them safer to use in the clinic. Cryopreservation is the most common long-term preservation method to maintain
the vitality and function of cells and tissues. But, the formation of ice crystals in cells and tissues is considered to be the main
mechanism that could harm cells and tissues during freezing and thawing. To reduce the formation of ice crystals, cryoprotective
agents (CPAs) must be added to the cells and tissues to achieve the cryoprotective effect. However, conventional cryopreservation of
cells and tissues often needs to use toxic organic solvents as CPAs. As a result, cryopreserved cells and tissues may need to go
through a time-consuming washing process to remove CPAs for further applications in translational medicine, and multiple valuable
cells are potentially lost or killed. Currently, trehalose has been researched as a nontoxic CPA due to its cryoprotective ability and
stability during cryopreservation. Nevertheless, trehalose is a nonpermeable CPA, and the lack of an effective intracellular trehalose
delivery method has become the main obstacle to its use in cryopreservation. This article illustrated the properties, mechanisms,
delivery methods, and applications of trehalose, summarized the benefits and limits of trehalose, and summed up the findings and
research direction of trehalose in biomedical cryopreservation.
KEYWORDS: trehalose, cryopreservation, cryoprotective agents, nontoxic, nonpermeable

1. INTRODUCTION
Cells and tissues are essential in translational medicine. Due to
negative storage effects on morphological and biochemical
features, the shelf life of red blood cells (RBCs) for transfusion
is short (less than 42 days) after donation, resulting in an
unwanted waste of 1.7 million units of blood in the U.S.1,2
Therefore, prolonging RBCs’ storage duration is essential to
their supply. The cryopreservation technology offers a new way
for long-term storage of RBCs. Additionally, ovarian tissue
(OT) cryopreservation can effectively preserve hundreds of
primordial follicles simultaneously. OT is carefully and slowly
frozen to −140 °C and is then kept in liquid nitrogen at −196
°C. During this process, no serious tissue deformation is
observed. Nowadays, ovarian tissue cryopreservation and
transplantation (OTC-T) offers a promising method for
preserving fertility, especially for postpubertal cancer patients
who need urgent treatment or who are ineligible for ovarian
stimulation.3 Cryopreservation is defined as the long-term
preservation of individual living cells and biological tissues at
extremely low temperatures, often at −196 °C for liquid
nitrogen. At this temperature, cells can remain genetically
stable for a very long period until they are required because
cellular processes are temporarily stopped. Meanwhile, it is the
best approach for preserving living tissues for a long time
because it is less expensive than other methods.4 However,
Received: October 18, 2022
Accepted: February 1, 2023
Published: February 13, 2023

© 2023 American Chemical Society https://doi.org/10.1021/acsbiomaterials.2c01225

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Table 1. Structure, Mechanism, and Application of Typical CPAsa,b,15,16,32−63

a
The structure of α,α-1,1-trehalose.64 bThe diagram shows the schematic structure of HES.15

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cells and tissues are vulnerable to damage in the freezing
environment. Mazur et al.5 proposed that freezing of water
damages cells in two different ways: Slow cooling rates will
expose the cells to high solute concentrations and cause
osmotic damage. But, if the cooling rate is too fast, intracellular
ice induced by the trapped water will lead to mechanical
damage to the cells. These two damages restrict the
development of cryopreservation.6 Thus, cryoprotectants
(CPAs) must be added to cells and tissues to minimize
freezing damage in cryopreservation.7 There are two kinds of
CPAs according to their functions and mechanisms: permeable
and impermeable.8 The former are mostly small molecules,
which penetrate into the cells to replace intracellular water and
ultimately prevent cell damage caused by intracellular ice
formation (IIF) and excessive cell shrinkage.9 In addition, they
can get inside cells to adjust osmotic pressure and decrease
osmotic damage.10 Permeable glycerol,11 dimethyl sulfoxide
(DMSO),12 and ethylene glycol (EG)13 fall under this
category. The latter act in extracellular to work by promoting
cell dehydration to inhibit the growth of intracellular ice and
also protect cells from extracellular ice crystals’ damage,9
including trehalose,14 hydroxyethyl starch (HES),15 polyam-
pholytes,16 poly(vinyl)alcohol (PVA),17 and so forth. The
properties, functions, and mechanisms of these typical CPAs
are described in more detail (listed in Table 1).
However, conventional cryopreservation of cells and tissues
needs to utilize toxic organic solvents (such as DMSO18 and
glycerol19) as CPAs. Therefore, cryopreserved cells and tissues
have to withstand a cumbersome washing process to eliminate
them for further applications. Moreover, this process may
cause osmotic shock, resulting in cellular damage.20,21
Conversely, trehalose, a nontoxic, nonpermeable CPA, can
replace the traditional CPAs that are highly toxic to cells and
tissues.22 Additionally, the trehalose-cryopreserved cells can be
employed without being washed, which should make cell-based
treatment more widely available.23 At present, trehalose has
been efficiently used for cryopreservation of murine spermato-
logical,24 adult hematopoietic,25,26 erythrocytes,27 human
adipose tissue-derived mesenchymal stromal cells,28 and
human transplantable hepatocytes.29 Additionally, it has also
been applied to human pancreatic islets30 and fetal skin.31
This article gives a thorough overview of the recent studies
on the use of trehalose in cryopreservation. It includes
probable mechanisms, delivery methods, applications, benefits
and limits, and the related findings and research direction of
trehalose in biomedical cryopreservation in detail. We hope
this review can help researchers fully understand the relevant
knowledge of trehalose in cryopreservation and promote the
development of translational medicine.
2. PROPERTIES OF TREHALOSE
Trehalose is a nonreducing disaccharide that is found in plants,
animals, microcells, and tissues.65 But, it is not found in
mammals.66 Although there are three possible anomers of
trehalose, namely, α,β-1,1-, β,β-1,1-, and α,α-1,1-, only the α,α-
trehalose, which is commonly known as trehalose, has been
isolated from and biosynthesized in living cells and tissues.67
Besides, trehalose comprises two glucopyranosyl units linked
by α-1,1-glycosidic bonds.68 And, the molecular formula of
trehalose is C12H22O11.69
Trehalose can be cleaved into two constituent D-glucose
units by trehalase, and its nature is very stable.70 Some
properties of it are listed in Table 2.67,71
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Table 2. Properties of Trehalose
property result
chemical formula C12H22O11
appearance white, odorless powder
sweetness (relative) 45% (as compared to that of sucrose)
toxicity nontoxic
molecular weight 342.30 g/mol
relative density 1.22 g/cm3 (at 25 °C and weight fraction of
0.5)
glass transition 117 °C
temperature
freezing temperature −197 °C (100 mg/mL water)
chemical nature nonreducing disaccharide
chemical nomenclature α-D-glucopyranosyl-α-D-glucopyranoside
three possible anomers α,β-trehalose, β,β-trehalose, α,α-trehalose
3. MECHANISMS OF CRYOPROTECTIVE EFFECTS OF
TREHALOSE
Trehalose is a nonpermeable CPA, whose high molecular
weight limits its permeability.72 Although the effects of
trehalose on biological molecules in cryopreservation have
been extensively studied, mechanisms of cryoprotective effects
of trehalose are extremely complex, and there are contradictory
conclusions in different articles. The hypotheses about the
main cryopreservation mechanisms of trehalose are summar-
ized as follows:
3.1. Preferential Exclusion Theory. The cellular
protective effect of trehalose is attributable to its preferential
repellency.73 According to this theory, trehalose does not
directly interact with biological macromolecules. It means that
water is ordered around trehalose rather than included in the
biomolecules’ solvation layer. Trehalose and biomolecules
compete for the available water as the concentration of
trehalose in a large volume of water increases. This
competition leads to water molecules being destructured
surrounding biomolecules and “structured” around trehalose,
which makes it become a kosmotrope or water structure-
maker.74,75 This indicates that trehalose and water interact
considerably more strongly than water and water does.71
Furthermore, the hydrated radius of trehalose is exceedingly
large, and nuclear magnetic resonance (NMR) relaxation time
measurements indicate that trehalose has the highest hydration
capacity of all investigated saccharides.76,77 The water
molecules are not uniformly distributed around the trehalose,
but trehalose can manipulate the water structure to become
organized around it in an oriented manner, with hydrogen
bonds in all orientations, which makes it possible for trehalose
to be involved in the bioprotection of cells. Moreover, a large
number of experiments, such as infrared and Raman spectros-
copies, NMR, ultrasound measurements,78,79 and inelastic
neutron scattering (INS) measurement,80 demonstrated the
destruction of the tetrahedral network of water molecules after
adding trehalose, thus hindering the crystallization of ice
efficiently.
For the protection ability of proteins, the addition of
trehalose in solution keeps water molecules away from
proteins, reducing their hydrated radius and improving their
compactness and stability to effectively prevent protein
molecules from denaturation. These properties make trehalose
become one of the best CPAs (Figure 1A).74 It is especially
noteworthy that trehalose stabilizes the protein’s partially
unfolded state rather than its native structure.81
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Figure 1. Illustration of the mechanisms of various theories of
trehalose on proteins during cryopreservation. (A) Preferential
exclusion theory. (B) Vitrification theory. (C) Water replacement
theory.
3.2. Vitrification Theory. Vitrification is the solidification
process from liquid to glass.70 Trehalose has a strong capacity
for intermolecular interactions, making it easy to build cluster
structures that can contain glass phase and have a modest
impact on neighboring and remote water molecules.82
Meanwhile, trehalose has excellent glass-forming properties
because of its different polymorphic forms.71 Besides, trehalose
is superior to other sugars for preserving cells and tissues in
certain circumstances because of its highest glass transition
temperature among all the disaccharides.83−85 And, the glass
transition temperature of the freeze-concentrated trehalose
solution is in the vicinity of −30 °C.31 Therefore, the solution
viscosity of trehalose rapidly increases during the freezing
process to form a stable glass matrix that acts like a cocoon,
which is another benefit as an increase in viscosity might lead
to reduced crystal growth. These benefits do not damage the
cell structure and protect the proteins and other biomolecules
(Figure 1B).72,86
3.3. Water Replacement Theory. Water replacement
theory refers to the direct interaction of trehalose molecules
with the protected biological structures via hydrogen bonds.87
This hypothesis could explain the protective effect of trehalose
on proteins and other bioactive macromolecules. The
protective membrane formed by hydrogen bonding between
trehalose molecules and proteins replaces the necessary water
molecules to maintain the three-dimensional structure during
the freezing process, allowing the biomolecules to retain the
integrity of cell membrane structure and function at very low
moisture levels to avoid the loss of intracellular components
(Figure 1C). Meanwhile, trehalose is a membrane stabilizer.88
During cryopreservation, trehalose and phospholipid polar
groups could replace the lost structural water by hydrogen
bonding. By delaying the onset of the phase transition from a
liquid crystal to a gel state, the membranes are prevented from
approaching each other, thus inhibiting membrane fusion to
stabilize the cell membrane whose fluidity declines during
temperature downshift (Figure 2).74,85,89,90 However, some
previous studies have indicated that the water replacement
theory is unable to explain the cryoprotective properties of
trehalose.47,91
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Figure 2. Illustration of the mechanism of lipid bilayer’ stabilization in
the freezing state by trehalose.
3.4. Active Protein Stabilizer. Trehalose is an active
protein stabilizer.92,93 During cryopreservation, it is speculated
that trehalose can prevent the inactivation and aggregation of
proteins at low temperatures.67,72 All the three hypotheses
above seem to explain the mechanism of protein protection by
trehalose. At the same time, the biological protection provided
by trehalose is not only to stabilize proteins but also to
nonspecifically stabilize intracellular nucleic acid and other
biological macromolecules, which leads to reducing the
appearance of intracellular ice during freezing.71,94 Because
trehalose acts on different types of biomolecular structures, the
protective mechanism of trehalose in cryopreservation is
described as a nonspecific process.
