
==== Front
RSC Med Chem
RSC Med Chem
MD
RMCSCX
RSC Medicinal Chemistry
2632-8682
RSC

d4md00174e
10.1039/d4md00174e
Chemistry
Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities
Murray Alex ab
Kilbride Peter c
https://orcid.org/0000-0002-8297-1278
A-7348-2010
Gibson Matthew I. abcde
a Department of Chemistry, University of Warwick CV4 7AL UK
b Division of Biomedical Sciences, Warwick Medical School, University of Warwick CV4 7AL UK
c Asymptote, Cytiva Chivers Way Cambridge CB24 9BZ USA
d Department of Chemistry, University of Manchester Oxford Road Manchester M13 9PL UK Matt.gibson@manchester.ac.uk

e Manchester Institute of Biotechnology, University of Manchester 131 Princess Street Manchester M1 7DN UK
19 7 2024
19 9 2024
19 7 2024
15 9 29802995
14 3 2024
8 7 2024
This journal is © The Royal Society of Chemistry
2024
RSC
https://creativecommons.org/licenses/by/3.0/ This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.
Cryopreservation is crucial to fields including immune and stem cell therapies, reproductive technology, blood banking, regenerative medicine and across all biotechnology. During cryopreservation, cryoprotectants are essential to protect cells from the damage caused by exposure to freezing temperatures. The most common penetrating cryoprotectants, such as DMSO and glycerol do not give full recovery and have a cytotoxicity limit on the concentration which can be applied. The non-reducing disaccharide trehalose has been widely explored and used to supplement these, inspired by its use in nature to aid survival at extreme temperatures and/or desiccation. However, trehalose has challenges to its use, particular its low membrane permeability, and how its protective role compares to other sugars. Here we review the application of trehalose and its reported benefit and seek to show where chemical tools can improve its function. In particular, we highlight emerging chemical methods to deliver (as cargo, or via selective permeation) into the intracellular space. This includes encapsulation, cell penetrating peptides or (selective) modification of hydroxyls on trehalose.

The state of the art, challenges and opportunities for chemists to engineer and deliver trehalose as a cryoprotectant to address challenges in biomedicine and biotechnology are reviewed.

Biotechnology and Biological Sciences Research Council 10.13039/501100000268 BB/M01116X/1 Royal Society 10.13039/501100000288 191037 H2020 European Research Council 10.13039/100010663 866056 pubstatusPaginated Article
==== Body
pmcIntroduction

Cryopreservation is the practice of storing biological materials at sub-zero temperatures to halt metabolism and degradation. Cryopreservation is deployed for a vast range of biological materials from proteins,1 cells,2 tissues,3,4 mRNA vaccines,5 and potentially organs.6,7 Cryopreservation is essential in cell culture as it allows for the routine storage of cell lines, reducing the need for continuous culture, which would otherwise lead to phenotype drift as well as being practically challenging. It also has medical applications such as transporting stem cells,8 and blood for transfusion.9 Current cryopreservation techniques, whilst successful, are not perfect: a proportion of cells always die during the process and their functionality is often reduced. One of the major limiting factors in cryopreservation is cryoprotectant toxicity; cryopreservation requires the use of cryoprotectants (including but not limited to solvents) such as DMSO or glycerol which are penetrating cryoprotectants. These can have adverse effects when the thawed cells are transplanted into patients.10 Cryoprotectant toxicity also limits the concentration of cryoprotectant that can be used. The mechanism of cryoprotectant action is concentration-dependent but there becomes a point where further increases in concentration lead to toxicity that outweighs any protective effects. This has motivated research into non-/lower toxicity cryoprotectants.

Trehalose (α-d-glucopyranosyl-(1 → 1)-α-d-glucopyranoside) is a water-soluble non-reducing disaccharide made up of two glucose subunits, joined by a 1,1-glycosidic bond.11 It has 8 hydrogen bond donors and 11 hydrogen bond acceptors. Trehalose is biosynthesised by a wide variety of non-mammalian organisms as an energy source and is used in some organisms to protect against freezing and desiccation, allowing them to survive over winter or in other harsh environments. These organisms include the rice water weevil Lissorhoptrus oryzophilus,12 the codling moth Cydia pomonella13 and some tardigrade species such as Macrobiotus richtersi.14 In the past, trehalose was expensive to make, but the development of an efficient manufacturing process in 1994 made this much cheaper, leading to increased research interest.15 Trehalose has two proposed protective mechanisms, which have been termed the ‘vitrification hypothesis’ and the ‘water replacement hypothesis’. These mechanisms are usually discussed in the context of preservation by desiccation or anhydrobiosis in nature but are also applicable to cryopreservation. It is crucial to note that trehalose (and other disaccharides) are non-penetrating cryoprotectants in contrast to the widely used DMSO or glycerol. Fig. 1 summarises the two key methods in cryopreservation relating to vitrification and slow-freezing, which necessitate different concentrations of cryoprotectant and freezing rates, and where problems can occur. Fig. 2 shows the chemical structure of trehalose compared to sucrose.

Fig. 1 Schematic of the vitrification verses slow freezing pathways, indicating where failure can occur.

Fig. 2 Chemical structure of trehalose and sucrose.

The vitrification hypothesis

The vitrification hypothesis holds that trehalose protects biological materials by forming a high-viscosity glass-like state. The transition of a liquid trehalose solution to a glassy state is achieved by concentrating the trehalose through either desiccation or freeze-induced dehydration. During cryopreservation by freezing, the cells survive within the vitrified fraction of the sample.16 Trehalose has several properties that contribute to the formation of this fraction: adding trehalose to a solution increases its glass transition temperature (Tg),17 resulting in vitrification at a higher temperature. Trehalose is also a kosmotrope,18 it orders water molecules around itself, altering the structure of the surrounding hydrogen bond network in such a way that it cannot form ice.19 This network is over three hydration shells wide,20 so each molecule of trehalose can prevent many molecules of water from freezing.21 Trehalose has been found to reduce the size of ice crystals in a concentration-dependent manner.22

The water replacement hypothesis

Under normal conditions, proteins and other cell components are stabilised/hydrated by bound water. The water replacement hypothesis holds that trehalose stabilises these components once the water content is sufficiently low by acting as a replacement for this water, Fig. 3.23,24 During cryopreservation, trehalose protects proteins from cold denaturation,25 and cryoprotectant toxicity.26 Trehalose also lowers the temperature at which the membrane gel-to-liquid crystal phase transition occurs.27,28 During desiccation, trehalose stabilises the cell membrane by hydrogen bonding to phospholipids,29 so trehalose may play a similar role in stabilising the cell membrane during cryopreservation. In addition to stabilisation, trehalose likely also counteracts solute concentration damage when it is used at sufficient concentrations, as would any biocompatible, water-soluble, small molecule.

Fig. 3 Vitrification compared to water replacement for protein storage using trehalose. The bottom panels show ice formation and protein denaturation in the absence of trehalose.

Trehalose is a polar molecule (log P ∼ −3.7), which alongside its large number of hydrogen bond donors/acceptors, means it does not cross the cell plasma membrane and hence cannot provide intracellular cryoprotection. Thus, it is usually used as an extracellular cryoprotectant, but recent advances now allow its intracellular delivery. This review will firstly discuss trehalose as a cryoprotectant for different cell types and provide a critical comparison to other sugars, highlighting cases where trehalose does not offer additional benefit. Secondly, methods to deliver trehalose into the intracellular space and the impact on cryopreservation will be explored.

Trehalose as a (primarily) extracellular cryoprotectant

The need for improved cryoprotectants has resulted in research into sugars as a supplement or replacement for traditional penetrating cryoprotectants such as DMSO, ethylene glycol, propylene glycol and glycerol. Trehalose has gained particular attention due to its unique properties among the sugars, which will be discussed in more detail later.

Trehalose has been tested as a cryoprotectant in an extensive range of cell types and other biological materials. In most studies, trehalose is supplemented into the standard penetrating cryoprotectant for that cell type – typically 10% DMSO. It is observed that this increases cell recovery and/or improves post-thaw proliferation and other functional outcomes. A recurring trend throughout these studies is that trehalose has an optimal concentration – usually between 100 mM to 400 mM – beyond which adding further trehalose becomes detrimental to post-thaw outcome.30–32 The detrimental effect of higher concentrations is likely due to osmotic pressure rather than through any specific trehalose toxicity. Importantly, trehalose can often lower the amount of penetrating cryoprotectant required for cryopreservation while providing an equivalent or better post-thaw outcome.

Trehalose is not cell penetrative and cannot normally cross the cell membranes and enter the intracellular space unaided, but occasionally, extracellular trehalose can be used without solvent cryoprotectants,33–35 suggesting the presence of intracellular trehalose, which can enter the cell through endocytosis or a membrane phase transition during cooling. Whilst this section is about the extracellular use of trehalose, small amounts of trehalose are likely to be present intracellularly in many of these studies. These concepts are discussed in later in this review. Fig. 4 shows an overview of this, and Fig. 5 and 6 demonstrate the individual mechanisms. A summary of trehalose supplementation to extracellular media and its impact is made below spanning a range of cryopreservation scenarios to allow the reader to compare and contrast the benefits.

Fig. 4 Schematic of localisation of trehalose compared to DMSO during cryopreservation (green hexagons = trehalose, grey dots = DMSO).

Fig. 5 Possible mechanisms by which trehalose can enter cells.

Fig. 6 Engineered and formulated trehalose increases passive uptake by membrane diffusion followed by esterase action.

Stem cell preservation

For murine (mouse) spermatogonial stem cells, adding 50 mM trehalose to the standard 10% DMSO freezing media improved viability after storage for one week (90% vs. 76%).36 Adding 200 mM trehalose did not improve viability, but did improve long-term proliferation (49% vs. 28%). For murine microencapsulated mesenchymal stem cells, 5% DMSO, 10%, glycerol, 10%, 5% trehalose, or any combination of trehalose and less than 10% DMSO, all resulted in lower recovery than 10% DMSO.37 For mesenchymal stem cells DMSO can be partially replaced with trehalose or poly(ethylene glycol) (PEG), although trehalose was not more effective than PEG, which might suggest that lower DMSO was tolerated in this model.38

Human pluripotent stem cells: cryopreservation can be improved by replacing the standard 10% DMSO with 500 mM trehalose and 10% glycerol. Relative viability was increased by approximately 20–30% depending on the exact cell type. Phenotype and functionality were maintained.39 This raises a particular point that DMSO-free cryopreservation is possible, but in many cases achieved by adding a different penetrating solvent in its place.

