The roles of reactive oxygen species and antioxidants in cryopreservation

Cryopreservation has facilitated advancement of biological research by allowing the storage of cells over prolonged periods of time. While cryopreservation at extremely low temperatures would render cells metabolically inactive, cells suffer insults during the freezing and thawing process. Among such insults, the generation of supra-physiological levels of reactive oxygen species (ROS) could impair cellular functions and survival. Antioxidants are potential additives that were reported to partially or completely reverse freeze-thaw stress-associated impairments. This review aims to discuss the potential sources of cryopreservation-induced ROS and the effectiveness of antioxidant administration when used individually or in combination.


Introduction
The ability to keep an organism alive while frozen and allowing it to survive for a prolonged period of time may sound like a scene lifted directly from a science fiction movie. Although freezing complex multicellular organisms remains challenging and often faced significant obstacles during the revival of the frozen organism [1][2][3][4], reviving single-cell organisms after a prolonged period of time has been a reality for several decades. Among many cases, the ability to revive single cell prokaryotic organisms such as Escherichia coli and Treponema pallidum were demonstrated in 1913 and 1954 respectively [5,6]. Such results were also obtained from the unicellular eukaryotic organism Saccharomyces cerevisiae in 1902 [7].
In the field of research involving mammalian cells, significant progress was made when Polge et al. [8] successfully revived frozen fowl spermatozoa in 1949 and Bos taurus spermatozoa cells in 1952 using glycerol as a cryoprotective agent (CPA) [9]. The subsequent use of dimethyl sulfoxide (DMSO) as CPA, which remarkably preserved erythrocytes, was first reported in the 1950s [10] and is now a common component of cryopreservation medium. Although the ability to allow cells to be transported across the world has fostered trans-global scientific collaborations as well as independent verifications of experimental results and clinical advancements, it is frequently taken for granted. One could only imagine the hindrance to scientific advancements if the cryopreservation techniques were absent. For the past few decades, although significant advancements were made in the cryopreservation field on mammalian cells, the technique is far from perfect. Many researchers face challenges such as poor recovery [11,12], loss of functional characteristics of specific cell types [4,13,14] and, in the case of stem cell research, the inability to retain pluripotency [15,16]. In this review, we focus on the role of reactive oxygen species (ROS), a product of cellular metabolism that can be damaging to cells and how ROS contributes to the undesirable results seen after cryopreservation. We further explore current advancements in using antioxidants to negate these undesirable effects observed in cryopreservation. (B) Sources of ROS, and localization of enzymes that counteracts ROS in the mitochondria, endoplasmic reticulum (ER), peroxisome, cytosol and the extracellular space. SOD1 is localized in both the mitochondria intermembrane space and cytosol, SOD3 is located extracellularly and SOD2 is found exclusively mostly in the mitochondria matrix. Catalase that reduces hydrogen peroxide (H 2 O 2 ) into H 2 O is mostly located in the peroxisomes. Glutathione peroxidase (GPx) is found in the mitochondria and cytosol. Peroxiredoxins (Prx) and thioredoxins (Trx) which constitute the Peroxiredoxin-Thioredoxin (Prx/Trx) system can be found in the nucleus, mitochondria, ER, peroxisome and the extracellular envi-  Ascorbic acid Sperm • ROS a (↓) [149] • Viability (↑) [149] • Motility (Weak ↑) [149] • MMP (↑) [149] • Apoptotic cells (↓) [149] • DNA damage (↓) [148,150] ∧ [149] • Motility(N/C) [ LN vapor phase (6.5-2 cm) at 10-15 min [148] LN vapor phase (10 cm) at 10 min [149], −20 • C at 10 min + LN vapor phase at 2 h [150] Mouse embryos • Percentage of intact embryos, blastocyst and number of hatching blastocyst (↑) [183] • Number of implantation sites (↑) [183] • Fetal development (N/C) [183] Vitrification and slow freezing [183] Antifreeze proteins (AFP)