3.5. Increase of Membrane Permeability. Trehalose
protects cells by increasing the permeability of membranes to
water at subzero temperatures and decreasing the amount of
cellular water before freezing, thus reducing the likelihood of
intracellular ice during freezing and preventing excessive
swelling and osmotic shock.31,72,95
To sum up, trehalose works by modulating osmotic stress
and inhibiting ice formation.96 Various experimental and
theoretical studies have revealed that the mechanisms of the
cryoprotective effects of trehalose on cells and tissues are
complicated, and none of them can be fully accepted since they
sometimes lead to contradictory conclusions. At the same time,
the stability of trehalose under various circumstances can be
explained by any one or all of these theories. They are not
necessarily mutually exclusive and may be unified. Therefore,
there is no doubt that we need to analyze the mechanism of
trehalose under different conditions using experiments and
theoretical foundations.
4. DELIVERY METHODS OF TREHALOSE DURING
CRYOPRESERVATION
Trehalose is a particularly attractive CPA because of its well-
known role in keeping cells’ structure and protecting cells from
various damages in cryopreservation.73 Trehalose must be
present in both intracellular and extracellular cells to supply
optimal protection against stress and damage during
cryopreservation.97 Whereas, trehalose is difficult to transport
across the cell membrane, so its cryoprotective activity is
limited.98,99 Consequently, the current research focuses on the
delivery methods of trehalose into cells. Janis et al.100 provided
evaluation criteria for new delivery methods: (1) the
efficiencies of loading trehalose to RBCs are comparable to
classic methods, including but not limited to electroporation
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and fluid-phase endocytosis and (2) Avoiding cellular
pressures that from high trehalose concentrations and long
incubation time. The criteria are important for us to find
promising new delivery methods for trehalose-loaded cells.
4.1. Phospholipid Phase Transition. One of the
methods of introducing trehalose into mammalian cells is to
enhance the permeability of cell membranes by inducing
phospholipid phase changes.30 So far, thermal, osmotic, and
electrical stress have been investigated as induction methods to
improve membrane permeability and permit trehalose to
deliver into cells.101
Beattie et al.30 used the lipid phase transition of islet cell
membranes to introduce trehalose into the cells before freezing
with a high concentration of trehalose outside. Furthermore,
when islet cells underwent a thermogenic lipid phase
transition, membrane permeability would increase. It allowed
trehalose to diffuse from the surrounding environment along
its concentration gradient to control the load.
Zhang et al.95 have recently shown that endocytic and
freezing-induced trehalose uptake have been combined for
cryopreservation of mammalian cells. The presence of ice
affected the permeability of water and nonpermeable
molecules. During cryopreservation, membranes became
permeable as a result of a freeze-induced membrane phase
transition and an osmotic driving force, resulting in the
delivery of trehalose into fibroblasts.102
Satpathy et al.103 proposed a method for loading RBCs with
trehalose. Through the combination of osmotic imbalance and
phospholipid phase transition, trehalose from the foreign
medium may be loaded into RBCs, which led to an
approximately 40 mM intracellular trehalose concentration.
And, trehalose was not likely to decompose in RBCs.
Therefore, it must be noted that RBCs were exposed to
hypertonic trehalose solution during loading, causing morpho-
logical changes and possible damages.
Electropermeabilization, which is also called electroporation
or electroinjection, has been taken to enhance membrane
permeability and deliver extracellular molecules into the
cytoplasm.104 Dovgan et al.105 cryopreserved human adipose-
derived stem cells (hADSCs) by combining reversible
electroporation with trehalose, which allowed for long-term
cryopreservation without DMSO. However, this method is
highly invasive and may seriously damage cells, causing further
freezing damages during cryopreservation.22,73
The advantage of this method is that the concentration
gradient can be manipulated to control the loading of
trehalose, and membrane permeability is increased. Never-
theless, membrane permeability is nonspecifically permitting
the entry of substances other than trehalose, and there are
problems about cytotoxicity and membrane stability caused by
thermal, osmotic, and electric shock to cells.101
4.2. Engineered Pores. Trehalose can be loaded into
mammalian cytoplasm by producing pores on the membranes
of cells.26 Eroglu et al.98 loaded low concentrations (0.2 M) of
trehalose into 3T3 fibroblasts and human keratinocytes
through exploiting a genetically engineered mutant of Staph-
ylococcus aureus α-hemolysin to create pores in cell membranes,
which could significantly improve the survival rate of cells in
cryopreservation. Moore et al.85 reported that trehalose could
be delivered into an isolated mammalian mitochondrial matrix
through the transient opening of the mitochondrial perme-
ability transition pore (MPTP). The MPTP is a megapore of
the inner membrane composed of ATP synthase dimers.
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However, they are invasive delivery methods and may seriously
damage cells, causing further freezing damages during
cryopreservation.22
4.3. Microinjection. Exogenous trehalose can be injected
into the cytoplasm of mammalian cells by microinjection.
Eroglu et al.106 showed that the microinjection technology
allowed for the introduction of trehalose into oocytes in a
controllable manner by the volumetric response. And,
trehalose was a unique CPA for oocytes, which quickly
disappeared during embryonic development.107 Obviously,
trehalose has been transported into mammalian oocytes with
the size of ∼100 μm in diameter and the number of dozens or
hundreds by using this method. However, it is often hard to
apply for smaller cells (usually less than 20 μm in diameter) or
massive needs (usually millions to billions) of mammalian
cells.23,108,109
4.4. TRET1 Transporter. Researchers have isolated the
trehalose transporter TRET1 from the anhydrobiotic larvae of
the African chironomid, which is called Polypedilum
vanderplanki. Uchida et al.110 conducted a comparative
cryopreservation experiment between the TRET1-expressing
CHO-K1 cells (CHO-TRET1) and the CHO-K1 cells
transfected with the empty vector (CHO-vector). The
conclusion showed that trehalose delivered into the cells
with TRET1 dramatically increased the cryoprotective
function. In summary, TRET1 enabled cells to deliver
trehalose specifically and effectively across the plasma
membrane, but the cells needed to be genetically modified first.
4.5. Polymers. Lynch et al.97 have investigated synthetic
polymers to help deliver trehalose into the cytoplasm by
interacting with the cell membrane and transiently enhancing
its permeability. More than 250 mM trehalose was introduced
to human RBCs using the membrane-permeabilizing polymer
PP-50, exceeding the estimated bioprotection threshold of
100−200 mM intracellular trehalose. PP-50 is noncytotoxic
and can be cleared from cell membranes through tiny changes
in pH. And, increased membrane permeability could be
reversed by washing in PBS.101 Sharp et al.111 and Slater et
al.112 expanded the application of PP-50 from RBCs to a
nucleated cell line that originated from human osteosarcoma
(SAOS-2). Biopolymer-mediated trehalose uptake could trans-
port high-concentration trehalose at relatively low osmolarities
but with a long incubation time.
4.6. Liposomes. Liposomes are thought to be potential
vesicles for delivering trehalose to mammalian cells.113 Stoll et
al.102 found that liposomes could play a role in the
cryopreservation of RBCs. It is thought that liposomes alter
cell membranes by exchanging lipid and cholesterol, thereby
changing the physical properties and making the cells more or
less stable at low temperatures. Nevertheless, the approach can
only load micromolar concentrations of trehalose within cells.
Moreover, the release of trehalose in vesicles is inefficient.22,101
4.7. Nanoparticles (NPs). Rao et al.23 synthesized pH
responsive genipin-cross-linked Pluronic F127-chitosan nano-
particles (GNPs) and encapsulated trehalose in GNPs to
obtain nanoparticles-encapsulated trehalose (nTre). Further-
more, GNPs, a vehicle, had superior biocompatibility and were
taken up by cells. The results proved that trehalose could be
effectively encapsulated in GNPs for intracellular transport,
enabling primary hADSCs to be frozen. As a consequence, it
was a noninvasive prospective strategy as it exploited a cell’s
natural feeding process called endocytosis to absorb trehalose
encapsulated in the NPs. Nevertheless, hADSCs were needed
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to be incubated with the trehalose-laden NPs for 1 day to
slowly release trehalose from the NPs that were absorbed by
cells, which caused a prolonged and tedious cryopreservation
process.
Under normal conditions, trehalose has low permeability to
RBCs. The mechanism by which apatite NP assists in the
transport of bioactive molecules in cells is usually on the basis
of cytosolic-mediated translocation. Whereas, endocytosis is
not present in mature mammalian RBCs,114 which implies that
with the assistance of apatite NPs, the transfer of trehalose to
RBCs should occur through different mechanisms, such as
local modification of RBCs’ physical properties. Stefanic et
al.115 reported for the first time a glycerol-free cryopreservation
method that used colloidal, bioinspired apatite NPs as
bioactive promoters of RBCs’ cryopreservation mediated by
trehalose. Instead of through the direct formation of transient
membrane holes, the translocation of trehalose to RBCs was
promoted by an indirect mechanism resulting from NP/bilayer
interactions. The findings implied that the transport of
trehalose by apatite NPs was through the local modification
of the three-dimensional structure of lipids in the interaction
with NPs and the temporary breaching of the lipid bilayer so as
to enhance the permeation of trehalose through the bilayers. In
addition, apatite NPs did not insert themselves into the lipid
bilayer core but rather adsorbed on it. Moreover, apatite NPs
were biocompatible with RBCs and suitable for blood
transfusion applications. The findings are in good agreement
with recent studies that reported good hemocompatibility of
apatite NPs, particularly in direct contact with RBCs.116,117
Zhang et al.22 utilized a cold-responsive polymer (poly(N-
isopropylacrylamide-co-butyl acrylate)) to synthesize NPs for
the encapsulation and intracellular transport of trehalose.
Trehalose could effectively absorb cold-responsive NPs
through endocytosis (or other mechanisms). The rapid and
irreversible disassembly of the NPs after cold treatment
allowed for the controlled and quick release of trehalose
from intracellular NPs. Cells with intracellular trehalose
conveyed using the NPs showed comparable survival after
cryopreservation compared to cells treated with DMSO. In
addition, the precipitated polymers resulting from the cooling
and warming of intracellular NPs could be excreted from the
cells via exocytosis if they were not degraded inside the cells. It
is also crucial to consider that the incubation time of the
trehalose encapsulated in the PLGA−pNIPAM-B−PF127 NPs
(nTre) must be optimized. There are two reasons. The former
is that the reduction of intracellular trehalose might be
insufficient to prevent the cells from cryodamage because of
the short incubation time with nTre NPs. The latter is that
excess of intracellular trehalose caused by excessive incubation
time with nTre NPs might increase intracellular osmotic stress
and allow extracellular water to enter the cells, ultimately
leading to an expansion in cell volume and subsequent cell
rupture. Hence, the incubation period with nTre NPs has to be
optimized to provide a suitable amount of intracellular
trehalose to avoid any injury to the cells prior to
cryopreservation. As mentioned above, delivery of trehalose
using GNPs requires 24 h of incubation to allow cells to take
up and release trehalose encapsulated in NPs,108 whereas cells
can be cryopreserved after 4 h of incubation with cold-
responsive NPs loaded with trehalose because cold-triggered
trehalose can be rapidly released from nTre NPs (within
several minutes), greatly reducing the preparation time of
cryopreserved cells.22
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Consequently, this method has advantages and disadvan-
tages. On the one hand, the method can specifically deliver
large amounts of trehalose into mammalian cells, and no
modification to the cells is needed. On the other hand, the
disadvantage of this method is the lengthy incubation period.
