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Cryoprotectant

A cryoprotectant is a used to protect biological tissues, cells, and organs from damage caused by formation during , enabling storage at cryogenic temperatures while preserving functionality upon thawing. These agents work by lowering the freezing point of , increasing solution , and stabilizing cellular structures through mechanisms such as , where the solution transitions to a glass-like state without crystals. Cryoprotectants are essential in fields like , , and reproductive , where they facilitate the long-term preservation of , embryos, cells, and tissues for transplantation or research. The primary mechanisms of cryoprotectants involve that reduce intracellular ice formation by dehydrating cells and moderating electrolyte concentrations, as well as direct stabilization of biomembranes via hydrogen bonding and water replacement. Penetrating cryoprotectants, such as (DMSO) and , diffuse across cell membranes to protect intracellular compartments, while non-penetrating types like and polyvinylpyrrolidone (PVP) act extracellularly to inhibit ice recrystallization and osmotic stress. Natural cryoprotectants, including antifreeze proteins from cold-adapted organisms and sugars like , mimic these effects by binding to ice surfaces or stabilizing lipid bilayers, often reducing oxidative damage from during freeze-thaw cycles. Common applications include cryopreservation of hematopoietic stem cells for cancer therapy, where combinations of 5-15% DMSO with sugars improve post-thaw viability to over 80%, and techniques for oocytes and embryos in assisted reproduction, minimizing chilling injury. However, challenges persist due to potential at high concentrations, such as DMSO-induced affecting , prompting into less toxic natural alternatives like and deep eutectic solvents for enhanced cytoprotection. Ongoing advancements focus on optimizing formulations to balance efficacy and safety across diverse biological systems.

Definition and Overview

Definition

Cryoprotectants are chemical compounds or mixtures added to aqueous solutions to protect biological structures, such as cells, tissues, and organs, from damage caused by ice formation during freezing processes in cryopreservation. These agents are essential for enabling the long-term storage of viable biological materials at ultra-low temperatures, typically below -130°C, by mitigating the physical and chemical stresses associated with phase transitions of water. The core functions of cryoprotectants include minimizing intracellular growth, which can puncture membranes and disrupt cellular architecture; reducing the effects of solute concentration increases during freezing, which otherwise lead to osmotic and ; and stabilizing biomolecular structures like proteins and membranes through interactions such as hydrogen bonding with molecules. By performing these roles, cryoprotectants help preserve cellular integrity and functionality upon thawing. Cryoprotectants are broadly classified into two categories based on their ability to penetrate membranes: permeable cryoprotectants, which cross cellular barriers to act intracellularly (e.g., and ), and non-permeable cryoprotectants, which remain extracellular and primarily influence the surrounding environment (e.g., sugars like and polymers like ). This classification guides their application in protocols to balance protection and potential toxicity. A key physical principle underlying their efficacy is the colligative property of lowering the freezing point of solutions to delay ice nucleation while elevating the temperature (Tg), which promotes the formation of a stable, amorphous state instead of crystalline during rapid cooling—a process known as .

Historical Development

The concept of cryoprotection emerged from early 20th-century investigations into the effects of low temperatures on biological materials. In , Basile J. Luyet and Paul M. Gehenio conducted pioneering studies on , demonstrating that rapid cooling could transform water in tissues into a glass-like state without formation, potentially preserving cellular integrity. Their work, detailed in publications such as Life and at Low Temperatures (1940), highlighted the detrimental role of intracellular and laid foundational ideas for avoiding freezing damage through non-crystalline solidification. A major breakthrough occurred in 1949 when Christopher Polge, Audrey U. Smith, and Alan S. Parkes accidentally discovered 's protective effects during experiments on spermatozoa. By adding glycerol to samples before freezing and thawing, they achieved rates exceeding 50% in multiple , marking the first reliable use of a permeable cryoprotectant to mitigate ice-induced . This serendipitous finding, stemming from contamination in a lab batch, revolutionized preservation and spurred broader applications in cell banking. The 1950s and 1960s saw expansion with the introduction of (DMSO) by James E. Lovelock and Mervyn W. H. Bishop in 1959, who demonstrated its efficacy in protecting human and bovine red blood cells from freezing injury by reducing concentration effects. Unlike , DMSO's high permeability allowed rapid penetration into cells impermeable to larger molecules, enabling survival rates of up to 80% post-thaw in erythrocytes. Concurrently, slow-freezing protocols were refined, optimizing cooling rates to 1°C per minute to minimize intracellular ice while leveraging cryoprotectants for extracellular stabilization. From the 1980s onward, techniques gained prominence, with William F. Rall and Gregory M. Fahy reporting in the successful ice-free of at -196°C using high-concentration mixtures of permeable cryoprotectants like DMSO, , and . This approach achieved over 90% embryo survival by inducing a glassy state, significantly reducing cryoprotectant through minimized exposure times compared to slow freezing. In the , efforts shifted toward organ , where challenges like cryoprotectant , osmotic stress, and uneven distribution persisted, limiting success to small tissues despite advances in screening. By the 2020s, research emphasized bio-based alternatives, such as and proteins from natural sources, which improved cell viability in and by inhibiting ice recrystallization with reduced —e.g., L-proline oligomers boosting survival to 99% alongside low-dose DMSO.