Hyper-polarization of the ψ can favor ROS generation [62], which is believed to be a result of a reduction in electron transfer [63]. Depolarization of ψ can be induced by ROS, which impairs oxidative phosphorylation and amplifies ROS generation [64]. Loss of ψ was reported in cryopreserved human oocytes [57], buffalo sperm [65,58], nucleus pulposus-derived mesenchymal stem cells [66], murine embryos [67], Meleagris gallopavo spermatozoa [68], koala spermatozoa [69] and porcine hepatocytes [59], although a transient elevation in ψ was reported in murine oocytes after freeze-thawing [70]. Opening of mtochondrial permeability transition pore (mPTP), which involves the formation of a 'hole' in the inner mitochondrial membrane (IMM) is known to lead to the dissipation of the ψ as well as an elevation in ROS levels [64]. Opening of mPTP leads to dissipation of the ψ, mitochondrial swelling, ATP depletion, relocalization of pro-apoptotic molecules and elevated ROS levels [71,72]. The involvement of mPTP in cryopreservation has been implicated in the study showing that inhibition of mPTP by bongkrekic acid successfully reduced cryopreservation-induced apoptosis in stallion spermatozoa [73]. mPTP opening has been known to enhance H 2 O 2 production through conformational alterations to complex I of ETC [74], depletion of ROS-scavengers as well as intensifying production of ROS from Krebs cycle oxidoreductases [75]. Opening of mPTP is known to be induced during oxidative stress and ROS-mediated alterations to mPTP regulators and components were suggested to be responsible for this. Indeed, it was found that the mild uncoupling agent 2,4-dinitrophenol, which normally reduces ROS, improved motility in sperm with low cryopreservability [76] while the antioxidant, monothioglycerol was found to reduce mitochondria ROS as well as increase fertility and the percentage of cells with the ability to undergo acrosome reaction [41]. ROS-mediated mitochondrial permeabilization involved oxidative attack on the protein thiol groups on the mitochondrial membrane. This may give rise to protein aggregates due to thiol groups cross-linking after being oxidized [77,78].

Protein folding in the endoplasmic reticulum and ROS production
Cryopreservation of cells was known to perturb the homeostasis of the endoplasmic reticulum (ER) [79][80][81][82] and ER is a known source of ROS [83,84]. The ER facilitates the proper folding and addition of some post-translational modifications to proteins in the secretory pathway. Accumulation of misfolded proteins in the ER could occur under conditions that perturb ER homeostasis, also known as ER stress. Increased protein synthesis is one such condition. The unfolded protein (UPR) response promotes an adaptive response against ER stress by increasing machineries for protein degradation and protein folding as part of an effort to restore ER homeostasis. The molecular mechanism on how UPR are activated has been comprehensively reviewed by [85,86]. UPR is activated via one of the three membrane-bound transducing receptors (ATF6, PERK, IRE1α), these three sensors thus constitute the three branches of the UPR signaling pathways [85,86]. It was observed that SOD1 and the ER stress marker ERP29 gene expression were significantly up-regulated in response to freeze-thaw stress in primate ovarian tissue [81]. In yeast, genes expression for protein chaperones such as SSA4, HSP26, HSP42 were found to be up-regulated in response to freeze-thaw stress when cells were frozen in the absence of cryoprotectants [87]. In mammals, all three arms of the UPR may be activated during cryopreservation. The XBP-1 protein levels were elevated in vitrified mice oocytes [79] and maturing oocytes exposed to delipidated serum were more susceptible to cryopreservation-induced ER stress [80]. Intriguingly, the handling of oocytes, itself, was sufficient to activate the IRE1α arm [88], highlighting the vulnerability of oocytes to cope with stress during the cryopreservation process.
In addition to its homeostatic role, sustained induction of the UPR in response to severe ER stress caused by a multitude of factors can antagonize cellular survival, resulting in cell death [89]. The activation of the UPR has been implicated in the production of at least two species of ROS: O 2 •− and H 2 O 2 [90][91][92] and these ROS were postulated to be an event preceding cellular death. The source of H 2 O 2 may be attributed to oxidative folding via the protein disulfide isomerase (PDI)-ER oxidoreductase (ERO) relay or the cytochrome P450 (CYP) family of enzymes. The PDI-ERO1 pathway has been demonstrated to produce ROS in the form of H 2 O 2 [93][94][95]. Activation of the PERK-arm of the UPR could lead to downstream ERO1α activation, H 2 O 2 production and mediates the feeding of calcium into the mitochondria which could promote O 2 •− production and apoptosis [92]. Interestingly, a yeast strain deleted for genes encoding for catalases and glutathione were hypersensitive to exogenous H 2 O 2 , but are not sensitive to ERO1 overexpression [96], indicating that there could be other, more potent sources of H 2 O 2 in the ER, or that cytosolic or mitochondria pool of H 2 O 2 are isolated from the ER. Studies have also indicated an ERO1-independent source of H 2 O 2 in the ER [97,98], suggesting the PDI-ERO1 pathway may not be the sole source of H 2 O 2 in the ER. Other possible pathway that could be activated through sustained UPR activation that may lead to mitochondrial ROS generation is through the dimerization of IRE1α, which activates the JNK-SAB axis to initiate cellular death [99].