4.8. Fluid-Phase Endocytosis. Fluid-phase endocytosis,
an inherent property of cells incorporating membrane-
nonpermeable molecules, can also be exploited for the
introduction of trehalose into cells, resulting in increased
intracellular trehalose contents.95 It is a nonspecific process.118
This mechanism involves the uptake of some substances from
the environment by the cells and their internalization through
a vesicle pinched off from the plasma membrane. This cellular
process has been utilized to deliver trehalose into mammalian
cells by simply culturing the cells in the existence of high
extracellular concentrations of trehalose.101 Jong et al.119 used
Paesun cells as experimental subjects. The researchers wanted
to see how cryoprotective media affected the cryopreservation
of Paesun cells using loading trehalose. The cryoprotective
media was a “Cryocool” solution, an intracellular-like cooling
solution for the treatment of the molecular-based cellular
damages (cryopreservation-induced delayed onset cell death)
during cryopreservation. The efficiency of cryopreservation via
loading trehalose was impacted by the cryoprotective media. In
this process, trehalose was loaded through fluid-phase
endocytosis of cells at physiological temperature. However,
fluid-phase endocytosis does not deliver sufficient amounts
(0.1 M or more) of trehalose to cells73 and a long incubation
time is required.101
4.9. Sonoporation. Janis et al.100 discovered that trehalose
may be effectively delivered into RBCs to promote long-term
cryopreservation by the transitory pore formation induced by
ultrasound and microbubbles (sonoporation). After freezing
and thawing, RBCs could maintain normal morphology.
Sonoporation occurred when ultrasound drove the oscillation
and collapse of microbubbles near the cell membrane, thereby
inducing transient pores formation, which could absorb
impermeant compounds including trehalose. This process
also contributed to the microstreaming in the surrounding
fluid, which enhanced the transport of compounds via formed
pores that reseal in a few seconds in a calcium-dependent
pathway. Therefore, in contrast to the method that relies on
passive diffusion, sonoporation can actively transport trehalose
into cells with higher efficiency through the plasma membrane
of RBCs. The advantage of sonoporation is that the loading of
trehalose can be completed in less than 1 min in combination
with a fluidics device, and the process could be simply
extended to the required yield. In addition, accurate loading of
the needed trehalose concentrations can be achieved by
changing the dosage of microbubbles, which is essential to
strike a balance between loading enough trehalose for efficient
cryopreservation and lessening the risk of osmotically induced
hemolysis during infusion. In summary, the use of sonopora-
tion to enhance the load of CPAs in RBCs is a promising
method to increase the storage time in cryopreservation.
However, it is worth noting that the membrane permeability is
not specific for trehalose, and ultrasound might also impair
activity or alter the morphology of cells.101
4.10. Other Methods. Abazari et al.120 investigated the
membrane permeability of engineered lipophilic derivatives of
trehalose. Trehalose conjugated with six acetyl groups
(trehalose hexaacetate or 6-O-Ac-Tre) exhibited more
excellent permeability in rat hepatocytes than regular trehalose.
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 Table 3. Summary of Various Methods for Delivery of Trehalose into Cells and Tissues during Cryopreservationa
incubation conditions of the experiment survival rate of the incubation conditions of the control survival rate of the control
cryopreserved samples delivery method group experiment group group group references
islets thermal shock 300 mmol/L trehalose + DMSO 92% DMSO alone 58% 199730
mouse embryonic fibroblast freezing-induced, fluid-phase DMEM with trehalose ∼56% (membrane intact DMEM without trehalose ∼9.3% (membrane intact cells) 201695
(3T3) cells endocytosis cells)
hADSCs electroporation electroporation at 1.5 kV/cm + 250 mM ∼83.8% 10% DMSO in 90% fetal bovine serum ∼91.5% 2017105
trehalose
human keratinocytes engineered pores 0.2 M intra- and extracellular trehalose ∼71.9% 0.2 M extracellular trehalose ∼43.3% 200098
oocytes microinjection 0.5 M extracellular trehalose + 0.15 M 63% (−15 °C), 0.5 M extracellular trehalose 22% (−15 °C), 2002106
intracellular trehalose
53% (−30 °C), all oocytes degenerated (−30
°C and-60 °C)
66% (−60 °C)
CHO-K1 cells TRET1 transporter CHO-TRET1 cells + 400 mM trehalose ∼80.6% (maximum CHO-vector cells + 250 mM trehalose ∼9.9% (maximum viabilities) 2007110
viabilities)
ACS Biomaterials Science & Engineering

human RBCs polymers 250 mM trehalose (intracellular ∼75% extracellular trehalose alone ∼60% 201197
concentration)
SAOS-2 cells polymers 25 μg/mL PP-50 + 0.2 M trehalose ∼60% (cell viability) 0.2 M trehalose ∼44% (cell viability) 2013111
RBCs liposomes liposomes + trehalose + HES ∼97.4% trehalose + HES ∼94.5% 2012102
hADSCs NPs 6.2 ± 0.2 mM nanoparticle-encapsulated ∼91.2% (cell viability) 10% (v/v) DMSO ∼88.2% (cell viability) 201523
trehalose
RBCs NPs 2.25 mg/mL apatite NP (pH = 6.5) + 0.35 ∼90.7% trehalose alone (pH = 7.4) ∼51.8% 2017115
M trehalose

1196
hADSCs NPs nTre ∼82.9% (cell viability) 10% (v/v) DMSO 86.3% (cell viability) 201922
MDA-MB-231 cancer cells NPs nTre ∼85.3% (cell viability) 10% (v/v) DMSO 90.2% (cell viability) 201922
Paesun cells fluid-phase endocytosis “Cryocool” + 30 mM trehalose ∼89.2% 10% FBS’s MEM + 30 mM trehalose ∼33.1% 2020119
RBCs sonoporation 5% (v/v) MB concentration + PBS + 200 ∼90% 0% (v/v) MB concentration + PBS + ∼35% 2021100
mM trehalose 200 mM trehalose
a
Abbreviations: DMSO = dimethyl sulfoxide, DMEM = Dulbecco’s modified Eagle medium, hADSCs = human adipose-derived stem cells, CHO-TRET1 cells = TRET1-expressing CHO-K1 cells,
CHO-vector = CHO-K1 cells transfected with the empty vector, RBCs = red blood cells, HES = hydroxyethyl starch, NP = nanoparticles, nTre = trehalose encapsulated in PNP nanoparticles, MB =
microbubble, PBS = phosphate buffered saline.
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Review

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Table 4. Cells and Tissues Cryopreserved Using Trehalose Alonea

cryopreserved concentrations of trehalose used for
samples cryopreservation effect of cryopreservation with trehalose references
platelets 300 mM trehalose significantly higher cryosurvival in trehalose compared to 5% DMSO 201247
yeast cells 0.5% trehalose within 5 days, exogenous trehalose could greatly increase the survival of yeast cells during 201748
cryopreservation
mouse sperm 0.3 M trehalose in skim milk 79% post thaw fertility in trehalose compared to 10% fertility in 0.3 M glycerol 200149
adipose tissues 0.25 M trehalose trehalose can enhance the adipose tissues’ long-term preservation 200550
MVs and PBS supplemented with 25 mM the morphology, integrity, and HSC-supportive potential of MVs and exosomes are well 202188
exosomes trehalose preserved using pTRE at 80 °C
a
Abbreviations: DMSO = dimethyl sulfoxide, MVs = microvesicles, PBS = phosphate buffered saline, pTRE = PBS supplemented with trehalose,
HSC = hematopoietic stem cell.

Once inside the cell, intracellular esterases hydrolyzed the 6-O-
Ac-Tre molecule, releasing the free trehalose into the
cytoplasm. The overall concentration of intracellular trehalose
(plus acetylated variants) achieved a suitable concentration for
applications in cryopreservation. This method of loading
trehalose controlled only the chemical properties of the
trehalose, not the cell, to elevate the membrane permeability.
Trehalose loading rate by 6-O-Ac-Tre was comparable to fluid-
phase endocytosis of platelets and the engineered TRET1
transporter protein but was typically slower than most other
methods involving perforation of the membrane. Compared to
other methods, the delivery efficiency of this method is
unprecedented. Overall, the engineering of the chemical
structure of trehalose is a harmless and cell-friendly way of
delivering trehalose. It has the potential to load trehalose in
various cell types and can promote the widespread use of
trehalose in cryopreservation.
Additionally, mammalian cells can endogenously synthesize
trehalose by genetic engineering. Whereas, this method needs
the application of adenoviral vectors at high infection
multiplicities, which can lead to substantial cytotoxicity. And,
it is mainly used in freeze-drying, so it may not be suitable for
cryopreservation.23,121
In conclusion, if trehalose is to be applied as an effective
CPA for the preservation of cells and tissues, the method of
loading should not markedly change cell structural or
functional aspects or lead to cellular harm or death. Methods
that permit selective access to trehalose are also the preferred
options since the influx or efflux of other small molecules that
may negatively affect cells is undesirable. In the meantime,
methods that allow for rapid uptake of trehalose are preferred
because incubation of cells typically needs to be completed
within 24 h. NPs or other methods seem promising because
they can specifically deliver trehalose without altering
important cellular structures. Moreover, the survival of cells
treated with trehalose-loaded NPs after cryopreservation is
similar to that treated with DMSO. Changing the NP design to
enable a faster and more efficient uptake of the cells is this
method’s future direction for improvement. All these methods
have proven the success of trehalose in cryopreserving cells,
but each method has its superiorities and limitations.
Here, we present a summary of the various methods that
have been studied to deliver trehalose into cells and tissues for
efficient cryopreservation, as listed in Table 3. It indicated that
the uptake of trehalose during cryopreservation can enor-
mously enhance the survival rates of cells and tissues by
comparing the survival rates of the experimental and control
groups. Moreover, extracellular and intracellular trehalose can
significantly increase the viability of the cells and tissues
compared to extracellular trehalose alone. Overall, these
1197
methods appear to be highly efficient for loading non-
permeable trehalose into cells, including but not limited to
electroporation, TRET1 transporter, liposomes, NPs, fluid-
phase endocytosis, sonoporation, and cryoprotective media,
which may significantly facilitate the application of trehalose as
an alternative and sole CPA for cells and tissues.
5. APPLICATIONS OF TREHALOSE IN
CRYOPRESERVATION
Trehalose has been widely used for cells and tissues in
cryopreservation. We will briefly discuss in this section the use
of trehalose alone and in combination with other CPAs.
5.1. Individual Use of Trehalose. Trehalose is commonly
used as a CPA.122,123 Using it as the sole CPA could also
protect cells and tissues even when it is not present in the
cell.96,124
Gläfke et al.47 investigated the application of trehalose in the
preservation of human platelets. Trehalose uptake could be
mediated by the phase transition of platelets’ membrane
induced by freezing during cryopreservation, thereby increas-
ing the survival rate of cryopreserved platelets. Yin et al.48
discovered that adding 0.5% trehalose preserved yeast cells in a
short time but was ineffective for cryopreservation for longer
than 5 days. It still indicated that yeast cells could survive with
a significantly higher survival rate in cryopreservation when
exogenous trehalose was present. Trehalose has also been used
alone for cryopreservation of sperm with excellent results.
Sztein et al.49 compared the cryoprotective ability of permeable
and nonpermeable compounds on freezing mouse sperm. Post-
freezing sperm viability studies showed that trehalose was more
protective than permeable CPA (e.g., DMSO or glycerol) for
mouse sperm.