Human umbilical cord blood stem cells: in Chen et al.30 2.5% DMSO and 30 mM trehalose was at least as good as 10% EG and 2% DMSO, or 10% DMSO and 2.0% dextran-40 at maintaining viability. Trehalose also slightly reduced the amount of post-thaw apoptosis. In Zhang et al.40 freezing mediums containing 146 mM trehalose + 10% or 5% DMSO resulted in proliferation that was as high as the non-frozen control group. Trehalose has also been successfully used in combination with taurine and catalase for cord blood stem cell preservation.41

Human peripheral blood stem cells: in Martinetti et al.35 the use of 1 M trehalose alone gave higher long- and short-term viability than DMSO alone, or DMSO combined with trehalose. The 1 M trehalose concentration used here is much higher than the typical optimal concentration of 100–400 mM in most cell types; it is possible that the slow addition of freezing media to the cells in this study allowed a higher concentration of trehalose to be reached without causing too much osmotic stress. For marrow and blood stem cells Scheinkönig et al.42 found that 500 mM trehalose provides a similar level of cryoprotection to DMSO.

Human adipose-derived stem cells: cryopreservation with 250 mM trehalose alone resulted in very poor viability compared to 5% DMSO (11% vs. 75%).43 In Pu et al.32 200 mM trehalose allowed the reduction of DMSO to 3.3% while maintaining good cryoprotection.

Spermatozoa

Spermatozoa are distinct from other cell types in that they have a much larger surface area to volume ratio, have a more densely packed cytoplasm, and contain less freezable water, which makes them less vulnerable to intracellular ice formation.44 Thus, optimal spermatozoa cryopreservation differs from that of more typical cells, with protocols often preferring fast freezing or vitrification to slow freezing.45 Trehalose has been added to the “freezing extender” (a term referring to a freezing media used for spermatozoa) and tested in the cryopreservation of spermatozoa from many different animals.

Rabbit: spermatozoa vitrification with 4% DMSO was improved with the addition of 100 mM trehalose as measured by increased motility (43% vs. 25%), membrane integrity (45% vs. 24%) and mitochondrial membrane integrity (53% vs. 28%). Trehalose also decreased ROS and improved catalase function.46

Ram: post-thaw outcome was improved in multiple studies, with the optimal trehalose concertation typically being 50–100 mM.47–53 Some studies found that higher concentrations of around 200 mM are ineffective,54,55 while others have found positive effects at higher concentrations.56,57 These differences may be due to the total osmolality of the solution; in an extender with a high initial osmolality, less trehalose can be added before the total osmolality becomes too high and dehydrates the cells. Trehalose has also been combined with low-density lipoprotein for use as an extender.58

Equine: trehalose has had mixed results in equine spermatozoa, one study reported a benefit to using a small amount of trehalose.59 Other studies reported that trehalose does not improve post-thaw outcome.60–62

Other animals: trehalose also improved cryopreservation outcomes for the spermatozoa of goats,63,64 carp,65 and oysters.66 Trehalose did not provide benefit for the cryopreservation of European brown hare spermatozoa.67

Tissue cryopreservation

Tissue cryopreservation is significantly more challenging than for individual cells (or e.g. spheroids) for several reasons. The large volume to surface area to ratio makes it difficult for a cryoprotectant to homogeneously permeate the tissue, and to ensure a uniform cooling rate. Extracellular ice formation is particularly damaging to the tissue macrostructure – unlike individual cells which can move freely within the media, allowing them to fit within the unfrozen portion of the sample. Several studies have found a cryoprotective benefit to using trehalose: in one study, the viability of adipose tissue was increased by 80% through the addition of 250 mM trehalose to an unspecified standard freezing media.68 In another study, the addition of 500 mM trehalose to 10% DMSO increased the post-thaw live cell count of human foetal skin (65% vs. 44%), improved post-thaw morphology, graft necrosis, and shrinking.69 Trehalose can also reduce post-thaw tissue pigmentation.70 Dog tracheas have been preserved for transplant in 10% DMSO and 100 mM trehalose, resulting in a 100% animal and graft survival rate (n = 6) after 184 days when transplanted. This study did not report cell recovery or viability.71 The viability of bovine calf testicular tissue, cryopreserved with 10% DMSO, was higher with increasing trehalose concentrations of up to 438 mM.72

Other cell types

Human: viability of hepatocytes was improved (63% vs. 47%) with the addition of 200 mM trehalose to 10% DMSO.73 Trehalose and salidroside increased the survival of red blood cells (RBCs) after cryopreservation. However, adding salidroside to glycerol is slightly more effective than replacing glycerol with trehalose 61% vs. 56% respectively.73 Trehalose and catalase protect the proteins on the surface of human hematopoietic cells.74

Non-human: in murine embryos, 1.5 M glycerol and 100 mM trehalose was the optimal cryoprotectant combination for freezing. Interestingly at 2.0 M glycerol, increasing trehalose concentration increases viability in a dose-dependent manner, even beyond the optimal trehalose concentration at optimal and suboptimal glycerol concentrations. This suggests that glycerol prevents damage by preventing dehydration damage, and/or trehalose protects cells from glycerol toxicity.75 For Trypanosoma brucei, studies have found that 400 mM trehalose in host blood, or 200 mM trehalose in vitro, without DMSO or glycerol, is optimal for cryopreservation. In these studies, the replacement of 7.5% DMSO with trehalose alone resulted in drastic increases in survival rate, depending on the form of the trypanosome.76,77 In porcine spermatogonia stem cells, 200 mM trehalose alone, compared to 10% DMSO, did not significantly increase the recovery of testis cells, but did increase proliferation capacity and the recovery of germ cells from thawed testis cells and tissue.78 In ecto-mycorrhizal basidiomycetes, trehalose addition to DMSO improves post-thaw viability of most sub-types.79 Finally, trehalose has been used to cryopreserve multiple cell types by pre-freeze dehydration.80

Comparison with other sugars

The key question raised from the above is to what extent is trehalose unique among the sugars? Has trehalose proven beneficial as a cryoprotectant only to the extent that any sugar can act as a cryoprotectant? A particular advantage that trehalose has, compared to other disaccharides is that it is non-reducing and does not have a hemiacetal/aldehyde equilibrium, hence it cannot undergo associated side reactions with e.g. protein side chains. Furthermore, the glycosidic bond of trehalose is more stable than that of sucrose and is less susceptible to hydrolysis.81 Disaccharides in general also appear to be superior cryoprotectants than monosaccharides.82,83 Although, this is not always the case: in some studies, the monosaccharides galactose84 and inositol85 have been more effective than disaccharides. One explanation for the relative success of disaccharides could be that, at any given molarity, disaccharides have more polar groups while exerting the same osmotic pressure. Thus one important consideration when comparing monosaccharides with disaccharides is that the experimental concentrations of sugars in cryoprotectant solutions are usually measured in terms of molarity, rather than % weight; comparing 100 mM monosaccharide with 100 mM disaccharide can be misleading because the former tend to be approximately half the molecular weight of the latter, and critically – contain just over half as many polar groups per mol, resulting in fewer hydrogen bonds with water.86 Trehalose is most often compared to sucrose – another non-reducing disaccharide. The two have a similar structure; trehalose being made up of two glucose subunits and sucrose of one glucose and one fructose subunit. They have equal molecular weights and an equal number of hydrogen bond donors and acceptors.

However, there are a number of ways in which trehalose appears to be a better cryoprotectant than sucrose: in solution, trehalose forms stronger hydrogen bonds with water than sucrose,87 and becomes associated with more molecules of unfrozen water, in this way each molecule of trehalose is able to disrupt the hydrogen bonding network of a greater number of water molecules, preventing them from forming ice.88,89 Trehalose has superior ice growth inhibiting properties compared with sucrose.90 Unlike sucrose, trehalose is able to displace water bound to the carbonyl groups of phospholipid membranes, displacing more water.91 Trehalose has a larger hydrated volume than sucrose, which is correlated with protein protection, and more sucrose is needed to provide the same level of protein protection as trehalose.21,92 Trehalose and sucrose cause a dynamic slowing effect on the surrounding water network, this effect is stronger in trehalose.93 Some of these effects may occur because sucrose has more intramolecular hydrogen bonding, resulting in fewer and/or weaker intermolecular hydrogen bonds to interact with water.88 Trehalose solutions have a higher glass transition temperature than sucrose solutions,94,95 however, a lower concentration of sucrose is needed to vitrify in aqueous solution at any given cooling rate.96 During freeze-drying where water content is very low and samples are stored at room temperature, trehalose would be the superior stabiliser as it would be more likely to remain above its glass transition temperature, even if it absorbed a small amount of water. Conversely, during cryopreservation, sucrose may be more useful (at least with regard to its vitrifying properties) in both vitrification, and in slow freezing where vitrification occurs due to freeze concentration. Sucrose also has greater solubility in water,97 making it less likely to precipitate out of solution at high concentrations and low temperatures. In some organisms that use trehalose as a cryoprotectant, antifreeze proteins may be used to mitigate this solubility problem.98 A summary of the comparative advantages of trehalose and sucrose can be seen in Table 1. While trehalose is – in theory – a better cryoprotectant than other sugars, the results of experimental cryopreservation studies where trehalose has been directly compared to other sugars are mixed. Discussed below are cryopreservation studies where trehalose has been tested and found to be a better, worse, or equivalent cryoprotectant compared to other sugars.