Nitric oxide synthase and NADPH oxidase
Although not considered ROS, nitric oxide (NO • ) and peroxynitrite (ONOO − ) are free radicals. NO • can diffuse through the cell membrane. In vivo, NO • is not highly reactive to most biomolecules. However, NO • can react with metal complexes to form metal nitrosyls. NO • reacts with O 2 •− to form more damaging species, such as ONOO − which is thought to occur mostly in the hydrophobic regions of the cell [100]. ONOO − can be detoxified by enzymes such as peroxiredoxins and glutathione peroxidase [101] (Figure 1). Unlike NO • , ONOO − are strong oxidants capable of causing oxidative damage, nitration and S-nitrosylation of proteins [102,103]. In vivo, nitric oxide (NO • ) is produced by the family of nitric oxide synthase (NOS) which consist of three isoforms: neuronal NOS or NOS1 ('neuronal' NOS/nNOS), NOS2 ('inducible' NOS/iNOS) and NOS3 ('endothelial' NOS/eNOS). NOS typically catalyzes the formation of NO • and citrulline from arginine and oxygen. Most NOS isoforms are usually regulated by calmodulin and calcium, and require the cofactors NADPH, FAD, Flavin mononucleotide (FMN) and tetrahydrobiopterin (BH4) [104,105]. Similar to ROS, NO • regulates cell death and survival [106][107][108][109][110]. NO • is essential for proper cellular physiological function such as vasodilation [111] as well as regulating immunosuppression [112] and tissue repair in mesenchymal stem cells (MSCs) [113]. Conversely, NO • , can interfere with hemopoiesis [114]. Moderate levels of NO • can initiate capacitation [115] and is essential for motile functions in sperm [116,117].
During cryopreservation, NOS activation or NO • production was observed in cryopreserved heart valves [118] and sperm [30,116,119]. While NO • itself has not been found to be significantly increased by freeze-thaw stress in RBC, the product of nitric oxide nitrosylation, S-nitrosohemoglobin was found to be increased by freeze-thaw stress [120]. At high levels of NO • , sperm functions can be antagonized [116,[121][122][123]. When cryopreserving sperms, the use of low concentrations of NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) [124], and anti-nitrosative agents such as hemoglobin [124] and lactoferrin [125] has been found to improve membrane functionality, survival and/or fertilization, indicating that reducing NO • may improve assisted reproductive technology outcomes especially since NO • was elevated in post-thawed cells. It should however be noted that the use of high concentrations of l-NAME was found to impair sperm function [124].
NADPH oxidases (NOXs) are a family of seven-membered enzymes that are highly regarded due to their role as a major non-mitochondrial ROS generator. NOX enzymes generate ROS, primarily O 2 •− , by catalyzing the transfer of one electron across the membrane from the electron-donating NADPH to the electron acceptor oxygen, thus reducing oxygen to form O 2 •− . Exceptions are NOX4, DUOX1 and DUOX2 of the NOX/DUOX family, which was documented to produce mainly H 2 O 2 [126]. While all seven members of the NOX family are found to be located to the plasma membrane, specific NOX isoforms such as NOX4, NOX5 and DUOX2 have also been detected at ER, with NOX4 residing at other subcellular locations including the mitochondria and the nuclear membrane [126,127]. Apart from the mitochondria and ER, the peroxisome is another source of intracellular ROS, which harbors pro-oxidant enzymes such as acyl CoA oxidase (ACOx) and xanthine oxidases (XOs) [128].