For the long-term preservation of living tissues, cryopre-
servation is also an efficient method. Pu et al.50 used 0.25 M
trehalose in the cryopreservation of adipose tissue. Compared
with the group without trehalose, in the trehalose-optimal
group, more viable adipocytes and greater cellular adipose
tissue function were observed.
Based on the results, trehalose can be used as a sole CPA to
effectively protect adipose tissue during cryopreservation.
In other aspects, Budgude et al.88 used PBS or PBS
supplemented with trehalose (pTRE) to cryopreserve extrac-
ellular vesicles (EVs)-microvesicles (MVs) and exosomes at
different temperatures, and they further investigated the impact
of cryopreserved EVs on the ex vivo expansion of
hematopoietic stem cells (HSCs). As a result, cryopreservation
at 80 °C in pTRE retained the morphology, integrity, and
HSC-supportive potential of both MVs and exosomes.
In summary, trehalose can significantly improve the survival
rate of cells and tissues compared with the group without
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Table 5. Cells and Tissues Cryopreserved Using Trehalose in Combination with Other CPAsa
cryopreserved incubation conditions of incubation conditions of trehalose and
samples trehalose alone survival rate other CPAs survival rate references
fetal skin 0.5 M Tre + 10% DMSO 65% (the membrane integrity, 200231
compared to 44% for 10% DMSO)
normal human 100 mM Tre 58.5% 10% (w/w) DMSO + 100 mM Tre 85.6% 201782
skin fibroblasts
ADSCs 1.0 M Tre 65 ± 1.10% 1.0 M Tre + 20% Gly 77 ± 1.72% 2020130
RBCs 0.2Tre ∼75% (hemolysis of 1.5Pro&0.2Tre 11.2 ± 2.73% 2019131
thawed RBCs)
(hemolysis of thawed RBCs)
RBCs 0.36 M Tre 78.3 ± 0.4% (pH 0.36 M Tre + 10 mg·mL−1 PKDF1T 89.2 ± 0.2% (pH 6.0) 2020132
6.0)
51.0 ± 5.1% (pH 81.9 ± 0.5% (pH 7.4)
7.4)
RBCs 290 mM Tre 77.8 ± 15.1% Tre + 24.5% (w/w) HES + 92.6−97.4% 2012102
0.5−7 mM liposomes
RBCs 100 mM Tre ∼40% 100 mM Tre, 100 mg·mL−1 97% 2021133
polyampholyte, 10% DMSO, in
water
RBCs 300 mM Tre + 100 μg/ 54.8 ± 1.7% 300 mM Tre +100 μg/mL polymer 61.2 ± 1.4% 2016135
mL polymer PP-50 PP-50 + 200 μM Sal
a
Abbreviations: DMSO = dimethyl sulfoxide, Tre = Trehalose, ADSCs = adipose-derived stem cells, Gly = glycerol, RBCs = red blood cells, 0.2Tre
= 0.2 mol of trehalose dissolve into 1 L of Dulbeco’s phosphate buffered saline (DPBS), 1.5Pro&0.2Tre = 1.5 mol of L-proline and 0.2 mol of
trehalose dissolve into 1 L of DPBS, PKDF1T = PEG-b-(Lys23-co-Asp45)-co-Phe6-g-Tre8, a kind of glycopeptide, HES = hydroxyethyl starch, Sal
= salidroside.

trehalose. And, the effects of cryopreservation with trehalose
on the different cells and tissues are summarized in Table 4.
5.2. Combination. Combining one CPA with another is a
common way to enhance the performance of CPAs.125
Trehalose is a nonpermeable CPA that is receiving increasing
concern owing to its capacity to protect against ice crystal
formation and exert cryoprotective influences.126,127 Never-
theless, trehalose may not inhibit the formation of intracellular
ice crystals because of its inability to pass through the cell
membrane. Consequently, using trehalose in combination with
other CPAs might be necessary to protect cells both inside and
outside. Previous studies have shown that this type of
combined treatment greatly enhances the protection of cells
or tissues during cryopreservation by a probable synergistic
mechanism.128,129
First, trehalose can be used with permeable CPAs for
protecting cells and tissues during cryopreservation. For
example, Erdag et al.31 found that trehalose and DMSO
working together as CPAs enhanced the cryopreservation of
fetal skin. Additionally, Kratochvilová́ et al.82 found that the
highest survival rate was obtained when DMSO was combined
with trehalose, which significantly altered the freezing process
in normal human skin fibroblasts (NHDF). The results were
compared with the samples that included control, AFP,
DMSO, and trehalose.
Trehalose and glycerol together are also a successful method
for cell cryopreservation. Zhang et al.130 postulated that the
combination of glycerol and trehalose could protect adipose-
derived stem cells (ADSCs) against cryodamage and preserve
the cells’ viability. In their study, they first determined the most
ideal concentration of trehalose and glycerol when supplied
alone and then evaluated the impact of trehalose and glycerol
combined on maintaining ADSCs’ viability and cell function.
The findings showed that 1.0 M Tre + 20% Gly could
cryopreserve ADSCs more effectively, making it potentially
useful for clinical applications.
Cryopreservation of RBCs has great potential benefits in
providing timely blood transfusions in emergencies. Dou et
1198
al.131 used natural CPA combinations of L-proline and
trehalose to obtain a low level of hemolysis (11.2 ± 2.73%)
after thawing. Moreover, Na+/K+-ATPase and Ca2+/Mg2+-
ATPase activities and the morphology of RBCs were well-
maintained. A further mechanistic study showed that L-proline
penetrated into the cells during freezing, which helped reduce
the freezing point and prevent the growth of ice crystals. In
contrast, trehalose prevents the growth of ice during freezing
and the recrystallization of ice during thawing. This simple
method offers a possible substitute for the current time-
consuming and labor-intensive RBCs’ cryopreservation meth-
od.
Second, with the help of nonpermeable CPAs alone or the
combination of nonpermeable and permeable CPAs, trehalose
can produce more positive results for cells and tissues during
cryopreservation. Liu et al.132 proposed biocompatible
synthetic glycopeptides to protect RBCs during cryopreserva-
tion. By using poly(ethylene glycol)-NH2 to induce the ring-
opening polymerization of N-carboxyanhydrides of lysine,
aspartic acid, and phenylalanine and then chemically anchoring
carboxylated trehalose to the pendant amino moieties, a series
of glycopeptides have been produced. The specially designed
glycopeptides with trehalose improved the cryopreservation
survival of sheep RBCs at pH 6.0 and pH 7.4 to 89.2 ± 0.3%
and 81.9 ± 0.3%, respectively. Synergistic cryopreservation of
RBCs was achieved through membrane stabilization of
glycopeptides by tuning water diffusion and inhibiting the ice
recrystallization of trehalose during cryopreservation. This
combination of synthetic glycopeptides and trehalose provides
a possible method for effective and glycerol-free cryopreserva-
tion of RBCs.
Stoll et al.102 used liposomes, trehalose, and HES to protect
RBCs synergistically during cryopreservation. After cryopre-
servation, trehalose was delivered to cells by freezing induction,
while liposomes could stabilize RBCs during cryopreservation.
HES is also a nonpermeable CPA, which could effectively
inhibit the formation of intracellular ice crystals and improve
cell survival rate.15 When cells were incubated with trehalose
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and liposomes and frozen in HES, the recovery was between
92.6% and 97.4%. In contrast, the recovery of RBCs incubated
and frozen in trehalose medium was 77.8%. Therefore, the
synergistic protective effect of trehalose, liposome, and HES
could significantly improve the survival rate of RBCs, which
was expected to become the future direction for development
of new methods for cryopreservation of RBCs. The benefit of
this method is that it does not require the removal of
protective compounds before blood transfusion, so as to avoid
a large number of time-consuming cleaning procedures.102
Murray et al.133 showed that combining CPAs with different
modes of action could result in a high post-thaw recovery of
RBCs. In this study, the results demonstrated that the most
effective formulation with a post-thaw recovery of 97%
contained 100 mg mL−1 polyampholyte, 10% DMSO, and
100 mM trehalose in water. The exact mechanism of the
cryoprotective ability of polyampholytes was not yet clear, but
the combination of intracellular (DMSO), extracellular
(trehalose), and polymer could lead to reduced hemolysis. In
terms of the exact mechanism of cryoprotective ability by
polyampholytes, Matsumura et al.134 did relevant research by
using solid-state NMR spectroscopy. This technique revealed
the mechanism of the cryoprotective ability of carboxylated
poly-L-lysines (COOH-PLLs), which could be summarized as
follows. First, under mild conditions, cell dehydration from the
outside inhibited IIF. This was accomplished by regulating
osmotic pressure by encasing water and salt molecules in the
polymer matrix. Second, preserving a viscous condition at
higher temperatures inhibited IIF caused by the influx of
extracellular ice.
Despite the protective effect of CPAs, freeze−thaw processes
can still alter cells and result in oxidative damage. This might
cause severe cellular damage by enhanced production of free
radical oxygen species (ROS).135 Antioxidants are able to
counteract oxidative stress and lessen ROS-related damage.
Consequently, the damage caused by cryopreservation can be
significantly reduced by using a combination of antioxidants
and CPAs.136 Alotaibi et al.135 evaluated the effects of a novel
CPA and a strong antioxidant named salidroside (Sal) on
sheep RBC biological functions during cryopreservation,
combining glycerol and trehalose/PP-50. The addition of Sal
improved survival by 7.6 ± 0.3% as compared to using
trehalose alone. Here, Sal can reduce the oxidative damage of
sheep RBCs during cryopreservation.
In summary, trehalose can effectively improve the survival
rate of cells and tissues by combining with other CPAs and can
be used as a new method in cryopreservation (Table 5).
6. BENEFITS AND LIMITS OF TREHALOSE IN
CRYOPRESERVATION
6.1. Benefits of Trehalose in Cryopreservation.
Trehalose is a particularly attractive biological CPA because
its role in protecting cells and tissues from freezing damages is
well-known.67,137 Conventional cryopreservation of cells and
tissues needs the utilization of toxic organic solvents (such as
DMSO) as CPAs, which cannot be used directly on
patients.138−140 As a result, cryopreserved cells have to
withstand cumbersome washing procedures to eliminate
organic solvents, resulting in significant cell loss.141,142
Conversely, as a sugar, trehalose is not toxic to cells and
tissues. Besides, intracellular trehalose can be delivered to the
extracellular space via exocytosis, which has been demonstrated
to remove cytoplasmic sucrose (also a disaccharide) from
1199
pubs.acs.org/journal/abseba Review
macrophages and fibroblasts in only a few hours at 37 °C.143
Meanwhile, Eroglu et al.106 proposed that the small number of
intracellular sugars could be metabolized after nonspecific
cleavage of their backbones, excreted by exocytosis, or even
more diluted by cell division. And, mammalian cells tolerate
intracellular trehalose of at least 0.2 M without damaging their
functions. Therefore, it does not have to be eliminated from
cryopreserved cells and tissues, and the effect of trehalose on
cells may be negligible.110,144 It greatly facilitates the
widespread use of emerging cellular medicine.23,50,102
6.2. Limits of Trehalose in Cryopreservation. It must
be noted that sugar also has the shortcomings of not being able
to penetrate cell membranes. And, since mammalian cells do
not metabolize trehalose, its use in mammalian cells is
limited.145 The nonpermeating trehalose provides the
cryoprotective ability, but to achieve the best protection for
cells, trehalose is supposed to exist inside and outside the
cells.72 Trehalose must be delivered into cells using various
methods or it must be combined with other CPAs.
However, in order to obtain higher intracellular trehalose
contents, cells must be incubated for a long time in the
existence of trehalose or at a higher trehalose concentration.