Comparison of the advantages of trehalose versus sucrose for cryopreservation

Advantages of trehalose	Advantages of sucrose	
Higher Tg at any given concentration (106 °C vs. 60 °C for the pure sugars)95	Lower Cv at any given volume (74% vs. 58% at 20 μl)96	
Higher hydration number (4.598 vs. 4.127 at 66% w/v)88	Greater solubility (68.1% vs. 52.3% at 30 °C)97	
Larger hydrated volume (62.5% vs. 87% water for a 1.5 M solution)21	Low concentration required to vitrify at a given cooling rate96	
Superior ice growth inhibiter (“approximately twice as effective”)90	—	
Displace water bound to the carbonyl groups of phospholipid membranes91	—	
Causes more dynamic slowing of the surrounding water network93	—	
More effective for protein stabilisation92	—	

Studies where trehalose outperforms other sugars

Cryopreservation of human embryonic stem cells with 10% DMSO + 200 mM trehalose, and adding trehalose to the recovery media, resulted in a greater number of undifferentiated cells surviving compared to DMSO alone. No improvement was seen with sucrose.99 In human amniotic fluid stem cells, trehalose was slightly better than sucrose in the absence of solvent, although both were inferior to standard cryoprotectants.100 In bovine spermatogonial stem cells, 20% DMSO + 200 mM trehalose improved stem cell recovery relative to DMSO alone, DMSO + sucrose, or to DMSO + PEG (19% vs. 7% vs. 5% respectively). The cells preserved with trehalose also showed the lowest post-thaw apoptosis.101 Trehalose was also better than sucrose for mouse oocyte vitrification,102 and improved post-thaw outcome relative to sucrose in human spermatozoa.103 In ram spermatozoa, trehalose was superior than sucrose and slightly better than raffinose at 100 mM.104 The presence of trehalose or sucrose in a cryoprotectant solution reduces solvent cryoprotectant toxicity to RBCs, but this effect is higher for trehalose.26 In mouse neuroblastoma cells, trehalose gave higher post-thaw survival than sucrose (35% vs. 20%). Cells frozen in trehalose benefit from trehalose preincubation, whereas cells frozen in sucrose do not benefit from preincubation with sucrose. The most effective cryoprotectant combination was trehalose + l-proline in a 1 : 1 ratio, at a total concentration 112.5 mM. This gave a post-thaw survival of 51%.105 Inositol is a non-reducing monosaccharide that can confer freeze tolerance to cricket cells, but not to crickets, while trehalose can confer freeze tolerance to both cricket cells and to whole crickets.106 Inositol is inferior to trehalose for protection during lyophilisation.107

Studies where other sugars outperform trehalose

Multiple sugars (trehalose, sucrose, sorbitol, glucose and mannitol) were tested for the cryopreservation of Catharanthus roseus cells, it was found that trehalose resulted in the lowest survival rate and sorbitol gave the highest.108 Sucrose was marginally better than trehalose for post-thaw survival in saltwater crocodile spermatozoa preservation.109 Mentioned above, Chaytor et al.84 measured the IRI activity of six different sugars (galactose, glucose, melibiose, lactose, trehalose, sucrose). They found that 220 mM lactose has the best IRI activity. Viability in a 200 mM solution of these sugars was then measured in human hepatocytes, both after incubation at 37 °C, and after freeze–thaw. Galactose and lactose gave the best viability after incubation at 37 °C. Galactose gave the best post-thaw viability, while glucose gave the worst. Trehalose was not better than sucrose. While this study does show that the selection of sugar can affect post-thaw outcome, the differences in post-thaw survival were not large (with the exception of glucose, which was by far the worst cryoprotectant here). Another study assessed the effect of multiple sugars (lactose, galactose, glucose, fructose, lactulose, melibiose, trehalose, sucrose) on post-thaw boar sperm quality. It was found that disaccharides were better than monosaccharides, and those disaccharides that contain glucose were better than does which did not. Trehalose increased membrane permeability in this study, resulting in worse post-thaw outcome than other sugars.84

Studies where trehalose and other sugars were equivalent

Rat hepatocyte viability after cryopreservation was slightly higher (80% vs. 75%) with 200 mM trehalose in the absence of DMSO, than with 5% DMSO. The addition of trehalose or sucrose to 5% DMSO did not improve viability, and the addition of glucose decreased viability.33 In mouse spermatogonial stem cells, trehalose did not significantly increase recovery, but almost doubled post-thaw proliferation. Trehalose was more effective than sucrose and maltose in this respect. Lactose was as effective as trehalose. Fructose, galactose, xylose, and raffinose did not increase proliferation compared to the control. Glucose and mannose did increase proliferation, but not as much as the disaccharides.83 In Rodrigues et al.31 post-thaw recovery with 5% DMSO + 146 mM trehalose was 85%, compared to 79% recovery with 10% DMSO for the preservation of human cord blood hematopoietic stem cells. Although there was no difference between using trehalose and sucrose in this study. Nynca et al.110 reports that sperm cryopreservation can be improved with glucose, sucrose, or trehalose, but trehalose was not more effective than other sugars for most Salmonidae species. The addition of 250 mM trehalose or 321 mM lactose to boar sperm freezing extender had no beneficial effects.111 In Woelders et al.112 bull sperm, isotonic trehalose and sucrose were equally beneficial at the optimal fast cooling rate, and in Chen et al.113 trehalose and sucrose provided equal benefit to bull spermatozoa motility. For the vitrification of human spermatozoa, trehalose and sucrose were equally effective at preserving motility, and were both slightly better than 3-O-methyl-d-glucopyranose, raffinose and stachyose.114 In dog spermatozoa vitrification, trehalose was not more effective than sucrose.115

Trehalose as an intracellular cryoprotectant

As discussed above in the context of cryopreservation performance, trehalose is most effective when it is in both the intracellular and extracellular space.116–118 In the intracellular space, it is able to inhibit intracellular ice, prevent cellular dehydration caused by both extracellular ice and extracellular cryoprotectants, and stabilise intracellular proteins. However, trehalose is a polar molecule, and does not cross the cell membrane by diffusion, nor are there any trehalose-specific transporters in most mammalian cells (e.g. a non-penetrating cryoprotectant). Various methods have been used to load trehalose into the intracellular space of cells which do not naturally produce it namely; electroporation, membrane permeabilising polymers, genetic engineering, microinjection, nanoparticles, liposomes, freeze-induced uptake, molecular engineering, and natural endocytosis through preincubation (Fig. 5). For an in-depth review of the topic, see Stewart and He.119 The ideal method for trehalose permeation would have low cytotoxicity, not be time-consuming or labour-intensive, and would allow for a high enough concertation of trehalose to be loaded that it could entirely replace penetrating cryoprotectants.

Preincubation, fluid-phase endocytosis, and osmotic manipulation

Fluid-phase endocytosis, or “cellular drinking” is where cells internalise the surrounding fluid using vesicles. This allows for the non-specific uptake of molecules from the surrounding environment. In this way, cells can be slowly loaded with trehalose by incubating them in a trehalose solution for a number of hours. Trehalose pre-incubation was first used in carrot and tobacco cells where it allowed for trehalose to be used as the sole cryoprotectant.120,121 This process is slow – cells have to be incubated for up to 24 hours before freezing – and the maximum concentration of trehalose that can be loaded is limited. Preincubation with trehalose has been used to improve the cryosurvival of mesenchymal stem cells,122 and is more effective than simply adding trehalose to the cryopreservation media without preincubation.123 In Stokich et al.124 human hepatocyte cell monolayer recovery was improved by a 24-hour preincubation with 100 mM trehalose in addition to the standard 10% DMSO, resulting in 39% and 10% viability respectively. In Campbell et al.125 preincubation with 200 mM trehalose before preservation with 400 mM trehalose greatly increased metabolic activity measured 7 days after thawing in bovine endothelial cells. In Hara et al.126 1.3 M trehalose in the absence of solvent increased proliferation compared to glycerol for human embryonic kidney cells (36% vs. 10%). For cattle ovarian granulosa, a 30-minute preincubation with 200–400 mM trehalose, before flash freezing with trehalose + 5% DMSO and 5% EG, greatly improved viability (70% vs. 15%).127 It is possible to force trehalose to cross the cell membrane by creating large osmotic pressure differences between the intracellular and extracellular spaces. However, it only allows for small amounts of trehalose to be loaded and it can damage cells. This has been attempted in red blood cells, allowing for up to 43 mM trehalose to be loaded.128,129 This has also been attempted in T-lymphocytes, resulting in 2.2 mM being loaded.130

Freeze-induced uptake

When trehalose is simply added to the extracellular space immediately prior to freezing, it can be mistakenly considered to only be an extracellular cryoprotectant. However, the phase change in the cell membrane during freezing, combined with freezing-induced osmotic effects and membrane damage, allows trehalose and other small molecules to enter the cell. The amount of trehalose loaded by freeze-induced uptake increases with freezing rate, resulting in a faster optimal cooling rate for cryoprotection from freeze-induced uptake than with DMSO.131 Beattie et al.132 was one of the earliest studies using intracellular trehalose to preserve mammalian cells. Here, human pancreatic islets were cryopreserved with 2 M DMSO + 300 mM trehalose, which resulted in improved cell recovery compared to 2 M DMSO + 11.1 mM glucose (94% vs. 58%). Insulin production was also greatly improved. Intracellular trehalose was measured and was found to increase as temperature was lowered. The threshold temperature for freeze-induced uptake appears to be 15 °C, as only very small amounts of intracellular trehalose were detected before this temperature was reached. In mouse neuroblastoma cells, both preincubation and freezing with trehalose contribute to increased cell recovery. Trehalose positively combines with l-proline at equal total concentration, even when l-proline was simply added to the extracellular freezing media, suggesting that l-proline protects cells through a mechanism other than inducing freeze tolerant metabolism.105 In Zhang et al.133 a 500 mM extracellular trehalose concentration was optimal for mouse embryonic fibroblasts, resulting in 50% survival. 160 mM trehalose could be detected inside cells after preservation. Preincubation with 25 mM trehalose had no positive effect. Freeze-induced uptake may also occur in platelets134 and spermatozoa.135

Electroporation

Electroporation is the application of an electric current to cells which causes the formation of pores, resulting in a temporary increase in membrane permeability. In Shirakashi et al.136 mouse myeloma cells were loaded with 100 mM trehalose from an extracellular trehalose concertation of 290 mM. Higher intracellular concentrations were achievable but required damaging voltages to be used. In Dovgan et al.137 human umbilical stem cells were electroporated and incubated with 200 mM trehalose for 25 minutes, which resulted in improved cell recovery relative to 10% DMSO (86% vs. 60%). For human adipose-derived stem cells, electroporation with trehalose increased recovery compared to trehalose incubation, as measured by trypan blue excision but not when measured by MTT assay.138