Our current understanding of the activation of NOX includes a collection of inducers which can be sorted into three main categories namely: chemical, biological and physical [129]. With respect to cryopreservation, NOX activation induced by chemical and physical inducers are particularly relevant and worth noting. Physical inducers are a broad collection of inducers including temperature [130], osmotic stress [131] and pH changes [132] which are documented NOX-inducers, that are coincidentally generated during cryopreservation [133]. During cryopreservation, the addition and removal of cryoprotectants, as well as freeze-thawing have been proven to subject cells to osmotic stress [134]. Extracellular ice formed during cryopreservation puts the cell through hypertonic conditions as the solute concentration elevates in the unfrozen extracellular portions. As a result, cells shrink as water leaves the cell to re-establish the equilibrium of solute concentration across the cell. The reverse is also true for thawing during cryopreservation, in which this time, cells are put through hypotonic conditions which lead to movement of water into the cell, consequently, cell swelling. Swelling of cells under hypotonic condition, however, was viewed as more pernicious due to the elevation in ROS levels following cryopreservation in the case of stallion sperm [135].
In astrocytes, hypo-osmotic swelling leads to an increase in ROS as well as phosphorylation of p47 phox and that, apocynin, an NOX inhibitor abrogated such effects [136]. Moreover, cortical brain slices of mice with p47 phox knockout failed to show elevated ROS levels as observed in wild-type mice suggesting hypo-osmotic swelling results in p47 phox -NOX-dependent generation of ROS [136]. In agreement with this, supporting evidence from skeletal muscles, in which osmotic stress leads to localized increase in Ca 2+ in the cytosol, termed as 'calcium spark' and an elevation in ROS levels have further substantiated this viewpoint. In addition to this, treatment of skeletal muscle cells with NOX inhibitors, apocynin and diphenyleneiodonium, reversed this effect. The exclusion of extracellular Ca 2+ restrained the increase in levels of ROS as well as calcium spark and the inhibition of Ca 2+ release from the sarcoplasmic reticulum by the inhibitors, ryanodine and thapsigargin were able to further reduce ROS levels [131].
Taken together, the above observations suggested that NOX activation via osmotic stress may be dependent on Ca 2+ release from the sarcoplasmic reticulum. The Ca 2+ could then influx into mitochondria from osmotic stress, leading to induction of NOX activity [131]. Thus, it could be inferred from the above studies that cryopreservation may induce NOX activation. Whether NOX inhibitors can abrogate the ROS generated in post-thawed cells remains to be investigated. Current findings indicate that NOX activation during cryopreservation could be a potential target to reduce ROS-induced damage in cells.

DNA damage, protein oxidation and lipid peroxidation in cryopreservation
Detection of oxidative damage in cryopreserved cells is a valuable measurement to determine the degree of damage. Many consequences of ROS-induced damages can be credited to lipid peroxidation [137], DNA damage [138] and protein oxidation [139,140] (Figure 2). Methods used for measurement of these damages are reviewed by [141].
Protein oxidation, as determined by protein carbonylation were detected in cryopreserved cells. Freezing stress was characterized to lead to the formation of carbonyl groups in intact and homogenized tissue [157]. RBCs cryopreserved with glycerol or trehalose were found to have increased ROS accumulation and protein oxidation. Supplementation of the antioxidant Salidroside ameliorated this effect [158]. Protein oxidation and increased ROS was also detected in cryopreserved sperm cells [159]. Lipid peroxidation can be due to the effect of • OH and ONOO − [160,161]. Increase in lipid peroxidation was observed in tissue specimens stored at −20 • C [162]. Lipid peroxidation as measured by either malondialdehyde (MDA) or 4-Hydroxynonenal (4-HNE) were detected in cryopreserved red blood cells [158], sperm [163][164][165][166] and hepatocytes [167]. Notably, the product of lipid peroxidation 4-HNE is extremely reactive, which allows it to react with DNA and proteins. Antioxidants such as iodixanol can reduce lipid peroxidation in cryopreserved buffalo semen [168].

Effectiveness of antioxidants in preventing cryoinjury: lesson learnt so far Endogenous defense mechanisms and effects of inhibitors on ROS-generating sources in cryopreservation
Transcriptomic studies have shown that many antioxidant genes such as SOD1, cytosolic catalase T (CTT1) and glutaredoxin-1 (GRX1) were induced in the model eukaryote Saccharomyces cerevisiae, also commonly known as the baker's yeast or brewer's yeast [169]. These studies indicated the importance of the role of antioxidants in mitigating freeze-thaw stress after cryopreservation [169]. Intriguingly, genetic screening of yeast mutants defective for different antioxidant genes highlighted that not all antioxidants contribute equally in their ability to protect cells from freeze-thaw stress [87,170]. It was found that yeast strains deleted for SOD1 and SOD2 were particularly sensitive to freeze-thaw stress, while single deletion of catalase and glutathione peroxidase were not as sensitive [170]. In mammals, both vitrification and slow freezing were found to up-regulate SOD gene expression and increase proteins levels in murine oocytes [171], embryos [172] and testicular tissue [142]. Furthermore, the addition of O 2 •− scavenging agent MnCl 2 rescued cells deleted for the SOD1 gene [170]. Collectively, these studies highlight the importance of the SOD genes in cryopreservation of various cell types.