But, if the preloading trehalose concentration is too high, the
cells will lose their viability due to osmotic shock.95 There are
two probabilities for cell responses to high osmotic pressure.
First, the cell membrane is unable to tolerate the movement of
the solution, leading to disruption of the cell membrane.
Second, when osmotic stress is imposed and the cell is overly
contracted, the deformation destroys the cell structure. An
improvement in trehalose concentration is associated with an
improvement in osmotic stress. Therefore, the incubation
period of trehalose has to be optimized to ensure a moderate
concentration of intracellular trehalose and an ideal balance
between trehalose’s protective and destructive impacts. To sum
up, the optimal trehalose concentration is needed to produce
finer ice crystals and reduce osmotic damage. Additionally, to
decrease the possibility of osmotic shocks, a high-capacity
trehalose transporter can be used. It can relatively quickly
reduce the trehalose gradient on the cell membrane.144
7. CONCLUSION
As more and more translational medicine products enter the
clinic, the need for advanced storage of cells and tissues is
increasing. One of the many factors considered in emerging
translational medicine is the safety of cryopreserving cells and
tissues. Therefore, the utility and demand for CPAs to prevent
or minimize damage from freezing are gradually improving.
Trehalose has gained interest in cryopreservation due to its
capacity to prevent ice crystal formation, protect proteins and
other biomolecules, stabilize cell membranes, and increase
membrane permeability. In this article, the properties,
mechanisms, delivery methods, applications, and benefits and
limits of trehalose are described comprehensively. There are
the following findings and research direction of trehalose in
biomedical cryopreservation.
(1) The effect of trehalose in cells is negligible. This
argument is based on relevant experiments with oocytes,
where Kikawada et al.144 incubated oocytes in a 105 mM
trehalose solution for 3 h and then discovered that the
elimination of intracellular trehalose from oocytes took
only about 2 h. Meanwhile, osmotically sensitive
mammalian oocytes were also capable of surviving
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ACS Biomaterials Science & Engineering
after being microinjected with 150 mM trehalose. For
oocytes, trehalose was a special CPA, which quickly
disappeared during embryonic development. Similarly,
although intracellular high trehalose might increase
intracellular osmotic pressure, there was no significant
effect on liver mononuclear cells (LMC) activity and
function after loading 240 mM trehalose. This might be
due to the ability of various tissue cells, including
hepatocytes and kidney cells, to activate cell volume
control mechanisms that adjusted cell volume according
to intraosmotic pressure over time.73 Consequently, the
effect of trehalose on cells during the incubation period
in a normal medium may be negligible.
(2) The use of trehalose in RBCs needn’t remove steps.
Janis et al. 100 delivered trehalose to RBCs by
sonoporation without removal steps, and the thawed
RBCs did not cause any adverse transfusion reactions.
Namely, trehalose in cells may not cause adverse blood
transfusion reactions and RBCs can be directly used for
clinical applications.
(3) More researches are needed to determine whether
trehalose must be delivered into the cells. As mentioned
above, trehalose must be present in both intracellular
and extracellular cells to supply maximum protection
against stress and damage during cryopreservation. But,
Crowe et al.124 expected trehalose to stabilize the cell
membrane through the pathway from extracellular to cell
membrane and membrane localization protein. This
study pointed out that, even when trehalose was not
present in the cell, it could still protect cells and enhance
liver-specific functions. Additionally, each delivery
method of trehalose has its superiorities and limitations.
Therefore, it is hard to evaluate whether trehalose must
be delivered into the cells.
(4) Exploring optimal concentration and proportion.
Trehalose has poor cell permeability, so other
supplementary CPAs are always added to provide
enough intracellular protection. When trehalose is
combined with other CPAs, the optimal concentration
and proportion should be explored first.
8. PROSPECT
Trehalose displays great promise as a nontoxic CPA that does
not require removal from cells and tissues before translational
medicine application, and much progress has been made in the
delivery of trehalose through the membrane to cells. In the
future, trehalose is hopeful to be applied in the cryopreserva-
tion of human tissues and organs to advance the development
of translational medicine.
■ AUTHOR INFORMATION
Corresponding Author
Songwen Tan − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China;
orcid.org/0000-0002-2138-7465; Email: songwen.tan@
csu.edu.cn
Authors
Yuying Hu − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China
Xiangjian Liu − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China
1200
pubs.acs.org/journal/abseba Review
Fenglin Liu − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China
Jingxian Xie − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China
Qubo Zhu − Xiangya School of Pharmaceutical Sciences,
Central South University, Changsha, Hunan 410013, China;
orcid.org/0000-0001-5202-7887
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsbiomaterials.2c01225
Author Contributions
All authors made contributions to the manuscript’s writing.
The published version of the manuscript has been read and
approved by all authors.
Funding
There was no external funding.
Notes
The authors declare no competing financial interest.
■ REFERENCES
(1) National Blood Collection & Utilization Survey; U.S. Department
of Health and Human Services, 2011.
(2) Janssen, S. J.; Braun, Y.; Wood, K. B.; Cha, T. D.; Schwab, J. H.
Allogeneic blood transfusions and postoperative infections after
lumbar spine surgery. The Spine Journal 2015, 15, 901.
(3) Lee, S.; Ozkavukcu, S.; Ku, S.-Y. Current and Future
Perspectives for Improving Ovarian Tissue Cryopreservation and
Transplantation Outcomes for Cancer Patients. Reproductive Sciences
2021, 28 (6), 1746−1758.
(4) Afreen, M. Significance of cryopreservation biotechnology for
protection of aquatic species. Eurasian Journal of Agricultural Research
2020, 4 (2), 64−71.
(5) Mazur, P.; Leibo, S. P.; Chu, E. H. A two-factor hypothesis of
freezing injury. Evidence from Chinese hamster tissue-culture cells.
Experimental cell research 1972, 71 (2), 345−55.
(6) Yeste, M. Sperm cryopreservation update: Cryodamage, markers,
and factors affecting the sperm freezability in pigs. Theriogenology
2016, 85 (1), 47−64.
(7) Gao, D.; Critser, J. K. Mechanisms of Cryoinjury in Living Cells.
ILAR Journal 2000, 41 (4), 187−196.
(8) Yurchuk, T.; Petrushko, M.; Fuller, B. Science of cryopreserva-
tion in reproductive medicine − Embryos and oocytes as exemplars.
Early Human Development 2018, 126, 6−9.
(9) Jahan, S.; Kaushal, R.; Pasha, R.; Pineault, N. Current and Future
Perspectives for the Cryopreservation of Cord Blood Stem Cells.
Transfusion Medicine Reviews 2021, 35 (2), 95−102.
(10) Yang, J.; Pan, C.; Zhang, J.; Sui, X.; Zhu, Y.; Wen, C.; Zhang, L.
Exploring the Potential of Biocompatible Osmoprotectants as Highly
Efficient Cryoprotectants. ACS Appl. Mater. Interfaces 2017, 9 (49),
42516−42524.
(11) Paul, R. K.; Kumar, D.; Singh, R. Carboxymethyl cellulose and
glycerol act synergistically as cryoprotectant during cryopreservation
of ram semen. Cryobiology 2021, 101, 61−66.
(12) Stubbs, C.; Bailey, T. L.; Murray, K.; Gibson, M. I.
Polyampholytes as Emerging Macromolecular Cryoprotectants.
Biomacromolecules 2020, 21 (1), 7−17.
(13) Keskin, N.; Erdogan, C.; Bucak, M. N.; Ozturk, A. E.; Bodu,
M.; Ili, P.; Baspinar, N.; Dursun, S. Cryopreservation effects on ram
sperm ultrastructure. Biopreservation and Biobanking 2020, 18 (5),
441−448.
(14) Ntai, A.; La Spada, A.; De Blasio, P.; Biunno, I. Trehalose to
cryopreserve human pluripotent stem cells. Stem Cell Research 2018,
31, 102−112.
(15) Stolzing, A.; Naaldijk, Y.; Fedorova, V.; Sethe, S. Hydrox-
yethylstarch in cryopreservation - mechanisms, benefits and problems.
Transfus Apher Sci. 2012, 46 (2), 137−47.
https://doi.org/10.1021/acsbiomaterials.2c01225
ACS Biomater. Sci. Eng. 2023, 9, 1190−1204
ACS Biomaterials Science & Engineering
(16) Rajan, R.; Hayashi, F.; Nagashima, T.; Matsumura, K. Toward a
Molecular Understanding of the Mechanism of Cryopreservation by
Polyampholytes: Cell Membrane Interactions and Hydrophobicity.
Biomacromolecules 2016, 17 (5), 1882−1893.
(17) Bachtiger, F.; Congdon, T. R.; Stubbs, C.; Gibson, M. I.; Sosso,
G. C. The atomistic details of the ice recrystallisation inhibition
activity of PVA. Nat. Commun. 2021, 12 (1), 1323.
(18) Shaluei, F.; Imanpoor, M. R.; Shabani, A.; Nasr-Esfahani, M. H.
Effect of different concentrations of permeable and non−permeable
cryoprotectants on the hatching rate of goldfish (Carassius auratus)
embryos. Asian Pacific Journal of Reproduction 2013, 2 (3), 185−188.
(19) Best, B. P. Cryoprotectant Toxicity: Facts, Issues, and
Questions. Rejuvenation Research 2015, 18 (5), 422.
(20) Ataseven, E.; Tufekci, O.; Yilmaz, S.; Guleryuz, H.; Oren, H.
Neurotoxicity Associated With Dimethyl Sulfoxide Used in Allogeneic
Stem Cell Transplantation. J. Pediatr. Hematol. Oncol. 2017, 39 (5),
E297−E299.
(21) Bakaltcheva, I. B.; Odeyale, C. O.; Spargo, B. J. Effects of
alkanols, alkanediols and glycerol on red blood cell shape and
hemolysis. Biochimica et Biophysica Acta (BBA) - Biomembranes 1996,
1280 (1), 73−80.
(22) Zhang, Y.; Wang, H.; Stewart, S.; Jiang, B.; Ou, W.; Zhao, G.;
He, X. Cold-Responsive Nanoparticle Enables Intracellular Delivery
and Rapid Release of Trehalose for Organic-Solvent-Free Cryopre-
servation. Nano Lett. 2019, 19 (12), 9051−9061.
(23) Rao, W.; Huang, H.; Wang, H.; Zhao, S.; Dumbleton, J.; Zhao,
G.; He, X. Nanoparticle-Mediated Intracellular Delivery Enables
Cryopreservation of Human Adipose-Derived Stem Cells Using
Trehalose as the Sole Cryoprotectant. ACS Appl. Mater. Interfaces
2015, 7 (8), 5017−5028.
(24) Lee, Y. A.; Kim, Y. H.; Kim, B. J.; Kim, B. G.; Kim, K. J.; Auh, J.
H.; Schmidt, J. A.; Ryu, B. Y. Cryopreservation in trehalose preserves
functional capacity of murine spermatogonial stem cells. PLoS One
2013, 8 (1), e54889.
(25) Rowley, S. D.; Anderson, G. L. Effect of DMSO exposure
without cryopreservation on hematopoietic progenitor cells. Bone
Marrow Transplant 1993, 11 (5), 389−393.
(26) Buchanan, S. S.; Gross, S. A.; Acker, J. P.; Toner, M.;
Carpenter, J. F.; Pyatt, D. W. Cryopreservation of stem cells using
trehalose: evaluation of the method using a human hematopoietic cell
line. Stem Cells Dev 2004, 13 (3), 295−305.
(27) Chen, S.; Wu, L.; Ren, J.; Bemmer, V.; Zajicek, R.; Chen, R.