Polymers

A polymer known as ‘PP-50’ (poly(l-lysine iso-phthalamide) grafted with l-phenylalanine) has been used to increase membrane permeability in a reversible pH-responsive manner.139 It can be used to permeabilise human red blood cells to trehalose resulting in 251 mM trehalose uptake from 700 mM solution after a 9 h incubation period. In ovine red blood cells, PP-50 allowed 123 mM trehalose loading from a 360 mM extracellular solution, with a 9-hour incubation. This resulted in 83% survival between the end of incubation and thawing. However, PP-50 causes some haemolysis, and this was not accounted for in the post-thaw haemolysis lysis assay, as RBCs were washed after incubation, which removed free haemoglobin. Elsewhere in the paper, it was found that 22% haemolysis occurs during loading, resulting in a probable final survival of around 61%. So, while this shows that the level of trehalose loading archivable with PP-50 greatly improves survival, it does not present PP-50 as a good way of achieving it. Nonetheless, PP-50 can be used to increase cryosurvival with trehalose uptake.116 The PP-50 trehalose combination was also used to improve the cryosurvival of osteosarcoma cells, although does not result in better cryosurvival than that with DMSO.140,141

Carrier peptides

A cargo peptide was developed by Wei et al. that non-covalently binds to trehalose and carries it across the cell membrane. The peptide was non-toxic, but only very small amounts of trehalose could be loaded into the cell.142

Gene expression for trehalose synthesis

Trehalose can be synthesised from glucose using trehalose 6-phosphate synthase and trehalose 6-phosphate phosphatase. Cells can be transfected with genes that encode these enzymes, but the intracellular trehalose concentration that can be achieved is low.143,144

Transporter proteins and channels

Most cell types have an endogenous receptor called P2Z (or P2X7) which forms pores when ATP is present extracellularly. In human hematopoietic stem cells, it allowed for 200 mM trehalose to be loaded from 200 mM extracellular trehalose after a 60-minute incubation period, resulting in 91% post-thaw viability as measured by proliferation. This is compared to 11% viability after freezing with 200 mM extracellular trehalose alone.117 Other pores and channels can be added artificially. For example, cells can be genetically engineered to express the trehalose transporter TRET1. In Chinese hamster ovary cells, this allowed 23 mM trehalose uptake after incubation in 400 mM trehalose for 4 hours. This did not affect the cells' proliferation.145 This technique was later used to protect Chinese hamster ovary cells during cryopreservation.146 H5 is a pore-forming agent which has been engineered from α-haemolysin. H5 forms pores in the cell membrane which can be opened and closed by altering zinc ion concentration. This allowed for 80% cryosurvival in murine fibroblasts, 70% cryosurvival for human keratinocytes,147 and 92% post-thaw viability for human hemopoietic stem cells.148 This approach allows for high concentrations of trehalose to be loaded quickly, but the potential of H5 to cause an immune response may limit its use in cells that are to be used clinically.

Microinjection

Microinjection allows for small volumes of liquid to be directly introduced into the intracellular space. It allows very high concentrations of trehalose to be loaded but it poses challenges when used on cells which are smaller than oocytes. In Eroglu et al.149 human oocytes were microinjected with 150 mM trehalose and frozen in 500 mM trehalose, resulting in 66% survival. In a later study, mouse oocytes were injected with 500 mM trehalose and frozen in 500 mM trehalose solution, giving 80% survival. Using 500 mM extracellular trehalose only gave just 15% survival. Reducing intracellular trehalose concentration to 80 mM and adding 500 mM DMSO (4% w/v) resulted in 97% survival. These oocytes were able to develop into embryos at the highest rate. One explanation for the increased cryosurvival in the presence of DMSO, is that DMSO crosses intracellular membranes and enter the organelles.150

Nanoparticles

Nanoparticles can encapsulate trehalose and deliver it into the cell. Thermally responsive nanoparticles can be used to load up to 300 mM trehalose into murine fibroblasts, from a 40 minute incubation followed by cold shock.151 In human adipose-derived stem cells, 24-hour incubation with pH-responsive nanoparticles and 200 mM extracellular trehalose allowed for a post-thaw viability equivalent to that given by 10% DMSO.152 Nanoparticles that increase membrane permeability have been used to load 51 mM trehalose into red blood cells from 350 mM extracellular trehalose, after a 7 hour incubation period, resulting in 91% post-thaw survival.153

Liposomes

Holovati and Acker154 developed liposomes that can be used to deliver trehalose into RBCs. These liposomes were used to carry a small volume of 300 mM trehalose and were incubated with red blood cells for 4 hours (for an estimated total of 15 mM intracellular trehalose), then frozen with 300 mM trehalose. This resulted in 67% post-thaw recovery, compared to 27% recovery when incubated in extracellular trehalose alone. The concentration of intracellular trehalose alone provided minimal cryoprotection, but surprisingly the liposomes themselves provided a very large degree of cryoprotection; cells incubated with liposomes containing no trehalose and frozen in 300 mM extracellular trehalose gave 60% recovery.155 The ability of liposomes to improve cryopreservation could cause misleading results when using them to deliver cryoprotectants. For example, Motta et al.156 used liposomes to assess the effect of intracellular and extracellular trehalose on umbilical cord blood stem cell preservation but did not control for the effects of the liposomes themselves. However, this cryoprotective ability is useful in its own right, and could be used as part of a multi-component cryoprotectant solution to deliver low concentrations of non-penetrating cryoprotectants such as antifreeze proteins. In Stoll et al.157 liposomes allowed for up to 80 mM trehalose to be loaded into human RBCs, dependent on liposome concentration. This slightly increased post-thaw recovery, but post-thaw recovery did not increase with increasing liposome concentration above 1 mM, which was the lowest liposome concentration used.

Ultrasound

In Zhang et al.158 ultrasound was used to induce non-specific permeability in human platelets. This technique allowed only 30 mM of trehalose to enter the cell after 30 minutes of treatment, which is insufficient for cryoprotection. This method also altered platelet morphology.

Modified trehalose

Trehalose itself can be chemically modified so that it can penetrate the cell membrane (Fig. 6). In Abazari et al.159 trehalose was acetylated at six of its hydroxyl groups, greatly increasing its hydrophobicity. Rat hepatocytes were incubated with 30 mM acetylated trehalose, resulting in an intracellular acetylated trehalose concentration of 80 mM within 1 hour, and of up to 300 mM after 8 hours. Trehalose concentration lagged behind this with a concentration of around 100 mM after 12 hours.

Trehalose removal

While trehalose has almost no direct toxicity at concentrations required for cryopreservation, trapped intracellular trehalose can cause osmotic damage when trehalose is removed from the extracellular solution.131 However, after thawing, trehalose will be eliminated from cells due to cell division, and possibly by exocytosis and/or conversion to glucose.160–162 Therefore, to maximise cell survival, the osmolarity of the recovery media can be progressively lowered to maintain osmotic balance, although this strategy is labour-intensive. Alternatively, using a lower intracellular concentration of trehalose in conjunction with a penetrating cryoprotectant can mitigate osmotic damage.150

Conclusions

This review has summarised the broad scope of trehalose in cryopreservation, highlighting specific areas where there were successes and also where the benefits were limited. We have also gathered the literature on solutions to overcome the limitations of trehalose's low membrane permeability, to show where chemistry can be deployed to delivery this inside cells: adapting trehalose to become a penetrating cryoprotectant. Whilst trehalose clearly shows benefits when added to the extracellular media, the delivery question challenge remains open to innovative solutions which may enable the reduction or removal of the need for conventional penetrating cryoprotectants (such as DMSO) whilst ensuring that post-thaw cells are viable and functional. With the rapid growth of cell-based therapies and biotechnology process, the need for tools to bank, transport and deliver cryopreserved cells (and other biological components) has never been higher. Any emerging tools will all need critical evaluation of the price, biocompatibility, and accessibility, compared to e.g. DMSO which is low cost, widely available and provides consistent results.

Data availability

This is a review article and no new data was generated.

Conflicts of interest

There is no conflict of interest to declare.

This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program grant agreement 866056. M. I. G. thanks the Royal Society for an Industry Fellowship (191037) joint with Cytiva. The Biotechnology and Biological Sciences Research Council (BBSRC) and the University of Warwick funded Midlands Integrative Biosciences Training Partnership (MIBTP) [grant number BB/M01116X/1] and Cytiva are thanked for support to A. M. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author-Accepted Manuscript version arising from this submission.
==== Refs
Notes and references