Besides SOD, the reduced GSH regeneration system or the pentose-phosphate shunt for NADPH production were up-regulated in S. cerevisiae during freeze-thaw [169]. Given that GSH is the most abundant antioxidant in almost all cell types [173], it is therefore not surprising that the glutathione cycle is required for freeze-thaw tolerance. Studies where spermatozoa were administered with GSH or thiols were demonstrated to modestly reduce ROS [174], increase the motility of spermatozoa [41,175] and the developmental competence of mouse oocytes [176]. In addition, the GSH and cysteine precursor, S-adenosylmethionine, increased the total GSH levels and the viability of cryopreserved cells. While the supplementation of S-adenosylmethionine lead to significantly lower MDA levels in cold-stored rat hepatocytes, it was however not determined in the cryopreserved cell group [177]. The proliferation of spermatogonial stem cells was however noted to be unaffected by administration of glutathione [178].
Interestingly, one of the more oxidizing environment in the cell is the ER, where the reduced GSH to oxidized glutathione (GSSG) ratio is 3:1 as compared with the cytosol where the ratio is 100:1 [179]. Perturbation of ER homeostasis was known to trigger the UPR. The induction of UPR coincides with a reduction in the developmental competence and modest reductions in survival of cryopreserved cells, which can be improved by supplementation of ER stress inhibitor TUDCA [79,80,82]. The use of Trolox, a water-soluble analog of vitamin E, increased antioxidant capacity, prevented ER stress and improved the viability of ovarian tissues. This indicates a role for both oxidative stress and ER stress during cryopreservation [81]. Intriguingly, it was found that an inhibitor that prevents ER stress-induced apoptosis, Salubrinal, did not improve development and viability of bovine blastocyst [180], indicating that preventing ER stress-induced cell death alone may not be sufficient to prevent cryopreservation-induced damage. However, it is worthy to note that in this specific case, the viability of the blastocyst is close to 100% in both control and Salubrinal treated groups [180].
Gene expression encoding for proteins which regulate or sequester the availability of Fenton reaction initiators are up-regulated in transcriptomic studies in freeze-tolerant animals or in yeast undergoing freezing stress [181,182]. As antioxidants, iron chelators such as deferoxamine, lactoferrin and transferrin were found to limit NO • production and improve cellular parameters affected by cryopreservation-induced oxidative stress [125,183,184]. Deferoxamine was found to prevent loss of highly unsaturated fatty acids in RBCs stored at −20 • C for a shorter period time as compared with the lipophilic free radical scavenger butylated hydroxytoluene (BHT) [184]. Interestingly, supplementation of transferrin, ascorbic acid and a combination of both compounds generally improved the percentage of intact embryos. However only when ascorbic acid was used alone did the number of hatching blastocysts appreciably increase [183]. These studies revealed the complexity of the outcome of cryopreserved cells when using antioxidants as a supplement for cryopreservation.

Apoptosis as an adversity after cryopreservation
The efficiency of the cryopreservation process is still partially compromised due to several factors. Reduced cell viability, increased senescence and impaired cellular functions are among the most widely reported adversities associated with oxidative stress generated during cryopreservation of cells. In spermatozoa, freeze-thawing during cryopreservation greatly reduced cell viability accompanied by a range of structural abnormalities and damages, presumed or found to be a consequence of oxidative stress [185][186][187]. Supplementation of antioxidants into the cryopreservation media generally yielded good cell viability, indicating that oxidative stress plays a role in inducing cellular death during cryopreservation [147,188,189]. Cryopreservation led to re-localization of phosphatidylserine from the inner to the outer leaflet of plasma membrane, a signal displayed by cells undergoing cell death [166,190]. Caspase activation, which is well-known to be involved in mediating the apoptotic cascade, were also observed in cryopreserved sperm cells [191][192][193]. The use of caspase inhibitors significantly improved the viability of hepatocytes and human embryonic stem cells after cryopreservation [194][195][196], indicating that preventing caspase activation can be a plausible approach to improve cell viability.