Mechanisms of Action

Vitrification and Glass Transition

refers to the process of rapidly cooling a to form a stable , or , rather than crystalline , thereby avoiding the mechanical damage caused by formation during . This biophysical mechanism is central to the protective action of cryoprotectants, which enable the transition to a vitreous state at practical cooling rates, preserving the structural integrity of biological samples. By suppressing and growth of , vitrification maintains the sample in a non-crystalline, supercooled liquid-like state that solidifies without . The temperature () is the critical point below which the supercooled liquid acquires the properties of a viscous, glassy solid, with dramatically reduced molecular mobility that inhibits formation. Cryoprotectants elevate the of aqueous solutions, allowing stabilization at temperatures above the homogeneous temperature of pure water, thus facilitating under controlled cooling conditions. For instance, permeable cryoprotectants like DMSO increase by disrupting water's hydrogen bonding network and forming a more rigid matrix. The Gordon-Taylor equation is commonly used to predict Tg in binary mixtures of cryoprotectants and water: T_g = \frac{w_1 T_{g1} k + w_2 T_{g2}}{w_1 k + w_2} where w_1 and w_2 are the weight fractions of the cryoprotectant and water, respectively, T_{g1} and T_{g2} are their respective glass transition temperatures, and k is an empirical constant reflecting intermolecular interactions. This model accurately describes how cryoprotectant addition shifts Tg, aiding the design of vitrification solutions. High concentrations of cryoprotectants, typically 40-50% w/v, are essential to achieve while preventing —recrystallization—during rewarming, as they sufficiently raise to suppress throughout the cycle. Factors such as molecular weight, with higher values generally increasing due to reduced chain mobility; hydrogen bonding capacity, which strengthens the glassy ; and interactions with , where cryoprotectants act as plasticizers or stabilizers, profoundly influence this elevation.

Ice Crystal Inhibition and Stabilization

Cryoprotectants inhibit ice crystal formation primarily through colligative effects that reduce the amount of free available for and . By increasing the solute concentration in solution, these agents lower the freezing point via , described by the equation \Delta T_f = K_f \cdot m, where \Delta T_f is the change in freezing temperature, K_f is the of the , and m is the of the solute. This colligative property, rooted in , disrupts the equilibrium between ice and liquid , thereby decreasing the temperature at which ice crystals initiate and expand. For instance, high concentrations of permeable cryoprotectants like (DMSO) form hydrogen bonds with molecules, further elevating the fraction of unfrozen and suppressing ice propagation. In addition to colligative mechanisms, cryoprotectants can adsorb directly onto nascent nuclei or surfaces, sterically hindering their growth. This adsorption-inhibition process limits the incorporation of molecules into the , effectively capping size and preventing the formation of sharp, damaging edges. Specialized -binding proteins, such as proteins (AFPs), exemplify this through their flat ice-binding faces that anchor to specific planes (e.g., basal or ) on , curving the - interface and inhibiting further advancement. Cryoprotectants also stabilize biomolecules against freeze-induced damage by preserving native structures and mitigating stress during changes. They maintain hydration shells around proteins, preventing denaturation by counteracting the loss of bound water that occurs as forms and concentrates extracellular solutes. Similarly, these agents reduce osmotic stress and inhibit deleterious transitions from to states, which could otherwise lead to leakage or rupture of bilayers. Non-permeating sugars like , for example, form a protective matrix that supports and integrity during dehydration. A critical distinction in ice formation lies between extracellular and intracellular locations, with cryoprotectants preferentially minimizing the latter to avoid mechanical disruption. Extracellular , which forms during slow cooling, induces cell shrinkage via but generally spares organelles; however, intracellular —arising from rapid freezing—produces sharp crystals that can puncture membranes and organelles, leading to . Permeable cryoprotectants penetrate cells to equilibrate osmotic gradients, thereby reducing the propensity for intracellular and growth while promoting controlled extracellular ice formation. Antifreeze proteins serve as potent, naturally evolved inhibitors of dynamics, exhibiting thermal hysteresis and recrystallization inhibition. Thermal hysteresis creates a gap between the and the non-equilibrium freezing point, where AFPs bind to ice surfaces and generate pinning sites that resist ice front propagation at temperatures below the . During thawing, these proteins further prevent recrystallization—the merging of small crystals into larger, more destructive ones—by adsorbing to grain boundaries and maintaining crystal multiplicity. This dual action, observed in polar and , has inspired biomimetic designs for enhanced . During freezing, cryoprotectant concentration gradients emerge as ice forms extracellularly, progressively concentrating solutes in the unfrozen channels and exacerbating the solute effect—increased and pH shifts that destabilize cells. To mitigate this, combinations of permeable and non-permeable cryoprotectants are employed, allowing intracellular equilibration to buffer osmotic imbalances and limit extreme solute buildup, often reducing required concentrations by up to 50% while preserving viability.