Deng J. Davies D. R. Wisedchaisri G. Wu M. Hol W. G. J. Mehlin C. An Improved Protocol for Rapid Freezing of Protein Samples for Long-Term Storage Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004 60 1 203 204 10.1107/S0907444903024491 14684931
Miyamoto Y. Ikeuchi M. Noguchi H. Hayashi S. Long-Term Cryopreservation of Human and Other Mammalian Cells at −80 °C for 8 Cell Med. 2018 10 215517901773314 10.1177/2155179017733148
Arav A. Friedman O. Natan Y. Gur E. Shani N. Rat Hindlimb Cryopreservation and Transplantation: A Step Toward “Organ Banking” Am. J. Transplant. 2017 17 11 2820 2828 10.1111/ajt.14320 28422434
Isachenko V. Dittrich R. Keck G. Isachenko E. Rahimi G. Van Der Ven H. Montag M. Hoffmann I. Müller A. Distler W. Beckmann M. W. Mallmann P. Cryopreservation of Ovarian Tissue: Detailed Description of Methods for Transport, Freezing and Thawing Geburtshilfe Frauenheilkd. 2012 72 10 927 932 10.1055/s-0032-1327812 25258453
Crommelin D. J. A. Anchordoquy T. J. Volkin D. B. Jiskoot W. Mastrobattista E. Addressing the Cold Reality of MRNA Vaccine Stability J. Pharm. Sci. 2021 110 3 997 1001 10.1016/j.xphs.2020.12.006 33321139
Gavish Z. Ben-Haim M. Arav A. Cryopreservation of Whole Murine and Porcine Livers Rejuvenation Res. 2008 11 4 765 772 10.1089/rej.2008.0706 18729808
Fahy G. M. Wowk B. Pagotan R. Chang A. Phan J. Thomson B. Phan L. Physical and Biological Aspects of Renal Vitrification Organogenesis 2009 5 3 167 10.4161/org.5.3.9974 20046680
Berz D. McCormack E. M. Winer E. S. Colvin G. A. Quesenberry P. J. Cryopreservation of Hematopoietic Stem Cells Am. J. Hematol. 2007 82 6 463 10.1002/ajh.20707 17266054
Lagerberg J. W. Cryopreservation of Red Blood Cells Methods Mol. Biol. 2015 1257 353 367 10.1007/978-1-4939-2193-5_17 25428017
Shu Z. Heimfeld S. Gao D. Hematopoietic SCT with Cryopreserved Grafts: Adverse Reactions after Transplantation and Cryoprotectant Removal before Infusion Bone Marrow Transplant. 2014 49 4 469 476 10.1038/bmt.2013.152 24076548
Teramoto N. Sachinvala N. D. Shibata M. Trehalose and Trehalose-Based Polymers for Environmentally Benign, Biocompatible and Bioactive Materials Molecules 2008 13 8 1773 1816 10.3390/molecules13081773 18794785
Lee K. Y. Chang Y. D. Kim Y. G. Trehalose, a Major Sugar Cryoprotectant of the Overwintering Rice Water Weevil, Lissorhoptrus Oryzophilus (Coleoptera: Curculionidae) J. Asia-Pac. Entomol. 2002 5 1 35 41 10.1016/S1226-8615(08)60130-2
Rozsypal J. Koštál V. Zahradníčková H. Šimek P. Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia Pomonella) PLoS One 2013 8 4 e61745 10.1371/journal.pone.0061745 23613923
Hengherr S. Heyer A. G. Köhler H. R. Schill R. O. Trehalose and Anhydrobiosis in Tardigrades – Evidence for Divergence in Responses to Dehydration FEBS J. 2008 275 2 281 288 10.1111/j.1742-4658.2007.06198.x 18070104
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 10.1016/S0278-6915(02)00011-X 12065209
Mazur P. Cole K. W. Roles of Unfrozen Fraction, Salt Concentration, and Changes in Cell Volume in the Survival of Frozen Human Erythrocytes Cryobiology 1989 26 1 1 29 10.1016/0011-2240(89)90030-8 2924590
Crowe L. M. Reid D. S. Crowe J. H. Is Trehalose Special for Preserving Dry Biomaterials? Biophys. J. 1996 71 4 2087 10.1016/S0006-3495(96)79407-9 8889183
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 174513 10.1063/1.1887167 15910051
Branca C. Magazù S. Maisano G. Migliardo P. α,α-Trehalose−Water Solutions. 3. Vibrational Dynamics Studies by Inelastic Light Scattering J. Phys. Chem. B 1999 103 8 1347 1353 10.1021/jp983470c
Liu Q. Brady J. W. Anisotropic Solvent Structuring in Aqueous Sugar Solutions J. Am. Chem. Soc. 1996 118 49 12276 12286 10.1021/ja962108d
Jain N. K. Roy I. Effect of Trehalose on Protein Structure Protein Sci. 2009 18 1 24 10.1002/pro.3 19177348
Solocinski J. Osgood Q. Wang M. Connolly A. Menze M. A. Chakraborty N. Effect of Trehalose as an Additive to Dimethyl Sulfoxide Solutions on Ice Formation, Cellular Viability, and Metabolism Cryobiology 2017 75 134 143 10.1016/j.cryobiol.2017.01.001 28063960
Lins R. D. Pereira C. S. Hünenberger P. H. Trehalose-Protein Interaction in Aqueous Solution Proteins 2004 55 1 177 186 10.1002/prot.10632 14997551
Crowe J. H. , Clegg J. S. and Crowe L. M. , Anhydrobiosis: The Water Replacement Hypothesis, The Properties of Water in Foods ISOPOW, 1998, vol. 6 , pp. 440–455
Tang X. Pikal M. J. The Effect of Stabilizers and Denaturants on the Cold Denaturation Temperatures of Proteins and Implications for Freeze-Drying Pharm. Res. 2005 22 7 1167 1175 10.1007/s11095-005-6035-4 16028018
Boutron P. Peyridieu J. F. Reduction in Toxicity for Red Blood Cells in Buffered Solutions Containing High Concentrations of 2,3-Butanediol by Trehalose, Sucrose, Sorbitol, or Mannitol Cryobiology 1994 31 4 367 373 10.1006/cryo.1994.1044 7924394
Leslie S. B. Teter S. A. Crowe L. M. Crowe J. H. Trehalose Lowers Membrane Phase Transitions in Dry Yeast Cells Biochim. Biophys. Acta, Biomembr. 1994 1192 1 7 13 10.1016/0005-2736(94)90136-8 8204652
Kandror O. DeLeon A. Goldberg A. L. Trehalose Synthesis Is Induced upon Exposure of Escherichia Coli to Cold and Is Essential for Viability at Low Temperatures Proc. Natl. Acad. Sci. U. S. A. 2002 99 15 9727 9732 10.1073/pnas.142314099 12105274
Rudolph A. S. Crowe J. H. Crowe L. M. Effects of Three Stabilizing Agents—Proline, Betaine, and Trehalose—on Membrane Phospholipids Arch. Biochem. Biophys. 1986 245 1 134 143 10.1016/0003-9861(86)90197-9 3947095
Chen G. Yue A. Ruan Z. Yin Y. Wang R. Ren Y. Zhu L. Comparison of the Effects of Different Cryoprotectants on Stem Cells from Umbilical Cord Blood Stem Cells Int. 2016 2016 1396783 26770201
Rodrigues J. P. Paraguassú-Braga F. H. Carvalho L. Abdelhay E. Bouzas L. F. Porto L. C. Evaluation of Trehalose and Sucrose as Cryoprotectants for Hematopoietic Stem Cells of Umbilical Cord Blood Cryobiology 2008 56 2 144 151 10.1016/j.cryobiol.2008.01.003 18313656
Pu L. L. Q. Cui X. Fink B. F. Cibull M. L. Gao D. Long-Term Preservation of Adipose Aspirates after Conventional Lipoplasty Aesthetic Surg. J. 2004 24 6 536 541 10.1016/j.asj.2004.09.002
Cardoso L. M. D. F. Pinto M. A. Henriques Pons A. Alves L. A. Cryopreservation of Rat Hepatocytes with Disaccharides for Cell Therapy Cryobiology 2017 78 15 21 10.1016/j.cryobiol.2017.07.010 28782503
Wen Y. Z. Su B. X. Lyu S. S. Hide G. Lun Z. R. Lai D. H. Trehalose, an Easy, Safe and Efficient Cryoprotectant for the Parasitic Protozoan Trypanosoma Brucei Acta Trop. 2016 164 297 302 10.1016/j.actatropica.2016.09.024 27686958
Martinetti D. Colarossi C. Buccheri S. Denti G. Memeo L. Vicari L. Effect of Trehalose on Cryopreservation of Pure Peripheral Blood Stem Cells Biomed. Rep. 2017 6 3 314 10.3892/br.2017.859 28451392
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 10.1371/journal.pone.0054889 23349986
Gurruchaga H. Ciriza J. Saenz Del Burgo L. Rodriguez-Madoz J. R. Santos E. Prosper F. Hernández R. M. Orive G. Pedraz J. L. Cryopreservation of Microencapsulated Murine Mesenchymal Stem Cells Genetically Engineered to Secrete Erythropoietin Int. J. Pharm. 2015 485 1–2 15 24 10.1016/j.ijpharm.2015.02.047 25708005
Liu Y. Xu X. Ma X. Liu J. Cui Z. Effect of Various Freezing Solutions on Cryopreservation of Mesenchymal Stem Cells from Different Animal Species CryoLetters 2011 32 5 425 435 22020465
Ntai A. La Spada A. De Blasio P. Biunno I. Trehalose to Cryopreserve Human Pluripotent Stem Cells Stem Cell Res. 2018 31 102 112 10.1016/j.scr.2018.07.021 30071393
Zhang X. B. Li K. Yau K. H. Tsang K. S. Fok T. F. Li C. K. Lee S. M. Yuen P. M. P. Trehalose Ameliorates the Cryopreservation of Cord Blood in a Preclinical System and Increases the Recovery of CFUs, Long-Term Culture-Initiating Cells, and Nonobese Diabetic-SCID Repopulating Cells Transfusion 2003 43 2 265 272 10.1046/j.1537-2995.2003.00301.x 12559024
Limaye L. S. Kale V. P. Cryopreservation of Human Hematopoietic Cells with Membrane Stabilizers and Bioantioxidants as Additives in the Conventional Freezing Medium J. Hematother. Stem Cell Res. 2001 10 5 709 718 10.1089/152581601753193931 11672518
Scheinkönig C. Kappicht S. Kolb H. J. Schleuning M. Adoption of Long-Term Cultures to Evaluate the Cryoprotective Potential of Trehalose for Freezing Hematopoietic Stem Cells Bone Marrow Transplant. 2004 34 6 531 536 10.1038/sj.bmt.1704631 15286692