Some antioxidants may exert their effects through modulation of genes responsible for pro-survival, apoptosis and/or oxidative stress. Melatonin, iodixanol, catalase and vitamin E can up-regulate anti-apoptotic genes such as Bcl2l1 (Bcl-xL) and Bcl-2, while down-regulating pro-apoptotic genes such as BAX/Bax [197][198][199][200]. With regard to melatonin, Deng et al. [199] and Chen et al. [198] observed different outcomes on BAX/Bax modulation. This difference could be attributed to the cell type used in each study. Apart from modulating genes responsible for cell survival, the pro-oxidative genes, ROMO1 (in canine) and NOX5 (in humans) have also been reported to be down-regulated by the administration of iodixanol [200] and melatonin, respectively [199]. The use of melatonin in cryopreservation has been noted to increase human antioxidant genes, such as NRF2 and SOD2 among others as shown in Table 1. It remains unclear if the up-regulation of antioxidant genes after antioxidant treatment provides direct benefit, if any, to protect cells against cryopreservation-induced ROS damage. Trehalose [201,202] and BHT [203] were reported to reduce lipid peroxidation [204] in testicular tissue and spermatozoa, respectively, generally enhance total antioxidant capacity, improve cellular viability [204][205][206][207] and reduce apoptosis [205,207]. Decrease in mitochondrial membrane potential ( ψ) has been observed in cells stimulated by apoptotic stimulus [208][209][210][211], which is also seen in thawed cells after cryopreservation [65]. The reduction in ψ is, however, prevented through antioxidant administration [212][213][214][215][216]. Administration of amino acid with antioxidant properties such as l-proline is one such case where it reduces ROS levels as well as increases ψ [217]. These studies indicate that supplementing antioxidants and/or factors that modulate the process of cell death can be a potential solution to reduce cryopreservation-induced cell death.

Context-dependent effects of antioxidants in cryopreservation
The effectiveness of antioxidants in ameliorating functional parameters during cryopreservation is also dependent on the cell type used as well as the integrity of the cells prior to cryopreservation. This could be observed in the cryopreservation of sperm cells from different organisms. In one example, Dong et al. reported that the beneficial effects of SOD administration were only seen in sperm with low post-thaw survivability [76]. This is also observed for other antioxidants namely coenzyme Q [218], resveratrol [219], zinc sulfate [220], ascorbic acid [150], catalase [76] and 2,4-dinitrophenol [76]. These studies concluded that the effectiveness of antioxidants was dependent on sperm quality. While vitamins C and E may generally reduce DNA damage of spermatozoa from human and Gilt-head seabream (Sparus aurata) [148,149,151], these antioxidants can increase DNA damage in cryopreserved sperm from European seabass (Dicentrarchus labrax) [148] suggesting the possibility that antioxidants ameliorate freeze-thaw stress in a species-dependent manner. In cases where trehalose was used to cryopreserve spermatogonial stem cells, while the proliferation capability of such cells was increased in vitro, this did not translate to a real improvement in the number of colonies formed when such cells were subsequently transplanted [207]. Such disagreement between in vitro and in vivo results can also be seen when mouse embryos were incubated with ascorbic acid prior and post-cryopreservation, where the number of normal fetuses was unchanged despite notable improvements such as embryo intactness as well as blastocyst stage [183]. Consistently, there have also been reports in the literature where cellular functionality saw no improvement after antioxidant supplementation for cryopreservation [41,178,219,221,222]. Table 1 is a summary of the effect of antioxidants on cryopreserving reproductive cells and embryos.

Potential practical applications of antioxidants and their effects on cellular function
Hemopoietic stem cells, hepatocytes and islet cells, all possess enormous potential when transplanted. In some studies, it has been reported that the ability to synthesize proteins [223] such as insulin [224,225] or albumin [226], metabolize xenobiotics [226,227], transplantation potential [226][227][228] and clonogenic potential [229][230][231] may either be lost or impaired via the process of cryopreservation. Such impairments or undesirable outcomes of cellular functionality have been partly improved through administration of antioxidants. These include ascorbic acid [221], astragalosides [232], taurine [222], hypotaurine [178], vitamin E [76], catalase [221], trehalose [205][206][207], combination of catalase with trehalose [233,234] as well as combination of BHT with ascorbic acid [235] to cell types such as mononuclear cells, pancreatic islets, germ cells, spermatozoa, dendritic cells, hepatocytes and hemopoietic cells (Table 1). Among these antioxidants, winter wheat lipocalins and peroxiredoxins obtained from wheat are especially notable. They were demonstrated to mollify cryopreservation-associated loss of attachment capacity of hepatocytes, as well as restoring the activity of CYP isoforms to the level similar from fresh, unfrozen murine hepatocytes [227,236]. Table 2 is a summary of the effect of antioxidants on non-reproductive cells.