Comb-like Pseudopeptides Enable Very Rapid and Efficient Intra-
cellular Trehalose Delivery for Enhanced Cryopreservation of
Erythrocytes. ACS Appl. Mater. Interfaces 2020, 12 (26), 28941−
28951.
(28) Fujita, Y.; Nishimura, M.; Komori, N.; Sawamoto, O.; Kaneda,
S. Protein-free solution containing trehalose and dextran 40 for
cryopreservation of human adipose tissue-derived mesenchymal
stromal cells. Cryobiology 2021, 100, 46.
(29) Katenz, E.; Vondran, F. W.; Schwartlander, R.; Pless, G.; Gong,
X.; Cheng, X.; Neuhaus, P.; Sauer, I. M. Cryopreservation of primary
human hepatocytes: the benefit of trehalose as an additional
cryoprotective agent. Liver Transpl 2007, 13 (1), 38−45.
(30) Beattie, G. M.; Crowe, J. H.; Lopez, A. D.; Cirulli, V.; Ricordi,
C.; Hayek, A. Trehalose: a cryoprotectant that enhances recovery and
preserves function of human pancreatic islets after long-term storage.
Diabetes 1997, 46 (3), 519−23.
(31) Erdag, G.; Eroglu, A.; Morgan, J. R.; Toner, M.
Cryopreservation of fetal skin is improved by extracellular trehalose.
Cryobiology 2002, 44 (3), 218−228.
(32) Zheng, X.; Liu, J.; Liu, Z.; Wang, J. Bio-inspired Ice-controlling
Materials for Cryopreservation of Cells and Tissues. Acta Chimica
Sinica 2021, 79 (6), 729.
(33) Jesus, A. R.; Meneses, L.; Duarte, A. R. C.; Paiva, A. Natural
deep eutectic systems, an emerging class of cryoprotectant agents.
Cryobiology 2021, 101, 95−104.
(34) Keros, V.; Rosenlund, B.; Hultenby, K.; Aghajanova, L.;
Levkov, L.; Hovatta, O. Optimizing cryopreservation of human
1201
pubs.acs.org/journal/abseba Review
testicular tissue: comparison of protocols with glycerol, propanediol
and dimethylsulfoxide as cryoprotectants. Hum. Reprod. 2005, 20 (6),
1676−1687.
(35) Newton, H.; Aubard, Y.; Rutherford, A.; Sharma, V.; Gosden,
R. Ovary and ovulation: Low temperature storage and grafting of
human ovarian tissue. Human reproduction 1996, 11 (7), 1487−1491.
(36) Bakhach, J. The cryopreservation of composite tissues:
principles and recent advancement on cryopreservation of different
type of tissues. Organogenesis 2009, 5 (3), 119−126.
(37) Zhang, P.-Q.; Tan, P.-C.; Gao, Y.-M.; Zhang, X.-J.; Xie, Y.;
Zheng, D.-N.; Zhou, S.-B.; Li, Q.-F. The effect of glycerol as a
cryoprotective agent in the cryopreservation of adipose tissue. Stem
Cell Res. Ther. 2022, 13 (1), 152.
(38) Lovelock, J. E.; Bishop, M. W. H. Prevention of Freezing
Damage to Living Cells by Dimethyl Sulfoxide. Nature 1959, 183
(4672), 1394−1395.
(39) Hornberger, K.; Yu, G.; McKenna, D.; Hubel, A.
Cryopreservation of Hematopoietic Stem Cells: Emerging Assays,
Cryoprotectant Agents, and Technology to Improve Outcomes.
Transfusion Medicine and Hemotherapy 2019, 46 (3), 188−196.
(40) Jia, X.; Yu, Y.; Li, D. Investigation on the effect of trehalose on
alpha-actinin in cryopreserved human skin. Zhongguo Xiu Fu Chong
Jian Wai Ke Za Zhi 2008, 22 (4), 446−449.
(41) Xia, Z.; Duan, X.; Murray, D.; Triffitt, J. T.; Price, A. J. A
method of isolating viable chondrocytes with proliferative capacity
from cryopreserved human articular cartilage. Cell and tissue banking
2013, 14 (2), 267−276.
(42) Keskin, N.; Erdogan, C.; Bucak, M. N.; Ozturk, A. E.; Bodu,
M.; Ili, P.; Baspinar, N.; Dursun, S. Cryopreservation Effects on Ram
Sperm Ultrastructure. Biopreserv Biobank 2020, 18 (5), 441−448.
(43) Oldenhof, H.; Gojowsky, M.; Wang, S.; Henke, S.; Yu, C.;
Rohn, K.; Wolkers, W. F.; Sieme, H. Osmotic Stress and Membrane
Phase Changes During Freezing of Stallion Sperm: Mode of Action of
Cryoprotective Agents1. Biol. Reprod. 2013, 88 (3), 1−11.
(44) Chen, S.-U.; Lien, Y.-R.; Chao, K.-H.; Lu, H.-F.; Ho, H.-N.;
Yang, Y.-S. Cryopreservation of mature human oocytes by vitrification
with ethylene glycol in straws. Fertility and Sterility 2000, 74 (4),
804−808.
(45) von Bomhard, A.; Elsässer, A.; Ritschl, L. M.; Schwarz, S.;
Rotter, N. Cryopreservation of Endothelial Cells in Various
Cryoprotective Agents and Media − Vitrification versus Slow
Freezing Methods. PLoS One 2016, 11 (2), No. e0149660.
(46) Chi, H. J.; Koo, J. J.; Kim, M. Y.; Joo, J. Y.; Chang, S. S.; Chung,
K. S. Cryopreservation of human embryos using ethylene glycol in
controlled slow freezing. Hum. Reprod. 2002, 17 (8), 2146−51.
(47) Gläfke, C.; Akhoondi, M.; Oldenhof, H.; Sieme, H.; Wolkers,
W. F. Cryopreservation of platelets using trehalose: the role of
membrane phase behavior during freezing. Biotechnol. Prog. 2012, 28
(5), 1347−54.
(48) Yin, H.; Wang, Y.; He, Y.; Xing, L.; Zhang, X.; Wang, S.; Qi, X.;
Zheng, Z.; Lu, J.; Miao, J. Cloning and expression analysis of tps, and
cryopreservation research of trehalose from Antarctic strain
Pseudozyma sp. 3 Biotech 2017, 7, 343.
(49) Sztein, J. M.; Noble, K.; Farley, J. S.; Mobraaten, L. E.
Comparison of permeating and nonpermeating cryoprotectants for
mouse sperm cryopreservation. Cryobiology 2001, 42 (1), 28−39.
(50) Pu, L. L.; Cui, X.; Fink, B. F.; Cibull, M. L.; Gao, D.
Cryopreservation of adipose tissues: the role of trehalose. Aesthet Surg
J. 2005, 25 (2), 126−31.
(51) Lionetti, F. J.; Hunt, S. M. Cryopreservation of human red cells
in liquid nitrogen with hydroxyethyl starch. Cryobiology 1975, 12 (2),
110−118.
(52) Lionetti, F. J.; Hunt, S. M.; Gore, J. M.; Curby, W. A.
Cryopreservation of human granulocytes. Cryobiology 1975, 12 (3),
181−191.
(53) Ashwood-Smith, M.; Warby, C.; Connor, K.; Becker, G. Low-
temperature preservation of mammalian cells in tissue culture with
polyvinylpyrrolidone (PVP), dextrans, and hydroxyethyl starch
(HES). Cryobiology 1972, 9 (5), 441−449.
https://doi.org/10.1021/acsbiomaterials.2c01225
ACS Biomater. Sci. Eng. 2023, 9, 1190−1204
ACS Biomaterials Science & Engineering
(54) Kenmochi, T.; Asano, T.; Maruyama, M.; Saigo, K.; Akutsu, N.;
Iwashita, C.; Ohtsuki, K.; Suzuki, A.; Miyazaki, M. Cryopreservation
of human pancreatic islets from non-heart-beating donors using
hydroxyethyl starch and dimethyl sulfoxide as cryoprotectants. Cell
transplantation 2008, 17 (1−2), 61−67.
(55) Luo, K.; Wu, G.; Wang, Q.; Sun, Y.; Liu, H. Effect of
dimethylsulfoxide and hydroxyethyl starch in the preservation of
fractionated human marrow cells. Cryobiology 1994, 31 (4), 349−354.
(56) Neveux, N.; De Bandt, J.; Charrueau, C.; Savier, E.; Chaumeil,
J.-C.; Hannoun, L.; Giboudeau, J.; Cynober, L. A. Deletion of
hydroxyethylstarch from University of Wisconsin solution induces cell
shrinkage and proteolysis during and after cold storage of rat liver.
Hepatology 1997, 25 (3), 678−682.
(57) Mabrut, J.; Adham, M.; Bourgeot, J.; Eljaafari, A.; DelaRoche,
E.; Ducerf, C. Mechanical and histological characteristics of human
trachea before and after cryopreservation: an opportunity for tracheal
tissue banking. Transplantation Proceedings 2001, 33, 609−611.
(58) Watanabe, H.; Kohaya, N.; Kamoshita, M.; Fujiwara, K.;
Matsumura, K.; Hyon, S.-H.; Ito, J.; Kashiwazaki, N. Efficient
production of live offspring from mouse oocytes vitrified with a
novel cryoprotective agent, carboxylated ε-poly-L-lysine. PLoS One
2013, 8 (12), e83613.
(59) Tariq, A.; Ahmad, M.; Iqbal, S.; Riaz, M. I.; Tahir, M. Z.;
Ghafoor, A.; Riaz, A. Effect of carboxylated poly l-Lysine as a
cryoprotectant on post-thaw quality and in vivo fertility of Nili Ravi
buffalo (Bubalus bubalis) bull semen. Theriogenology 2020, 144, 8−
15.
(60) Matsumura, K.; Hyon, S. H. Polyampholytes as low toxic
efficient cryoprotective agents with antifreeze protein properties.
Biomaterials 2009, 30 (27), 4842−9.
(61) Nakayama, K.; Yamanaka, T.; Tamada, Y.; Hirabayashi, M.;
Hochi, S. Supplementary cryoprotective effect of carboxylated ε-poly-
l-lysine during vitrification of rat pancreatic islets. Cryobiology 2019,
88, 70−74.
(62) Inada, T.; Lu, S.-S. Inhibition of Recrystallization of Ice Grains
by Adsorption of Poly(Vinyl Alcohol) onto Ice Surfaces. Cryst.
Growth Des. 2003, 3 (5), 747−752.
(63) Bachtiger, F.; Congdon, T. R.; Stubbs, C.; Gibson, M. I.; Sosso,
G. C. The atomistic details of the ice recrystallisation inhibition
activity of PVA. Nat. Commun. 2021, 12 (1), 1323.
(64) Nunes, S. C. C.; Jesus, A. J. L.; Moreno, M. J.; Eusébio, M. E. S.
Conformational preferences of α,α-trehalose in gas phase and aqueous
solution. Carbohydr. Res. 2010, 345 (14), 2048−2059.
(65) Zhang, S.; Wang, H.; Luo, J.; Yu, W.; Xiao, Y.; Peng, F. Peach
PpSnRK1α interacts with bZIP11 and maintains trehalose balance in
plants. Plant Physiology and Biochemistry 2021, 160, 377−385.
(66) Stolz, J.; Luyckx; Baudouin, C. Trehalose: an intriguing
disaccharide with potential for medical application in ophthalmology.
Clin Ophthalmol 2011, 5, 577−581.
(67) Elbein, A. D.; Pan, Y. T.; Pastuszak, I.; Carroll, D. New insights
on trehalose: a multifunctional molecule. Glycobiology 2003, 13 (4),
17R−27R.