Toxicity Considerations

Cryoprotectants can induce various forms of toxicity during , primarily arising from their chemical interactions with cellular components, osmotic imbalances, and exposure to subzero temperatures. These adverse effects limit the concentrations usable for effective ice prevention, particularly in protocols requiring high levels. Chemical toxicity manifests through direct disruption of cellular structures, such as (DMSO) extracting from bilayers, which decreases thickness, forms transient pores at 10-20% concentrations, and destroys bilayer integrity at 25-30%. This solvent-like action compromises membrane permeability and function, leading to leakage of intracellular contents. , while less disruptive, can depolymerize filaments in at concentrations above 1.5%, impairing cytoskeletal stability. Osmotic toxicity occurs due to rapid volume changes in cells during CPA addition or removal; hypertonic solutions cause shrinkage, while hypotonic conditions lead to swelling and potential , as seen with glycerol's low permeability exacerbating extracellular . Chilling injury below 0°C further compounds this, as without ice formation triggers phase transitions in membranes and proteins, promoting instability even in the presence of CPAs. Toxicity is highly dose-dependent, with concentrations exceeding 10% v/v often inducing via activation or through membrane blebbing and ultrastructural damage; for instance, DMSO above 1.41 M (about 10% v/v) reduces in rats and triggers mitochondrial permeability transition. Exposure time and temperature modulate these effects, as prolonged contact at warmer temperatures accelerates chemical reactions, while lower temperatures slow permeation but prolong osmotic stress. Species-specific sensitivities influence vulnerability, with mammalian cells generally more susceptible than cells owing to differences in ; mammalian plasma membranes, rich in unsaturated , undergo greater perturbations from CPAs like DMSO, whereas cells maintain rigidity through sterols and natural solutes, enhancing tolerance. For example, embryos exhibit higher glycerol toxicity compared to bacterial cells, highlighting phylogenetic variations in CPA . Basic mitigation involves stepwise addition and removal of CPAs to minimize osmotic shock, allowing gradual equilibration and reducing peak intracellular concentrations. Antioxidants, such as ascorbic acid, counteract (ROS) generated by CPAs like DMSO during freezing, which oxidize and proteins, thereby preserving cell viability. Key toxicity metrics include LD50 values; for , the oral LD50 in mice is approximately 4.1 g/kg, indicating relatively low acute systemic risk compared to DMSO's 17.4 g/kg in rats, though cellular effects dominate in cryopreservation contexts.