Collectively, the different studies examined in this review indicated that the effectiveness of antioxidant supplement for cryopreservation very much depends on the cell type, organism as well as the specific antioxidant used. Table 1 provides a summary of the type of the antioxidant used, the cell type and organism as well as the effectiveness of the antioxidant based on the parameters measured.

Moving forward
Oxidative stress is inevitably generated in the cryopreservation process and has been widely cited as the causative factor for some of the cryoinjuries inflicted on the cell [163,165,187,237]. Therefore, administering antioxidants in an effort to counter these deleterious effects on cells during cryopreservation is a plausible solution. Indeed, the use of antioxidants has undoubtedly conferred protection to certain cell type by improving several cellular function parameters and general cryopreservation outcome in specific circumstances as those indicated in the sections above and in Tables 1 and 2. Although effective in some circumstances, antioxidants can be ineffective or even deleterious for some cells. Antioxidants consist of a broad class of substances and molecules with varying physio-chemical properties that dictate their specificity, localization and/or ROS-scavenging roles [238,239]. Mitochondria-targeted antioxidants, MitoTEMPO [214] and melatonin [240,241] are potent antioxidants that prevent oxidative stress-associated damages encountered during cryopreservation. Melatonin in particular, has performed unexpectedly well by exerting its ROS-ameliorating properties through its multi-faceted mechanisms [242]. While the administration of melatonin improved the generation and survival of somatic cell nuclear transferred (SCNT) murine embryos from vitrified oocytes, whether melatonin directly affects ROS or inhibits apoptosis remains to be elucidated [243]. As such, the use of antioxidant in different combinations for the different cell types for cryopreservation may prove to be more effective in countering cryopreservation-induced ROS damage.
There are evidences to indicate that the use of different antioxidants in combination could provide additive protective effect when compared with those administered individually (Tables 1 and 2). In reproductive cells, catalase and low concentration of SOD have been reported to have no effect on oocyte survivability and fertility when used alone. However, when the same dose of SOD was co-administered with catalase, significant improvement in oocyte survivability was observed [124]. For the case of sperm cryopreservation, supplementation of SOD alone has no effect on the general sperm parameters such as motility (total and progressive motility), viability and percentage of sperm with high MMP, while supplementation of catalase alone was beneficial only at high concentrations [244]. Notably, when catalase and SOD were used in combination, sperm parameters such as total motility was greatly improved as compared with individual use of them at the respective dose [244]. In another example, sperm cells frozen in SOD and catalase, or vitamins C and E led to significantly improved parameters such as reduced ROS [215] and increased lateral head displacement of the sperm cells [245] whereas previously such parameters were not improved when the antioxidants were used individually [215,245].
Not all antioxidants used in combination yield additional benefits. For example, single administration of trehalose or catalase improved clonogenic parameters such as burst-forming unit erythroid and colony-forming unit granulocyte-monocyte in fetal liver hematopoietic cells and umbilical cord blood, respectively. However, when trehalose and catalase were administered in combination, no significant improvements in these parameters were observed [246]. Hence, use of different antioxidants in combination does not always imply additional improvement in cellular parameters.
Based on the studies examined in this review, it is notable that antioxidant supplement for cryopreservation can be effective. However, the effectiveness of the specific antioxidants depends on the cell type that undergoes the cryopreservation process. It is therefore important to consider supplementing cryopreservation media with specific antioxidants according to the specific species, cell type, quality and integrity of cells prior to cryopreservation. Additionally, several studies determined the efficacy of the antioxidants on cryopreservation by measuring functional parameters of the cryopreserved cells in an ex vivo setting. Given that many applications for cryopreservation are in the area of reproductive and regenerative medicine, future studies should be attempted to investigate the recovery and efficacy of the cryopreserved cells after transplantation in vivo to better understand the efficacy of the antioxidant used in this process.