(68) Furze, C. M.; Delso, I.; Casal, E.; Guy, C. S.; Seddon, C.;
Brown, C. M.; Parker, H. L.; Radhakrishnan, A.; Pacheco-Gomez, R.;
Stansfeld, P. J.; Angulo, J.; Cameron, A. D.; Fullam, E. Structural basis
of trehalose recognition by the mycobacterial LpqY-SugABC
transporter. J. Biol. Chem. 2021, 296, 100307.
(69) Heczko, D.; Grelska, J.; Jurkiewicz, K.; Spychalska, P.;
Kasprzycka, A.; Kamiński, K.; Paluch, M.; Kamińska, E. Anomalous
narrowing of the shape of the structural process in derivatives of
trehalose at high pressure. The role of the internal structure. J. Mol.
Liq. 2021, 336, 116321.
(70) Richards, A. B.; Krakowka, S.; Dexter, L. B.; Schmid, H.;
Wolterbeek, A. P. M.; Waalkens-Berendsen, D. H.; Shigoyuki, A.;
Kurimoto, M. Trehalose: a review of properties, history of use and
human tolerance, and results of multiple safety studies. Food Chem.
Toxicol. 2002, 40 (7), 871−898.
(71) Jain, N. K.; Roy, I. Trehalose and protein stability. Curr. Protoc
Protein Sci. 2010, 59, 1−12.
1202
pubs.acs.org/journal/abseba Review
(72) Diaz-Dussan, D.; Peng, Y.-Y.; Sengupta, J.; Zabludowski, R.;
Adam, M. K.; Acker, J. P.; Ben, R. N.; Kumar, P.; Narain, R.
Trehalose-Based Polyethers for Cryopreservation and Three-Dimen-
sional Cell Scaffolds. Biomacromolecules 2020, 21 (3), 1264−1273.
(73) Wang, B.; Liu, G.; Balamurugan, V.; Sui, Y.; Wang, G.; Song, Y.;
Chang, Q. Apatite nanoparticles mediate intracellular delivery of
trehalose and increase survival of cryopreserved cells. Cryobiology
2019, 86, 103−110.
(74) Jain, N. K.; Roy, I. Effect of trehalose on protein structure.
Protein Sci. 2008, 18 (1), 24−36.
(75) Ajito, S.; Hirai, M.; Iwase, H.; Shimizu, N.; Igarashi, N.; Ohta,
N. Protective action of trehalose and glucose on protein hydration
shell clarified by using X-ray and neutron scattering. Physica B:
Condensed Matter 2018, 551, 249−255.
(76) Kawai, H.; Sakurai, M.; Inoue, Y.; Chûjô, R.; Kobayashi, S.
Hydration of oligosaccharides: Anomalous hydration ability of
trehalose. Cryobiology 1992, 29 (5), 599−606.
(77) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.;
Wolkers, W.; Tablin, F. The Trehalose Myth Revisited: Introduction
to a Symposium on Stabilization of Cells in the Dry State. Cryobiology
2001, 43 (2), 89−105.
(78) Lerbret, A.; Bordat, P.; Affouard, F.; Descamps, M.; Migliardo,
F. How Homogeneous Are the Trehalose, Maltose, and Sucrose
Water Solutions? An Insight from Molecular Dynamics Simulations. J.
Phys. Chem. B 2005, 109 (21), 11046−11057.
(79) Lerbret, A.; Bordat, P.; Affouard, F.; Guinet, Y.; Hédoux, A.;
Paccou, L.; Prévost, D.; Descamps, M. Influence of homologous
disaccharides on the hydrogen-bond network of water: complemen-
tary Raman scattering experiments and molecular dynamics
simulations. Carbohydr. Res. 2005, 340 (5), 881−887.
(80) Branca, C.; Maccarrone, S.; Magazu, S.; Maisano, G.;
Bennington, S. M.; Taylor, J. Tetrahedral order in homologous
disaccharide-water mixtures. J. Chem. Phys. 2005, 122 (17), 174513.
(81) Sola-Penna, M.; Ferreira-Pereira, A.; Lemos, A. d. P.; Meyer-
Ferwandes, J. R. Carbohydrate protection of enzyme structure and
function against guanidinium chloride treatment depends on the
nature of carbohydrate and enzyme. Eur. J. Biochem. 1997, 248 (1),
24−29.
(82) Kratochvílová, I.; Golan, M.; Pomeisl, K.; Richter, J.; Sedláková,
S.; Š ebera, J.; Mičová, J.; Falk, M.; Falková, I.; Ř eha, D.; Elliott, K. W.;
Varga, K.; Follett, S. E.; Š imek, D. Theoretical and experimental study
of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide
cryoprotection mechanism: correlation with cryopreserved cell
viability. RSC Adv. 2017, 7 (1), 352−360.
(83) Crowe, L. M.; Reid, D. S.; Crowe, J. H. Is trehalose special for
preserving dry biomaterials? Biophysical journal 1996, 71 (4), 2087−
93.
(84) Green, J. L.; Angell, C. A. Phase relations and vitrification in
saccharide-water solutions and the trehalose anomaly. J. Phys. Chem.
1989, 93 (8), 2880−2882.
(85) Moore, D. S.; Hand, S. C. Cryopreservation of lipid bilayers by
LEA proteins from Artemia franciscana and trehalose. Cryobiology
2016, 73 (2), 240−7.
(86) Corradini, D.; Strekalova, E. G.; Stanley, H. E.; Gallo, P.
Microscopic mechanism of protein cryopreservation in an aqueous
solution with trehalose. Sci. Rep. 2013, 3 (1), 1218.
(87) Lins, R. D.; Pereira, C. S.; Hünenberger, P. H. Trehalose−
protein interaction in aqueous solution. Proteins: Struct., Funct., Bioinf.
2004, 55 (1), 177−186.
(88) Budgude, P.; Kale, V.; Vaidya, A. Cryopreservation of
mesenchymal stromal cell-derived extracellular vesicles using trehalose
maintains their ability to expand hematopoietic stem cells in vitro.
Cryobiology 2021, 98, 152−163.
(89) Ohtake, S.; Schebor, C.; de Pablo, J. J. Effects of trehalose on
the phase behavior of DPPC−cholesterol unilamellar vesicles.
Biochimica et Biophysica Acta (BBA) - Biomembranes 2006, 1758
(1), 65−73.
https://doi.org/10.1021/acsbiomaterials.2c01225
ACS Biomater. Sci. Eng. 2023, 9, 1190−1204
ACS Biomaterials Science & Engineering
(90) Rudolph, A. S.; Crowe, J. H. Membrane stabilization during
freezing: The role of two natural cryoprotectants, trehalose and
proline. Cryobiology 1985, 22 (4), 367−377.
(91) Zhang, M.; Oldenhof, H.; Sieme, H.; Wolkers, W. F. Freezing-
induced uptake of trehalose into mammalian cells facilitates
cryopreservation. Biochim. Biophys. Acta 2016, 1858 (6), 1400−9.
(92) Mitchell, D. E.; Fayter, A. E.; Deller, R. C.; Hasan, M.;
Gutierrez-Marcos, J.; Gibson, M. I. Ice-recrystallization inhibiting
polymers protect proteins against freeze-stress and enable glycerol-
free cryostorage. Materials horizons 2019, 6 (2), 364−368.
(93) Lee, J.; Lin, E.-W.; Lau, U. Y.; Hedrick, J. L.; Bat, E.; Maynard,
H. D. Trehalose Glycopolymers as Excipients for Protein Stabiliza-
tion. Biomacromolecules 2013, 14 (8), 2561−2569.
(94) Bosch, S.; de Beaurepaire, L.; Allard, M.; Mosser, M.;
Heichette, C.; Chretien, D.; Jegou, D.; Bach, J. M. Trehalose prevents
aggregation of exosomes and cryodamage. Sci. Rep 2016, 6, 36162.
(95) Zhang, M.; Oldenhof, H.; Sieme, H.; Wolkers, W. F.
Combining endocytic and freezing-induced trehalose uptake for
cryopreservation of mammalian cells. Biotechnol. Prog. 2017, 33 (1),
229−235.
(96) Yoshida, K.; Ono, F.; Chouno, T.; Perocho, B. R.; Ikegami, Y.;
Shirakigawa, N.; Ijima, H. Cryoprotective enhancing effect of very low
concentration of trehalose on the functions of primary rat
hepatocytes. Regenerative Therapy 2020, 15, 173−179.
(97) Lynch, A. L.; Slater, N. K. H. Influence of intracellular trehalose
concentration and pre-freeze cell volume on the cryosurvival of
rapidly frozen human erythrocytes. Cryobiology 2011, 63 (1), 26−31.
(98) Eroglu, A.; Russo, M. J.; Bieganski, R.; Fowler, A.; Cheley, S.;
Bayley, H.; Toner, M. Intracellular trehalose improves the survival of
cryopreserved mammalian cells. Nat. Biotechnol. 2000, 18 (2), 163−
167.
(99) Zhang, T. Y.; Tan, P. C.; Xie, Y.; Zhang, X. J.; Zhang, P. Q.;
Gao, Y. M.; Zhou, S. B.; Li, Q. F. The combination of trehalose and
glycerol: an effective and non-toxic recipe for cryopreservation of
human adipose-derived stem cells. Stem Cell Res. Ther. 2020, 11 (1),
460.
(100) Janis, B. R.; Priddy, M. C.; Otto, M. R.; Kopechek, J. A.;
Menze, M. A. Sonoporation enables high-throughput loading of
trehalose into red blood cells. Cryobiology 2021, 98, 73−79.
(101) Stewart, S.; He, X. Intracellular Delivery of Trehalose for Cell
Banking. Langmuir 2019, 35 (23), 7414−7422.
(102) Stoll, C.; Holovati, J. L.; Acker, J. P.; Wolkers, W. F.
Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for
cryopreservation of human erythrocytes. Biotechnol. Prog. 2012, 28
(2), 364−371.
(103) Satpathy, G. R.; Török, Z.; Bali, R.; Dwyre, D. M.; Little, E.;
Walker, N. J.; Tablin, F.; Crowe, J. H.; Tsvetkova, N. M. Loading red
blood cells with trehalose: a step towards biostabilization. Cryobiology
2004, 49 (2), 123−136.
(104) Shirakashi, R.; Köstner, C. M.; Müller, K. J.; Kürschner, M.;
Zimmermann, U.; Sukhorukov, V. L. Intracellular delivery of trehalose
into mammalian cells by electropermeabilization. J. Membr. Biol. 2002,
189 (1), 45−54.
(105) Dovgan, B.; Barlič, A.; Knežević, M.; Miklavčič, D.
Cryopreservation of Human Adipose-Derived Stem Cells in
Combination with Trehalose and Reversible Electroporation. J.
Membr. Biol. 2017, 250 (1), 1−9.
(106) Eroglu, A.; Toner, M.; Toth, T. L. Beneficial effect of
microinjected trehalose on the cryosurvival of human oocytes. Fertility
and Sterility 2002, 77 (1), 152−158.
(107) Eroglu, A.; Elliott, G.; Wright, D. L.; Toner, M.; Toth, T. L.
Progressive elimination of microinjected trehalose during mouse
embryonic development. Reproductive BioMedicine Online 2005, 10
(4), 503−510.
(108) Bhowmick, P.; Eroglu, A.; Wright, D. L.; Toner, M.; Toth, T.
L. Osmometric behavior of mouse oocytes in the presence of different
intracellular sugars. Cryobiology 2002, 45 (2), 183−187.
1203
pubs.acs.org/journal/abseba Review
(109) Eroglu, A.; Lawitts, J. A.; Toner, M.; Toth, T. L. Quantitative
microinjection of trehalose into mouse oocytes and zygotes, and its
effect on development. Cryobiology 2003, 46 (2), 121−34.