Yong K. W. Pingguan-Murphy B. Xu F. Abas W. A. B. W. Choi J. R. Omar S. Z. Azmi M. A. N. Chua K. H. Safwani W. K. Z. W. Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-Derived Stem Cells Sci. Rep. 2015 5 9596 10.1038/srep09596 25872464
Morris G. J. Rapidly Cooled Human Sperm: No Evidence of Intracellular Ice Formation Hum. Reprod. 2006 21 8 2075 2083 10.1093/humrep/del116 16613884
Isachenko E. Isachenko V. Katkov I. I. Dessole S. Nawroth F. Vitrification of Mammalian Spermatozoa in the Absence of Cryoprotectants: From Past Practical Difficulties to Present Success Reprod. BioMed. Online 2003 6 2 191 200 10.1016/S1472-6483(10)61710-5 12675999
Zhu Z. Fan X. Pan Y. Lu Y. Zeng W. Trehalose Improves Rabbit Sperm Quality during Cryopreservation Cryobiology 2017 75 45 51 10.1016/j.cryobiol.2017.02.006 28223021
Cirit Ü. Baǧiş H. Demir K. Agca C. Pabuccuoǧlu S. Varişli Ö. Clifford-Rathert C. Agca Y. Comparison of Cryoprotective Effects of Iodixanol, Trehalose and Cysteamine on Ram Semen Anim. Reprod. Sci. 2013 139 1–4 38 44 10.1016/j.anireprosci.2013.03.010 23602486
Aisen E. G. Medina V. H. Venturino A. Cryopreservation and Post-Thawed Fertility of Ram Semen Frozen in Different Trehalose Concentrations Theriogenology 2002 57 7 1801 1808 10.1016/S0093-691X(02)00653-2 12041684
Aisen E. G. Medina V. H. Venturino A. Cryopreservation and Post-Thawed Fertility of Ram Semen Frozen in Different Trehalose Concentrations Theriogenology 2002 57 7 1801 1808 10.1016/S0093-691X(02)00653-2 12041684
Bittencourt R. F. Oba E. de Almeida Biscarde C. E. Azevedo H. C. Bittencourt M. V. de Menezes G. F. O. da Silva Lima A. da Mata Fuchs K. de Lisboa Ribeiro Filho A. Dimethylacetamide and Trehalose for Ram Semen Cryopreservation Cryobiology 2018 85 1 6 10.1016/j.cryobiol.2018.10.266 30391233
Berlinguer F. Leoni G. G. Succu S. Mossa F. Galioto M. Madeddu M. Naitana S. Cryopreservation of European Mouflon (Ovis Gmelini Musimon) Semen during the Non-Breeding Season Is Enhanced by the Use of Trehalose Reprod. Domest. Anim. 2007 42 2 202 207 10.1111/j.1439-0531.2006.00753.x 17348979
Aisen E. Quintana M. Medina V. Morello H. Venturino A. Ultramicroscopic and Biochemical Changes in Ram Spermatozoa Cryopreserved with Trehalose-Based Hypertonic Extenders Cryobiology 2005 50 3 239 249 10.1016/j.cryobiol.2005.02.002 15925576
Bucak M. N. Ateşşahin A. Varişli Ö. Yüce A. Tekin N. Akçay A. The Influence of Trehalose, Taurine, Cysteamine and Hyaluronan on Ram Semen Microscopic and Oxidative Stress Parameters after Freeze-Thawing Process Theriogenology 2007 67 5 1060 1067 10.1016/j.theriogenology.2006.12.004 17280711
Aisen E. G. Alvarez H. L. Venturino A. Garde J. J. Effect of Trehalose and EDTA on Cryoprotective Action of Ram Semen Diluents Theriogenology 2000 53 5 1053 1061 10.1016/S0093-691X(00)00251-X 10798483
Valente S. S. Pereira R. M. Baptista M. C. Marques C. C. Vasques M. I. Pereira M. V. C. S. Horta A. E. M. Barbas J. P. In Vitro and in Vivo Fertility of Ram Semen Cryopreserved in Different Extenders Anim. Reprod. Sci. 2010 117 1–2 74 77 10.1016/j.anireprosci.2009.04.007 19482446
Matsuoka T. Imai H. Kohno H. Fukui Y. Effects of Bovine Serum Albumin and Trehalose in Semen Diluents for Improvement of Frozen-Thawed Ram Spermatozoa J. Reprod. Dev. 2006 52 5 675 683 10.1262/jrd.18033 16905877
Akhtarshenas B. Karami Shabankareh H. Hajarian H. Bucak M. N. Abdolmohammadi A. R. Dashtizad M. The Protease Inhibitor Antipain Has a Beneficial Synergistic Effect with Trehalose for Ram Semen Cryopreservation Reprod. Domest. Anim. 2018 53 6 1359 1366 10.1111/rda.13253 30011087
Tonieto R. A. Goularte K. L. Gastal G. D. A. Schiavon R. S. Deschamps J. C. Lucia T. Cryoprotectant Effect of Trehalose and Low-Density Lipoprotein in Extenders for Frozen Ram Semen Small Rumin. Res. 2010 93 2–3 206 209 10.1016/j.smallrumres.2010.05.003
Pérez-Marín C. C. Requena F. D. Arando A. Ortiz-Villalón S. Requena F. Agüera E. I. Effect of Trehalose- and Sucrose-Based Extenders on Equine Sperm Quality after Vitrification: Preliminary Results Cryobiology 2018 80 62 69 10.1016/j.cryobiol.2017.12.002 29229561
Vafaei F. Kohram H. Zareh-Shahne A. Ahmad E. Seifi-Jamadi A. Influence of Different Combinations of Permeable and Nonpermeable Cryoprotectants on the Freezing Capacity of Equine Sperm J. Equine Vet. Sci. 2019 75 69 73 10.1016/j.jevs.2019.01.014 31002096
De Oliveira R. A. Budik S. Aurich C. Influence of Partial or Total Replacement of Glycerol by Alternative Cryoprotectants in Ghent Freezing Extender on Post-Thaw Sperm Quality in Stallions Reprod. Domest. Anim. 2017 52 5 715 721 10.1111/rda.12970 28326634
Squires E. L. Keith S. L. Graham J. K. Evaluation of Alternative Cryoprotectants for Preserving Stallion Spermatozoa Theriogenology 2004 62 6 1056 1065 10.1016/j.theriogenology.2003.12.024 15289047
Khalili B. Farshad A. Zamiri M. J. Rashidi A. Fazeli P. Effects of Sucrose and Trehalose on the Freezability of Markhoz Goat Spermatozoa Asian-Australas. J. Anim. Sci. 2009 22 12 1614 1619 10.5713/ajas.2009.90286
Aboagla E. M. E. Terada T. Trehalose-Enhanced Fluidity of the Goat Sperm Membrane and Its Protection During Freezing Biol. Reprod. 2003 69 4 1245 1250 10.1095/biolreprod.103.017889 12801983
Franěk R. Marinović Z. Lujić J. Urbányi B. Fučíková M. Kašpar V. Pšenička M. Horváth Á. Cryopreservation and Transplantation of Common Carp Spermatogonia PLoS One 2019 14 4 e0205481 10.1371/journal.pone.0205481 30998742
Lyons L. Jerry R. Southgate P. C. Cryopreservation of Black-Lip Pearl Oyster (Pinctada Margaritifera, L.) Spermatozoa: Effects of Cryoprotectants on Spermatozoa Motility J. Shellfish Res. 2005 24 4 1187 1190
Kozdrowski R. The Effect of Trehalose on Post-Thaw Viability and Fertility of European Brown Hare (Lepus Europaeus Pallas, 1778) Spermatozoa Anim. Reprod. Sci. 2009 116 3–4 326 334 10.1016/j.anireprosci.2009.02.012 19304414
Pu L. L. Q. Cui X. Fink B. F. Cibull M. L. Gao D. Cryopreservation of Adipose Tissues: The Role of Trehalose Aesthetic Surg. J. 2005 25 2 126 131 10.1016/j.asj.2005.01.003
Erdag G. Eroglu A. Morgan J. R. Toner M. Cryopreservation of Fetal Skin Is Improved by Extracellular Trehalose Cryobiology 2002 44 3 218 228 10.1016/S0011-2240(02)00023-8 12237087
Kang X. L. Shen H. Pigmentation of Skin Graft Is Improved by Cryopreservation of Human Skin with Trehalose Kouqiang Hemian Waike Zazhi 2012 70 6 1464 1472
Yokomise H. Inui K. Wada H. Hasegawa S. Ohno N. Hitomi S. Reliable Cryopreservation of Trachea for One Month in a New Trehalose Solution J. Thorac. Cardiovasc. Surg. 1995 110 2 382 385 10.1016/S0022-5223(95)70234-2 7637356
Zhang X. G. Wang Y. H. Han C. Hu S. Wang L. Q. Hu J. H. Effects of Trehalose Supplementation on Cell Viability and Oxidative Stress Variables in Frozen-Thawed Bovine Calf Testicular Tissue Cryobiology 2015 70 3 246 252 10.1016/j.cryobiol.2015.03.004 25818604
Katenz E. Vondran F. W. R. 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 10.1002/lt.20921 17154395
Sasnoor L. M. Kale V. P. Limaye L. S. Supplementation of Conventional Freezing Medium with a Combination of Catalase and Trehalose Results in Better Protection of Surface Molecules and Functionality of Hematopoietic Cells J. Hematother. Stem Cell Res. 2003 12 5 553 564 10.1089/152581603322448268 14594512
Honadel T. E. Killian G. J. Cryopreservation of Murine Embryos with Trehalose and Glycerol Cryobiology 1988 25 4 331 337 10.1016/0011-2240(88)90041-7 3409707
Wang H. Y. Wen Y. Z. Lun Z. R. Lu S. S. Visual Observation of African Trypanosomes during Cryopreservation Biopreserv. Biobanking 2014 12 4 265 268 10.1089/bio.2014.0011
Wen Y. Z. Su B. X. Lyu S. S. Hide G. Lun Z. R. Lai D. H. Trehalose, an Easy, Safe and Efficient Cryoprotectant for the Parasitic Protozoan Trypanosoma Brucei Acta Trop. 2016 164 297 302 10.1016/j.actatropica.2016.09.024 27686958
Lee Y. A. Kim Y. H. Ha S. J. Kim K. J. Kim B. J. Kim B. G. Choi S. H. Kim I. C. Schmidt J. A. Ryu B. Y. Cryopreservation of Porcine Spermatogonial Stem Cells by Slow-Freezing Testis Tissue in Trehalose J. Anim. Sci. 2014 92 3 984 995 10.2527/jas.2013-6843 24504041