Types of Cryoprotectants

Permeable Cryoprotectants

Permeable cryoprotectants are small molecules with molecular weights typically below 500 Da that can diffuse across membranes to provide intracellular protection during . These agents primarily function by colligatively depressing the freezing point of , inhibiting intracellular ice formation, and stabilizing biomolecules through interactions like bonding. Key properties include high aqueous , relatively low to facilitate mixing and penetration, and the ability to form bonds with molecules, which disrupts ice crystal lattice formation. However, they often exhibit drawbacks such as potential at higher concentrations, arising from osmotic or chemical interactions with cellular components. Among the most widely used permeable cryoprotectants is (1,2,3-propanetriol), a that penetrates cell membranes slowly but effectively protects against freezing damage. Discovered as a cryoprotectant in by Polge, Smith, and Parkes during experiments on spermatozoa, has since become a standard for slow-freezing protocols in . Its efficacy stems from its nature, enabling strong hydrogen bonding with water to prevent ice nucleation. Dimethyl sulfoxide (DMSO) is another prominent example, known for its rapid membrane penetration due to its amphiphilic structure. Introduced by Lovelock and in as a protective for red blood cells during slow cooling, DMSO is commonly employed at concentrations of 10-15% v/v for mammalian cells and tissues. It excels in applications by quickly equilibrating intracellularly, but its higher toxicity compared to necessitates careful post-thaw removal to avoid adverse effects like . Ethylene glycol serves as a less toxic alternative to DMSO, particularly in and vitrification, where it penetrates efficiently and minimizes osmotic injury. With a molecular weight of 62 Da, it allows for higher concentrations without excessive , often used in mixtures to balance efficacy and safety. In usage protocols, permeable cryoprotectants like these are typically added stepwise to achieve equilibrium distribution across cell membranes during slow freezing at rates of 0.3-1°C/min, with final concentrations ranging from 5-20% v/v to optimize and solute while limiting . Comparatively, DMSO offers superior performance for rapid cooling scenarios due to its faster , achieving better post-thaw viability in sensitive cells than , which requires longer equilibration times. However, generally exhibits lower , making it preferable for applications where prolonged exposure is unavoidable, such as in .

Non-Permeable Cryoprotectants

Non-permeable cryoprotectants are solutes that do not cross cell membranes, including both small compounds lacking membrane transporters and large polymers, thereby acting primarily in the to create osmotic gradients that promote cellular and prevent intracellular formation during freezing. These agents remain confined to the extracellular compartment, aiding in by stabilizing the external environment. Key examples include (PVP), a synthetic used as a cryoprotectant in and cell preservation due to its ability to inhibit ice recrystallization extracellularly. and , non-permeable disaccharides, stabilize biomolecules by replacing water molecules in hydrogen bonding networks, with exhibiting superior glass formation owing to its unique α-1,1-glycosidic linkage that yields a higher temperature compared to . (HES), a modified , serves as an effective extracellular stabilizer in organ and cryopreservation, enhancing viability by promoting and reducing growth. These cryoprotectants are typically employed at concentrations of 10-30% w/v, such as 2-6% HES to permeable agents or 0.3-1 M (approximately 10-34%) for and , balancing efficacy with practicality in handling viscous solutions. 's di-saccharide structure contributes to its effectiveness at these levels by forming a more stable amorphous glass state during cooling. Non-permeable cryoprotectants offer advantages including lower toxicity relative to permeable counterparts, as they avoid intracellular accumulation, and are frequently combined with penetrating agents in protocols to optimize osmotic balance and overall outcomes.

Natural Cryoprotectants

In Insects

employ cryoprotectants to either avoid freezing by body fluids or tolerate extracellular ice formation while protecting intracellular compartments. Freeze-avoiding species accumulate polyhydric alcohols like and to depress the freezing point and enhance capacity. Freeze-tolerant , such as the larvae of the gall fly Eurosta solidaginis, rely on polyhydric alcohols like and (up to 0.5 M in northern populations), alongside disaccharides like and such as at elevated levels (up to several hundred mM) to stabilize membranes and proteins during controlled extracellular freezing. These species often produce nucleators to initiate freezing at higher subzero temperatures ( points around -10°C to -20°C), allowing controlled formation while cryoprotectants limit damage. Some freeze-tolerant also have -binding proteins that inhibit recrystallization, thereby limiting intracellular damage. The synthesis of these cryoprotectants occurs via rapid enzymatic conversion from stored reserves during acclimation, often triggered by decreasing temperatures in autumn. This involves activation of and the , leading to seasonal shifts in composition where polyols and sugars dominate in winter. Representative examples illustrate the diversity of insect adaptations. The Antarctic midge Belgica antarctica accumulates (along with glucose, , and ) to up to 100 mM or more, supporting freeze tolerance in larvae exposed to -15°C or lower during the austral summer. In bark beetles like Cucujus clavipes, serves as a non-toxic cryoprotectant, accumulating to high levels (often >100 mM) to stabilize proteins and contribute to without the toxicity risks of polyols. These strategies provide adaptive benefits, with particularly effective in lowering the freezing point colligatively (approximately 1.86°C per mole) while also stabilizing molecular structures, allowing survival at -20°C to -30°C without in many .