(110) Uchida, T.; Furukawa, M.; Kikawada, T.; Yamazaki, K.;
Gohara, K. Intracellular trehalose via transporter TRET1 as a method
to cryoprotect CHO-K1 cells. Cryobiology 2017, 77, 50−57.
(111) Sharp, D. M. C.; Picken, A.; Morris, T. J.; Hewitt, C. J.;
Coopman, K.; Slater, N. K. H. Amphipathic polymer-mediated uptake
of trehalose for dimethyl sulfoxide-free human cell cryopreservation.
Cryobiology 2013, 67 (3), 305−311.
(112) Mercado, S.A.; Slater, N. K. H. Increased cryosurvival of
osteosarcoma cells using an amphipathic pH-responsive polymer for
trehalose uptake. Cryobiology 2016, 73, 175.
(113) Holovati, J. L.; Gyongyossy-Issa, M. I. C.; Acker, J. P. Effects
of trehalose-loaded liposomes on red blood cell response to freezing
and post-thaw membrane quality. Cryobiology 2009, 58 (1), 75−83.
(114) Bonazzi, M.; Cossart, P. Bacterial entry into cells: A role for
the endocytic machinery. FEBS Lett. 2006, 580 (12), 2962−2967.
(115) Stefanic, M.; Ward, K.; Tawfik, H.; Seemann, R.; Baulin, V.;
Guo, Y.; Fleury, J. B.; Drouet, C. Apatite nanoparticles strongly
improve red blood cell cryopreservation by mediating trehalose
delivery via enhanced membrane permeation. Biomaterials 2017, 140,
138−149.
(116) Choimet, M.; Hyoung-Mi, K.; Jae-Min, O.; Tourrette, A.;
Drouet, C. Nanomedicine: Interaction of biomimetic apatite colloidal
nanoparticles with human blood components. Colloids Surf., B 2016,
145, 87−94.
(117) Han, Y.; Wang, X.; Dai, H.; Li, S. Nanosize and Surface
Charge Effects of Hydroxyapatite Nanoparticles on Red Blood Cell
Suspensions. ACS Appl. Mater. Interfaces 2012, 4 (9), 4616−4622.
(118) Rauch, C.; Pluen, A.; Foster, N.; Loughna, P.; Mobasheri, A.;
Lagadic-Gossmann, D.; Counillon, L. On Some Aspects of the
Thermodynamic of Membrane Recycling Mediated by Fluid Phase
Endocytosis: Evaluation of Published Data and Perspectives. Cell
Biochem. Biophys. 2010, 56 (2), 73−90.
(119) Jong, K. S.; Hui, Y. L.; Yu, C. M.; Ki, S. Y.; Kim, S. H.; Pak, H.
H. The impact of cryoprotective media on cryopreservation of cells
using loading trehalose. Cryobiology 2020, 92, 258−259.
(120) Abazari, A.; Meimetis, L. G.; Budin, G.; Bale, S. S.; Weissleder,
R.; Toner, M. Engineered Trehalose Permeable to Mammalian Cells.
PLoS One 2015, 10 (6), No. e0130323.
(121) Guo, N.; Puhlev, I.; Brown, D. R.; Mansbridge, J.; Levine, F.
Trehalose expression confers desiccation tolerance on human cells.
Nat. Biotechnol. 2000, 18 (2), 168−71.
(122) Shinde, P.; Khan, N.; Melinkeri, S.; Kale, V.; Limaye, L.
Freezing of dendritic cells with trehalose as an additive in the
conventional freezing medium results in improved recovery after
cryopreservation. Transfusion 2019, 59 (2), 686−696.
(123) Susa, F.; Bucca, G.; Limongi, T.; Cauda, V.; Pisano, R.
Enhancing the preservation of liposomes: The role of cryoprotectants,
lipid formulations and freezing approaches. Cryobiology 2021, 98, 46−
56.
(124) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Rudolph, A. S.;
Wistrom, C. A.; Spargo, B. J.; Anchordoguy, T. J. Interactions of
sugars with membranes. Biochim. Biophys. Acta 1988, 947 (2), 367−
84.
(125) Cai, L.; Nian, L.; Cao, A.; Zhang, Y.; Li, X. Effect of
Carboxymethyl Chitosan Magnetic Nanoparticles Plus Herring
Antifreeze Protein on Conformation and Oxidation of Myofibrillar
Protein From Red Sea Bream (Pagrosomus major) After Freeze-Thaw
Treatment. Food Bioprocess Technol. 2020, 13, 355.
(126) Shen, L.; Guo, X.; Ouyang, X.; Huang, Y.; Gao, D.; Zhao, G.
Fine-tuned dehydration by trehalose enables the cryopreservation of
RBCs with unusually low concentrations of glycerol. J. Mater. Chem. B
2021, 9 (2), 295−306.
(127) Kamalifar, S.; Azarpira, N.; Sadeghi, L.; Ghorbani-Dalini, S.;
Nekoei, S. M.; Aghdaie, M. H.; Esfandiari, E.; Azarpira, M. R. ROCK
Y-27632 Inhibitor, Ascorbic Acid, and Trehalose Increase Survival of
https://doi.org/10.1021/acsbiomaterials.2c01225
ACS Biomater. Sci. Eng. 2023, 9, 1190−1204
ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Review

Human Wharton Jelly Mesenchymal Stem Cells After Cryopreserva- (145) Campbell, L. H.; Brockbank, K. G. M. Culturing with
tion. Exp Clin Transplant 2020, 18 (4), 505−511. trehalose produces viable endothelial cells after cryopreservation.
(128) Stokich, B.; Osgood, Q.; Grimm, D.; Moorthy, S.; Cryobiology 2012, 64 (3), 240−244.
Chakraborty, N.; Menze, M. A. Cryopreservation of hepatocyte
(HepG2) cell monolayers: Impact of trehalose. Cryobiology 2014, 69
(2), 281−290.
(129) Kang, X. L.; Shen, H. Pigmentation of skin graft is improved
by cryopreservation of human skin with trehalose. J. Oral Maxillofac
Surg 2012, 70 (6), 1464−72.
(130) Zhang, T.-Y.; Tan, P.-C.; Xie, Y.; Zhang, X.-J.; Zhang, P.-Q.;
Gao, Y.-M.; Zhou, S.-B.; Li, Q.-F. The combination of trehalose and
glycerol: an effective and non-toxic recipe for cryopreservation of
human adipose-derived stem cells. Stem Cell Res. Ther. 2020, 11 (1),
460.
(131) Dou, M.; Lu, C.; Sun, Z.; Rao, W. Natural cryoprotectants
combinations of l-proline and trehalose for red blood cells
cryopreservation. Cryobiology 2019, 91, 23−29.
(132) Liu, B.; Zhang, L.; Zhang, Q.; Gao, S.; Zhao, Y.; Ren, L.; Shi,
W.; Yuan, X. Membrane Stabilization of Poly(ethylene glycol)-b-
polypeptide-g-trehalose Assists Cryopreservation of Red Blood Cells.
ACS Appl. Bio Mater. 2020, 3 (5), 3294−3303.
(133) Murray, A.; Congdon, T. R.; Tomás, R. M. F.; Kilbride, P.;
Gibson, M. I. Red Blood Cell Cryopreservation with Minimal Post-
Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotect-
ing Polyampholyte with DMSO/Trehalose. Biomacromolecules 2022,
23, 467.
(134) Matsumura, K.; Hayashi, F.; Nagashima, T.; Rajan, R.; Hyon,
S.-H. Molecular mechanisms of cell cryopreservation with poly-
ampholytes studied by solid-state NMR. Commun. Mater. 2021, 2 (1),
15.
(135) Alotaibi, N. A. S.; Slater, N. K. H.; Rahmoune, H. Salidroside
as a Novel Protective Agent to Improve Red Blood Cell
Cryopreservation. PLoS One 2016, 11 (9), No. e0162748.
(136) Liu, X.; Xu, Y.; Liu, F.; Pan, Y.; Miao, L.; Zhu, Q.; Tan, S. The
feasibility of antioxidants avoiding oxidative damages from reactive
oxygen species in cryopreservation. Front. Chem. 2021, 9, 64. Recommended by ACS
(137) Crowe, J. H.; Crowe, L. M. Preservation of mammalian cells -
Learning nature’s tricks. Nat. Biotechnol. 2000, 18 (2), 145−146. Human Natural Killer Cells Cryopreserved without DMSO
(138) Zambelli, A.; Poggi, G.; Da Prada, G.; Pedrazzoli, P.; Cuomo, Sustain Robust Effector Responses
A.; Miotti, D.; Perotti, C.; Preti, P.; Robustelli della Cuna, G. Clinical
Soumyajit Das, Sandro Matosevic, et al.
toxicity of cryopreserved circulating progenitor cells infusion.
JANUARY 17, 2024
Anticancer Res. 1998, 18 (6B), 4705.
MOLECULAR PHARMACEUTICS READ
(139) Ferrucci, P. F.; Martinoni, A.; Cocorocchio, E.; Civelli, M.;
Cinieri, S.; Cardinale, D.; Peccatori, F. A.; Lamantia, G.; Agazzi, A.;
Corsini, C.; Tealdo, F.; Fiorentini, C.; Cipolla, C. M.; Martinelli, G. Physicochemical Mechanisms of Protection Offered by
Evaluation of acute toxicities associated with autologous peripheral Agarose Encapsulation during Cryopreservation of
blood progenitor cell reinfusion in patients undergoing high-dose Mammalian Cells in the Absence of Membrane-Penetrati...
chemotherapy. Bone Marrow Transplant 2000, 25 (2), 173−7. Mian Wang, Alptekin Aksan, et al.
(140) Chen-Plotkin, A. S.; Vossel, K. A.; Samuels, M. A.; Chen, M. MAY 22, 2023
H. Encephalopathy, stroke, and myocardial infarction with DMSO use ACS APPLIED BIO MATERIALS READ
in stem cell transplantation. Neurology 2007, 68 (11), 859−61.
(141) Antonenas, V.; Shaw, P. J.; Bradstock, K. F. Infusion of Engineered Test Tissues: A Model for Quantifying the Effects
unwashed umbilical cord blood stem cells after thawing for allogeneic of Cryopreservation Parameters
transplantation. Bone Marrow Transplant 2004, 34 (8), 739.
Jonian Grosha, Marsha W. Rolle, et al.
(142) Perotti, C. G.; Fante, C. D.; Viarengo, G.; Papa, P.; Rocchi, L.;
OCTOBER 06, 2023
Bergamaschi, P.; Bellotti, L.; Marchesi, A.; Salvaneschi, L. A new ACS BIOMATERIALS SCIENCE & ENGINEERING READ
automated cell washer device for thawed cord blood units. Transfusion
2004, 44 (6), 900−906.
(143) Besterman, J. M.; Airhart, J. A.; Woodworth, R. C.; Low, R. B. Investigation of Rapid Rewarming Chips for
Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for Cryopreservation by Joule Heating
rapid turnover and compartmentation. J. Cell Biol. 1981, 91 (3), 716− Hengxin Han, Yi Xu, et al.
727. JULY 27, 2023
(144) Kikawada, T.; Saito, A.; Kanamori, Y.; Nakahara, Y.; Iwata, K.- LANGMUIR READ
i.; Tanaka, D.; Watanabe, M.; Okuda, T. Trehalose transporter 1, a
facilitated and high-capacity trehalose transporter, allows exogenous
Get More Suggestions >
trehalose uptake into cells. Proc. Natl. Acad. Sci. U. S. A. 2007, 104
(28), 11585.

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