Sato M. Inaba S. Sukenobe J. Sasaki T. Inoue R. Noguchi M. Nakagiri A. A Modified Perlite Protocol with a Mixed Dimethyl Sulfoxide and Trehalose Cryoprotectant Improves the Viability of Frozen Cultures of Ectomycorrhizal Basidiomycetes Mycologia 2019 111 1 161 176 10.1080/00275514.2018.1520035 30714878
Huang H. Zhao G. Zhang Y. Xu J. Toth T. L. He X. Predehydration and Ice Seeding in the Presence of Trehalose Enable Cell Cryopreservation ACS Biomater. Sci. Eng. 2017 3 8 1758 1768 10.1021/acsbiomaterials.7b00201 28824959
O'Brien J. Stability of Trehalose, Sucrose and Glucose to Nonenzymatic Browning in Model Systems J. Food Sci. 1996 61 4 679 682 10.1111/j.1365-2621.1996.tb12180.x
Lee Y. A. Kim Y. H. Ha S. J. Kim B. J. Kim K. J. Jung M. S. Kim B. G. Ryu B. Y. Effect of Sugar Molecules on the Cryopreservation of Mouse Spermatogonial Stem Cells Fertil. Steril. 2014 101 4 1165 1175 10.1016/j.fertnstert.2013.12.033 24462063
Gómez-Fernández J. Gómez-Izquierdo E. Tomás C. Mocé E. De Mercado E. Effect of Different Monosaccharides and Disaccharides on Boar Sperm Quality after Cryopreservation Anim. Reprod. Sci. 2012 133 1–2 109 116 10.1016/j.anireprosci.2012.06.010 22771077
Chaytor J. L. Tokarew J. M. Wu L. K. Leclre M. Tam R. Y. Capicciotti C. J. Guolla L. Von Moos E. Findlay C. S. Allan D. S. Ben R. N. Inhibiting Ice Recrystallization and Optimization of Cell Viability after Cryopreservation Glycobiology 2012 22 1 123 133 10.1093/glycob/cwr115 21852258
Bender M. A. Tran P. T. Smith L. H. Preservation of Viable Bone Marrow Cells by Freezing J. Appl. Physiol. 1960 15 520 524 10.1152/jappl.1960.15.3.520 13798783
Deller R. C. Congdon T. Sahid M. A. Morgan M. Vatish M. Mitchell D. A. Notman R. Gibson M. I. Ice Recrystallisation Inhibition by Polyols: Comparison of Molecular and Macromolecular Inhibitors and Role of Hydrophobic Units Biomater. Sci. 2013 1 5 478 485 10.1039/C3BM00194F 32482011
Magazu S. Villari V. Migliardo P. Maisano G. Telling M. T. F. Diffusive Dynamics of Water in the Presence of Homologous Disaccharides: A Comparative Study by Quasi Elastic Neutron Scattering. IV J. Phys. Chem. B 2001 105 9 1851 1855 10.1021/jp002155z
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: Complementary Raman Scattering Experiments and Molecular Dynamics Simulations Carbohydr. Res. 2005 340 5 881 887 10.1016/j.carres.2005.01.036 15780254
Kawai H. Sakurai M. Inoue Y. Chûjô R. Kobayashi S. Hydration of Oligosaccharides: Anomalous Hydration Ability of Trehalose Cryobiology 1992 29 5 599 606 10.1016/0011-2240(92)90064-9 1424716
Sei T. Gonda T. Arima Y. Growth Rate and Morphology of Ice Crystals Growing in a Solution of Trehalose and Water J. Cryst. Growth 2002 240 1–2 218 229 10.1016/S0022-0248(02)00875-8
Del M. Amalfa F. Nuñez A. M. Díaz S. Biondi De Lopez A. C. Disalvo E. A. Effect of Trehalose and Sucrose on the Hydration and Dipole Potential of Lipid Bilayers Biophys. J. 2000 78 5 2452 10.1016/S0006-3495(00)76789-0 10777741
Sola-Penna M. Meyer-Fernandes J. R. Stabilization against Thermal Inactivation Promoted by Sugars on Enzyme Structure and Function: Why Is Trehalose More Effective than Other Sugars? Arch. Biochem. Biophys. 1998 360 1 10 14 10.1006/abbi.1998.0906 9826423
Shiraga K. Adachi A. Nakamura M. Tajima T. Ajito K. Ogawa Y. Characterization of the Hydrogen-Bond Network of Water around Sucrose and Trehalose: Microwave and Terahertz Spectroscopic Study J. Chem. Phys. 2017 146 10 105102 10.1063/1.4978232 28298096
Kuleshova L. L. MacFarlane D. R. Trounson A. O. Shaw J. M. Sugars Exert a Major Influence on the Vitrification Properties of Ethylene Glycol-Based Solutions and Have Low Toxicity to Embryos and Oocytes Cryobiology 1999 38 2 119 130 10.1006/cryo.1999.2153 10191035
Roe K. D. Labuza T. P. Glass Transition and Crystallization of Amorphous Trehalose-Sucrose Mixtures Int. J. Food Prop. 2005 8 3 559 574 10.1080/10942910500269824
Berejnov V. Husseini N. S. Alsaied O. A. Thorne R. E. Effects of Cryoprotectant Concentration and Cooling Rate on Vitrification of Aqueous Solutions J. Appl. Crystallogr. 2006 39 2 244 251 10.1107/S0021889806004717
Lammert A. M. Schmidt S. J. Day G. A. Water Activity and Solubility of Trehalose Food Chem. 1998 61 1–2 139 144 10.1016/S0308-8146(97)00132-5
Wen X. Wang S. Duman J. G. Fnu Arifin J. Juwita V. Goddard W. A. Rios A. Liu F. Kim S. K. Abrol R. DeVries A. L. Henling L. M. Antifreeze Proteins Govern the Precipitation of Trehalose in a Freezing-Avoiding Insect at Low Temperature Proc. Natl. Acad. Sci. U. S. A. 2016 113 24 6683 6688 10.1073/pnas.1601519113 27226297
Wu C. F. Tsung H. C. Zhang W. J. Wang Y. Lu J. H. Tang Z. Y. Kuang Y. P. Jin W. Cui L. Liu W. Cao Y. L. Improved Cryopreservation of Human Embryonic Stem Cells with Trehalose Reprod. BioMed. Online 2005 11 6 733 739 10.1016/S1472-6483(10)61692-6 16417738
De Lara Janz F. De Aguiar Debes A. De Cássia Cavaglieri R. Duarte S. A. Romão C. M. Morón A. F. Zugaib M. Bydlowski S. P. Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation J. Biomed. Biotechnol. 2012 2012 649353 22665987
Kim K. J. Lee Y. A. Kim B. J. Kim Y. H. Kim B. G. Kang H. G. Jung S. E. Choi S. H. Schmidt J. A. Ryu B. Y. Cryopreservation of Putative Pre-Pubertal Bovine Spermatogonial Stem Cells by Slow Freezing Cryobiology 2015 70 2 175 183 10.1016/j.cryobiol.2015.02.007 25732704
Lestari S. W. Ilato K. F. Pratama M. I. A. Fitriyah N. N. Pangestu M. Pratama G. Margiana R. Sucrose “versus” Trehalose Cryoprotectant Modification in Oocyte Vitrification: A Study of Embryo Development Biomed. Pharmacol. J. 2018 11 1 97 104
Schulz M. Risopatrón J. Matus G. Pineda E. Rojas C. Isachenko V. Isachenko E. Sánchez R. Trehalose Sustains a Higher Post-Thaw Sperm Motility than Sucrose in Vitrified Human Sperm Andrologia 2017 49 9 e12757 10.1111/and.12757 28543267
Jafaroghli M. Khalili B. Farshad A. Zamiri M. J. The Effect of Supplementation of Cryopreservation Diluents with Sugars on the Post-Thawing Fertility of Ram Semen Small Rumin. Res. 2011 96 1 58 63 10.1016/j.smallrumres.2010.11.010
Bailey T. L. Wang M. Solocinski J. Nathan B. P. Chakraborty N. Menze M. A. Protective Effects of Osmolytes in Cryopreserving Adherent Neuroblastoma (Neuro-2a) Cells Cryobiology 2015 71 3 472 480 10.1016/j.cryobiol.2015.08.015 26408850
Toxopeus J. Koštál V. Sinclair B. J. Evidence for Non-Colligative Function of Small Cryoprotectants in a Freeze-Tolerant Insect Proc. R. Soc. B 2019 286 1899 20190050 10.1098/rspb.2019.0050
Groan S. C. Lyophilization of Hypha-Forming Tropical Wood-Inhabiting Basidiomycotina Mycologia 2019 92 4 810 817
Chen T. H. H. Kartha K. K. Leung N. L. Kurz W. G. W. Chatson K. B. Constabel F. Cryopreservation of Alkaloid-Producing Cell Cultures of Periwinkle (Catharanthus Roseus) Plant Physiol. 1984 75 3 726 731 10.1104/pp.75.3.726 16663695
Johnston S. D. Qualischefski E. Cooper J. McLeod R. Lever J. Nixon B. Anderson A. L. Hobbs R. Gosálvez J. López-Fernández C. Keeley T. Cryopreservation of Saltwater Crocodile (Crocodylus Porosus) Spermatozoa Reprod., Fertil. Dev. 2017 29 11 2235 2244 10.1071/RD16511 28356183
Nynca J. Judycka S. Liszewska E. Dobosz S. Grudniewska J. Arai K. Fujimoto T. Ciereszko A. Utility of Different Sugar Extenders for Cryopreservation and Post-Thaw Storage of Sperm from Salmonidae Species Aquaculture 2016 464 340 348 10.1016/j.aquaculture.2016.07.014
Silva C. G. Cunha E. R. Blume G. R. Malaquias J. V. Báo S. N. Martins C. F. Cryopreservation of Boar Sperm Comparing Different Cryoprotectants Associated in Media Based on Powdered Coconut Water, Lactose and Trehalose Cryobiology 2015 70 2 90 94 10.1016/j.cryobiol.2015.01.001 25595634
Woelders H. Matthijs A. Engel B. Effects of Trehalose and Sucrose, Osmolality of the Freezing Medium, and Cooling Rate on Viability and Intactness of Bull Sperm after Freezing and Thawing Cryobiology 1997 35 2 93 105 10.1006/cryo.1997.2028 9299101
Chen Y. Foote R. H. Brockett C. C. Effect of Sucrose, Trehalose, Hypotaurine, Taurine, and Blood Serum on Survival of Frozen Bull Sperm Cryobiology 1993 30 4 423 431 10.1006/cryo.1993.1042 8403993
Liu J. Tanrikut C. Wright D. L. Lee G. Y. Toner M. Biggers J. D. Toth T. L. Cryopreservation of Human Spermatozoa with Minimal Non-Permeable Cryoprotectant Cryobiology 2016 73 2 162 167 10.1016/j.cryobiol.2016.08.004 27498216