In Amphibians and Other Vertebrates

Amphibians and certain other vertebrates have evolved natural cryoprotective strategies to survive subzero temperatures, primarily through the accumulation of endogenous cryoprotective agents (CPAs) that enable either freeze or avoidance. In temperate zone amphibians, freeze is a prominent , allowing partial extracellular freezing while protecting intracellular fluids. This contrasts with polar , such as certain , which employ freeze-avoidance mechanisms to maintain all body fluids in a state below the freezing point. These strategies reflect evolutionary divergences: temperate favor to endure episodic freezes during , whereas polar prioritize avoidance to prevent any formation in perpetually cold environments. The wood frog (Rana sylvatica), a classic example of freeze tolerance in amphibians, accumulates massive amounts of glucose as its primary CPA during winter hibernation. Triggered by extracellular ice nucleation, the liver rapidly mobilizes glucose from glycogen stores, reaching plasma concentrations exceeding 200 mM and up to 300 mM in vital organs like the heart and liver. This hyperosmotic response draws water out of cells via cryoprotective dehydration, minimizing intracellular ice formation and stabilizing cellular structures during freezing episodes that can reach -16°C in subarctic populations. Glucose mobilization is organ-specific, with high levels prioritized in the brain, heart, and liver to preserve essential functions, while peripheral tissues rely on lower concentrations. In some Australian burrowing frogs, such as species in the genus Cyclorana, urea serves as a key permeable , accumulating to levels of 50-100 mM or higher during periods of environmental stress like and . As a small-molecule , urea permeates cell membranes to counteract and ionic imbalances, enhancing survival in both dry and cold conditions by stabilizing proteins and membranes without the osmotic extremes of glucose. This adaptation is particularly vital in arid-temperate regions where frogs face combined and frost risks. Among other vertebrates, polar fish like Antarctic notothenioids and Arctic cod produce glycoproteins (AFGPs), which are repetitive glycopeptides secreted into the blood to inhibit growth and lower the freezing point of bodily fluids by 1-2°C. These AFGPs bind to surfaces, preventing recrystallization and enabling freeze avoidance in that is -1.9°C. In contrast, freeze-tolerant reptiles such as the wood turtle (Glyptemys insculpta) employ glucose accumulation similar to amphibians, combined with upregulated defenses like and to mitigate oxidative damage from ischemia-reperfusion during thaw. Mechanisms underlying these adaptations include organ-specific mobilization of CPAs, often regulated by hormonal signals like norepinephrine in amphibians, and cryoprotective dehydration facilitated by aquaporins—water channel proteins that selectively permit extracellular ice-driven water efflux while retaining intracellular solutes. In wood frogs, aquaporin expression in skin and bladder aids controlled dehydration, limiting body water loss to 65% during freezing and preventing lethal intracellular icing. This aquaporin-mediated process underscores the integration of membrane transport with osmotic protection across freeze-tolerant vertebrates.

Applications

Cryopreservation of Cells and Tissues

Cryopreservation of cells and tissues relies on cryoprotectants to mitigate damage from ice formation, osmotic , and chilling during cooling to subzero temperatures and subsequent thawing. In biomedical applications, these agents enable the long-term of viable biological materials for clinical use, such as fertility preservation and . Protocols typically involve either slow freezing, which allows controlled ice nucleation outside cells, or , which achieves an amorphous state to prevent entirely. Success depends on optimizing cryoprotectant concentration, cooling/warming rates, and post-thaw recovery to maintain cellular . Slow freezing protocols for cells like hematopoietic stem cells commonly use permeable cryoprotectants such as 10% (DMSO) in a stepwise manner, with cooling rates of 1-2°C/min down to -80°C before transfer to . This method promotes extracellular ice formation while minimizing intracellular ice, achieving post-thaw viabilities exceeding 80% for mesenchymal stem cells when combined with appropriate serum or polymers. For instance, rat mesenchymal stem cells cryopreserved with 10% DMSO at 1°C/min exhibited immediate post-thaw viability around 85%, with sustained proliferation over days. Vitrification, in contrast, employs flash-freezing with high-concentration mixtures of 20-40% permeable and non-permeable cryoprotectants, such as combined with , to induce a glass-like state without crystals. For oocytes, a multi-step protocol equilibrates cells in increasing concentrations of (up to 12.5 mol/kg) plus 0.75 mol/kg , followed by plunging into , yielding high survival rates by avoiding ice-induced damage. This approach has become standard for delicate structures like oocytes, where post-thaw integrity is critical for fertilization potential. Key applications include and banking, established since the 1950s for fertility preservation, and cryopreservation of hematopoietic cells for transplants, where 10% DMSO enables engraftment rates comparable to fresh cells. In tissue engineering, cryoprotectants facilitate storage of skin grafts, maintaining viability for up to years in allogeneic models to support burn treatment and . These methods ensure off-the-shelf availability of cells and tissues for therapies. Recent advances as of 2025 include strategies for cryopreserving biofabricated tissues using matrices as cryoprotective supports. Standard protocols encompass cryoprotectant agent () loading through stepwise exposure to minimize toxicity, controlled cooling or , rapid warming to prevent recrystallization, and gradual unloading to avoid osmotic swelling. For example, loading occurs at 0-4°C in incremental concentrations, followed by warming at 37°C in a water bath and dilution with isotonic media to remove CPAs safely. These steps are essential to preserve integrity and metabolic activity across types. Post-thaw recovery rates serve as primary success metrics, often exceeding 80% for isolated cells but declining for thicker tissues due to uneven CPA penetration and thermal gradients. Challenges in scaling to whole organs persist, as seen in kidney trials using VMP vitrification solution, where rat models achieved functional recovery after 100-day storage via nanowarming, though initial dysfunction and ice formation limit human translation. Ongoing refinements aim to enhance uniformity for clinical viability.

Food Preservation and Processing

Cryoprotectants play a crucial role in by mitigating the adverse effects of freezing, such as formation that leads to cellular damage, drip loss, and protein denaturation. These additives stabilize structures during freeze-thaw cycles, preserving , , and nutritional quality in products like meats, , , and fruits. By lowering the freezing point and inhibiting large growth, cryoprotectants enable efficient industrial freezing processes while minimizing quality degradation over extended storage periods. Emerging bio-based cryoprotectants, such as natural sugars and proteins from or microbes, are gaining traction for sustainable applications as of 2025. In seafood processing, sugars such as , a non-permeable cryoprotectant, are widely used at concentrations of 1-5% to prevent drip loss and protein denaturation in fish fillets. For instance, in frozen fillets treated with 5% , thawing loss was reduced from 9.18% (control) to 6.60% by maintaining integrity and limiting ice recrystallization during storage. This stabilization is particularly effective in and fillets, where at 5-8% concentrations enhances water-holding capacity and retards myofibrillar protein changes, ensuring better gel-forming ability upon thawing. Glycerol serves as a common permeable cryoprotectant in frozen dairy products like to promote smoothness by reducing size and preventing coarsening during storage. This addition lowers the freezing point of the mixture, resulting in a creamier and decreased , which enhances sensory appeal without significantly altering . In fruit preservation, polymers are employed to further reduce formation, protecting cellular structures and minimizing structural collapse in frozen berries and other produce. Industrial techniques like air-blast freezing, which circulates cold air at -30°C to -40°C over , are often combined with cryoprotective agents to accelerate freezing rates and limit dehydration in products such as fillets. For higher-quality preservation, cryogenic methods using achieve ultra-rapid freezing, and when enhanced by polymers, they form smaller ice crystals that preserve the integrity of delicate foods like and . These approaches reduce and maintain product weight, with air-blast methods yielding typical losses of 1-2% in unpacked . The use of cryoprotectants extends for meats and to 6-12 months at -18°C to -25°C by inhibiting microbial growth and oxidative changes, as seen in treated fish mince that retains functional properties far longer than untreated samples. Nutritionally, they support retention of key vitamins, such as in , where rapid freezing with protectants inactivates degradative enzymes and preserves substantial levels of initial content after months of storage. Regulatory oversight ensures safe application, with the U.S. Food and Drug Administration (FDA) approving cryoprotectants like , , and at specified levels to meet safety standards without health risks. The is shifting toward bio-based cryoprotectants, such as natural sugars and proteins derived from plants or microbes, to replace synthetics and align with consumer demand for cleaner labels and .