Caturla-Sánchez E. Sánchez-Calabuig M. J. Pérez-Gutiérrez J. F. Cerdeira J. Castaño C. Santiago-Moreno J. Vitrification of Dog Spermatozoa: Effects of Two Cryoprotectants (Sucrose or Trehalose) and Two Warming Procedures Cryobiology 2018 80 126 129 10.1016/j.cryobiol.2017.11.001 29126865
Lynch A. L. Chen R. Slater N. K. H. PH-Responsive Polymers for Trehalose Loading and Desiccation Protection of Human Red Blood Cells Biomaterials 2011 32 19 4443 4449 10.1016/j.biomaterials.2011.02.062 21421265
Buchanan S. S. Menze M. A. Hand S. C. Pyatt D. W. Carpenter J. F. Cryopreservation of Human Hematopoietic Stem and Progenitor Cells Loaded with Trehalose: Transient Permeabilization via the Adenosine Triphosphate-Dependent P2Z Receptor Channel Cell Preserv. Technol. 2006 3 4 212 222 10.1089/cpt.2005.3.212
Diniz-Mendes L. Bernardes E. Araujo P. S. Panek A. Paschoalin V. Preservation of Frozen Yeast Cells by Trehalose Biotechnol. Bioeng. 1999 65 5 572 578 10.1002/(SICI)1097-0290(19991205)65:5<572::AID-BIT10>3.0.CO;2-7 10516583
Stewart S. He X. Intracellular Delivery of Trehalose for Cell Banking Langmuir 2019 35 23 7414 7422 10.1021/acs.langmuir.8b02015 30078320
Bhandal I. S. Hauptmann R. M. Widholm J. M. Trehalose as Cryoprotectant for the Freeze Preservation of Carrot and Tobacco Cells Plant Physiol. 1985 78 2 430 10.1104/pp.78.2.430 16664260
Oliver A. E. Jamil K. Crowe J. H. Tablin F. Loading Human Mesenchymal Stem Cells with Trehalose by Fluid-Phase Endocytosis Cell Preserv. Technol. 2004 2 1 35 49 10.1089/153834404322708745
Kusuma I. Hadi R. S. Kiranadi B. Boediono A. Trehalose Preincubation Increases Mesenchymal (CD271+) Stem Cells Post-Cryopreservation Viability Med. J. Indones. 2016 25 3 128 135 10.13181/mji.v25i3.1273
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 Human Wharton Jelly Mesenchymal Stem Cells After Cryopreservation Exp. Clin. Transplant. 2018 18 4 505 511 10.6002/ect.2017.0101 29957164
Stokich B. Osgood Q. Grimm D. Moorthy S. Chakraborty N. Menze M. A. Cryopreservation of Hepatocyte (HepG2) Cell Monolayers: Impact of Trehalose Cryobiology 2014 69 2 281 290 10.1016/j.cryobiol.2014.08.001 25127872
Campbell L. H. Brockbank K. G. M. Culturing with Trehalose Produces Viable Endothelial Cells after Cryopreservation Cryobiology 2012 64 3 240 244 10.1016/j.cryobiol.2012.02.006 22366172
Hara J. Tottori J. Anders M. Dadhwal S. Asuri P. Mobed-Miremadi M. Trehalose Effectiveness as a Cryoprotectant in 2D and 3D Cell Cultures of Human Embryonic Kidney Cells Artif. Cells, Nanomed., Biotechnol. 2017 45 3 609 616 10.3109/21691401.2016.1167698 27050441
Zheng Y. X. Ma L. Z. Liu S. J. Zhang C. T. Meng R. Chen Y. Z. Jiang Z. L. Protective Effects of Trehalose on Frozen-Thawed Ovarian Granulosa Cells of Cattle Anim. Reprod. Sci. 2019 200 14 21 10.1016/j.anireprosci.2018.11.005 30472065
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 10.1016/j.cryobiol.2004.06.001 15351684
Zhou X. He H. Liu B. Hua T. Loading Trehalose into Red Blood Cells by Improved Hypotonic Method Cell Preserv. Technol. 2008 6 2 119 122 10.1089/cpt.2008.0001
Reuss R. Ludwig J. Shirakashi R. Ehrhart F. Zimmermann H. Schneider S. Weber M. M. Zimmermann U. Schneider H. Sukhorukov V. L. Intracellular Delivery of Carbohydrates into Mammalian Cells through Swelling-Activated Pathways J. Membr. Biol. 2004 200 2 67 81 10.1007/s00232-004-0694-7 15520905
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 1409 10.1016/j.bbamem.2016.03.020 27003129
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 3 519 523 10.2337/diab.46.3.519 9032112
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 10.1002/btpr.2399 27802564
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 1354 10.1002/btpr.1600 22837111
Oldenhof H. Zhang M. Narten K. Bigalk J. Sydykov B. Wolkers W. F. Sieme H. Freezing-Induced Uptake of Disaccharides for Preservation of Chromatin in Freeze-Dried Stallion Sperm during Accelerated Aging Biol. Reprod. 2017 97 6 892 901 10.1093/biolre/iox142 29121172
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 10.1007/s00232-002-1003-y 12202951
Dovgan B. , Dermol J. , Barlič A. , Knežević M. and Miklavčič D. , Cryopreservation of Human Umbilical Stem Cells in Combination with Trehalose and Reversible Electroporation, IFMBE Proc., 2016, vol. 53 , pp. 307–310
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 10.1007/s00232-016-9916-z 27383230
Lynch A. L. Chen R. Dominowski P. J. Shalaev E. Y. Yancey R. J. Slater N. K. H. Biopolymer Mediated Trehalose Uptake for Enhanced Erythrocyte Cryosurvival Biomaterials 2010 31 23 6096 6103 10.1016/j.biomaterials.2010.04.020 20471082
Mercado S. A. Slater N. K. H. Increased Cryosurvival of Osteosarcoma Cells Using an Amphipathic PH-Responsive Polymer for Trehalose Uptake Cryobiology 2016 73 2 175 180 10.1016/j.cryobiol.2016.08.002 27497662
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 10.1016/j.cryobiol.2013.09.002 24045066
Wei Y. Li C. Zhang L. Xu X. Design of Novel Cell Penetrating Peptides for the Delivery of Trehalose into Mammalian Cells Biochim. Biophys. Acta 2014 1838 7 1911 1920 10.1016/j.bbamem.2014.02.011 24583082
Guo N. Puhlev I. Brown D. R. Mansbridge J. Levine F. Trehalose Expression Confers Desiccation Tolerance on Human Cells Nat. Biotechnol. 2000 18 2 168 171 10.1038/72616 10657122
García de Castro A. Tunnacliffe A. Intracellular Trehalose Improves Osmotolerance but Not Desiccation Tolerance in Mammalian Cells FEBS Lett. 2000 487 2 199 202 10.1016/S0014-5793(00)02340-1 11150509
Chakraborty N. Menze M. A. Malsam J. Aksan A. Hand S. C. Toner M. Cryopreservation of Spin-Dried Mammalian Cells PLoS One 2011 6 9 e24916 10.1371/journal.pone.0024916 21966385
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 10.1016/j.cryobiol.2017.05.008 28552273
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 10.1038/72608 10657121
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 10.1089/154732804323099226 15186725
Eroglu A. Toner M. Toth T. L. Beneficial Effect of Microinjected Trehalose on the Cryosurvival of Human Oocytes Fertil. Steril. 2002 77 1 152 158 10.1016/S0015-0282(01)02959-4 11779606
Eroglu A. Bailey S. E. Toner M. Toth T. L. Successful Cryopreservation of Mouse Oocytes by Using Low Concentrations of Trehalose and Dimethylsulfoxide Biol. Reprod. 2009 80 1 70 10.1095/biolreprod.108.070383 18815355
Zhang W. Rong J. Wang Q. He X. The Encapsulation and Intracellular Delivery of Trehalose Using a Thermally Responsive Nanocapsule Nanotechnology 2009 20 27 275101 10.1088/0957-4484/20/27/275101 19528681
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 10.1021/acsami.5b00655 25679454
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 10.1016/j.biomaterials.2017.06.018 28649014
Holovati J. L. Acker J. P. Spectrophotometric Measurement of Intraliposomal Trehalose Cryobiology 2007 55 2 98 107 10.1016/j.cryobiol.2007.06.003 17659270
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 10.1016/j.cryobiol.2008.11.002 19059392
Motta J. P. R. Paraguassú-Braga F. H. Bouzas L. F. Porto L. C. Evaluation of Intracellular and Extracellular Trehalose as a Cryoprotectant of Stem Cells Obtained from Umbilical Cord Blood Cryobiology 2014 68 3 343 348 10.1016/j.cryobiol.2014.04.007 24769312
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 10.1002/btpr.1519 22275294
Zhang S. Z. Fan J. L. Xu X. G. Chen G. M. Zhu F. M. Yan L. X. An Experimental Study of the Use of Ultrasound to Facilitate the Loading of Trehalose into Platelets Cryobiology 2009 59 2 135 140 10.1016/j.cryobiol.2009.06.003 19538954
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 e0130323 10.1371/journal.pone.0130323 26115179
Eroglu A. Elliott G. Wright D. L. Toner M. Toth T. L. Progressive Elimination of Microinjected Trehalose during Mouse Embryonic Development Reprod. BioMed. Online 2005 10 4 503 510 10.1016/S1472-6483(10)60828-0 15901459
Fahy G. M. Analysis of “Solution Effects” Injury. Equations for Calculating Phase Diagram Information for the Ternary Systems NaCl-Dimethylsulfoxide-Water and NaCl-Glycerol-Water Biophys. J. 1980 32 2 837 10.1016/S0006-3495(80)85019-3 7260303
Alfieri R. R. Petronini P. G. Hyperosmotic Stress Response: Comparison with Other Cellular Stresses Pfluegers Arch. 2007 454 2 173 185 10.1007/s00424-006-0195-x 17206446