Challenges and Future Directions

Toxicity Mitigation Strategies

One effective strategy to mitigate cryoprotectant involves stepwise equilibration, where cryoprotective agents (CPAs) are added gradually to or tissues to minimize osmotic stress and intracellular concentration spikes. For instance, a two-step procedure for addition begins with exposure to 0.87 mol/kg combined with 0.08 Osm/kg nonpermeating solute for 12 minutes at 37°C, followed by a rapid shift to 20 mol/kg with 0.2 Osm/kg nonpermeating solute for 1 minute at 4°C. This approach keeps volume within tolerable limits (0.2–2 times volume), reducing exposure compared to direct high-concentration loading. CPA cocktails, combining multiple agents at reduced individual concentrations, further lower toxicity while maintaining cryoprotective efficacy. A common formulation pairs (DMSO) with ; for example, 10% DMSO supplemented with 50 mM enhances post-thaw viability of murine spermatogonial stem cells to 90%, compared to 76% with 10% DMSO alone, by stabilizing membranes and mitigating oxidative damage. Similarly, 5% DMSO with 146 mM supports of umbilical cord blood stem cells equivalent to non-frozen controls, allowing a 50% reduction in DMSO dosage without compromising protection. Incorporating antioxidants and buffers into CPA solutions addresses (ROS) generation and pH fluctuations during exposure. , at 200 U/ml, scavenges in cryoprotective media containing 10% or , preserving membrane integrity in sperm at 95% during 10-minute pre-freeze equilibration (versus 70–75% without ). Alternative delivery methods enhance precision and reduce systemic exposure to toxic CPAs. Polymeric nanoparticles facilitate targeted intracellular release of agents like , bypassing membrane impermeability and minimizing extracellular high concentrations of permeable CPAs such as DMSO, thereby lowering overall toxicity while improving low-temperature viability. induces endogenous production of protective defenses; for example, inactivating class-I PI3K in C. elegans via the age-1(mg44) boosts survival to 94% after 5-minute exposure to 75% M22 CPA at 20°C, compared to 7.5% in wild-type, by upregulating cellular stress responses. These strategies collectively enable 20–50% reductions in effective concentrations, such as halving DMSO from 10% to 5% in combinations, while improving post-thaw viability from as low as 10% to over 80% in endothelial cells.

Emerging Bio-Based and Synthetic Advances

Recent advancements in bio-based cryoprotectants have focused on derivatives and analogs derived from bacterial sources to enhance organ preservation outcomes. For instance, sulfoxide-functionalized has been developed as a DMSO-free alternative, enabling of peripheral blood mononuclear cells (PBMCs) with preserved expansion potential, , and comparable to traditional methods. Plant-derived options, such as peptides, have emerged as effective cryoprotectants by mimicking proteins (AFPs), inhibiting recrystallization and maintaining high post-thaw viability of bulgaricus. These bio-based agents address concerns while promoting , with polyphenols from plants like exhibiting AFP-like thermal hysteresis to stabilize networks in frozen doughs. Synthetic innovations include -based ice blockers and fluorinated compounds designed for reduced and enhanced penetration. antifreeze agents, such as those derived from bovine , provide dual cryoprotection by adsorbing to surfaces and preserving rheological properties. In 2024 research, spontaneous coatings on surfaces delayed freezing by 5°C, offering potential for scalable protection without genetic modification. These synthetics enable with minimized formation, as seen in polyampholyte formulations that boost post-thaw recovery in mammalian cells by 20-30%. A pivotal development in 2024 involved the of human-scale volumes using the M22 solution, achieving successful glass formation in 3-liter bags without cracking during nanowarming, a step toward clinical banking. Computational models have optimized mixtures, such as those guided by mass transport simulations, reducing exposure times by approximately 20% while maintaining tissue viability above 85% in rat models. In September 2025, researchers at reported advancements in achieving a glass-like state for , potentially improving viability in and biological sample storage without relying on toxic CPAs. These advances tackle scalability challenges, with bio-based options mitigating environmental impacts from synthetic fluorocarbons. Looking ahead, integration of such CPAs with promises enhanced recovery of engineered tissues, potentially securing regulatory approvals for routine use by 2030 as protocols mature.