A perennial activity of humankind since the oldest recorded times was to prevent the spoilage of food, especially meat. To that end, methods have been continuously developed, such as cooking, sun drying, smoking, fermenting, or salting. In regions with a warm climate, cooling and freezing also emerged as methods, albeit they implied costly efforts with harvesting the ice and storing it in built pits, cellars, or icehouses, therefore cryopreservation of food and drinks remaining accessible mainly to the privileged classes. Ice also helped people to cool down in the scorching heat, as our species always strived for pleasant living conditions. There is recorded evidence that, 2,000–4,000 years ago, processing and using ice was an organized human activity in the ancient Mesopotamia (Ellison, 1978; Sasson, 1984; Biot, 1851; Li, 2022). Similar pursuits were recorded in Egypt, India, Greece and Roman Empire (Love, 2009). Interestingly, artificial cooling was proposed in Egypt for medical applications, as described in the Edwin Smith Surgical Papyrus. Written in the period 3000 to 2500 BCE, and re-copied around 1700 BCE, this manuscript is considered one of the most valuable ancient medical texts to ever be brought to light and translated into a modern language (Atta, 1999; Feldman and Goodrich, 1999). In the original text (Breasted, 1930), the process of cooling was mentioned as a stage in certain surgical protocols, most likely as a remedy against inflammation, while the cooling technique was not described.

Coming much closer to our times, it is known that Francis Bacon (1561–1626), a philosopher, jurist and scientist considered one of the creators of the modern world, has shown interest in the application of low temperatures for preservation of meat (Jardine and Stewart, 1998). He was also seeking an explanation of the effects of cooling in general, rather than aiming only at a simple application for food conservation. Freezing temperatures in his experiments were achieved by using snow, ice or ice-salt slurries. Bacon’s interest in low temperatures was part of his hypothesis regarding the prolongation of life by the conservation and induration of tissues caused by exposure to low temperatures. At that time, the term ‘induration’ referred to hardening or congealing, somewhat different from today’s meaning (i.e., thickening and hardening of soft tissues due to inflammatory processes; e.g., sclerosis). One of Bacon’s experiments carried out with an eviscerated chicken carcass, stuffed and packed with snow, might have led eventually to his death as he caught pneumonia during the outdoor experiment (Clark, 1898; Francis Bacon, 1993). The episode was first described (Clark, 1898), based on the information provided by Bacon’s friend, the great philosopher Thomas Hobbes. As the historian Thomas Macaulay later stated, “The great apostle of experimental philosophy was destined to be its martyr” (Macaulay, 1882). Although Bacon himself wrote before his death a letter mentioning the chicken experiment, which “succeeded excellently,” there is still skepticism among historians regarding the veracity of this particular episode. There is no doubt, however, that Bacon was a true pioneer of the conservation of tissues at low temperatures (Jardine and Stewart, 1998).

Robert Boyle (1627–1691), a chemist, physicist and inventor, regarded as the founder of modern chemistry, studied the direct action of low temperatures on vegetal and animal tissues. The results were published in a book (Boyle, 1665), mainly dedicated to his “doctrine” of cold, including aspects like freezing techniques and applications to various liquid substances, expansive force of freezing water, relation water-air during freezing, and thawing processes. He also investigated the freezing of plants, animal flesh and excised animal organs (eyes, livers, brains and tongues), using snow-salt mixtures. Employing occasionally a microscope, Boyle discovered that upon freezing the biological fluids (“alimental juices”) transformed into ice crystals (“icy corpuscles”) and separated from the remaining solid matrix. Showing a remarkable prescient insight, he hypothesized that the ice crystals could cause damage to the tissue, and thus accelerating the decomposition of biological matter during and after thawing. To avoid premature decay, Boyle recommended that the thawing should be done slowly (“by degrees”) in an aqueous medium, rather than direct exposure to external heating.

Notable work on the effect of low temperatures was carried out on living matter by Lazzaro Spallanzani (1729–1799) (Spallanzani, 1776; Sandler, 1973; Bwanga, 1991; Sztein et al., 2018), a biologist and a polymath who contributed to the development of many fields of science including reproductive medicine, cryobiology, paleontology, physiology, and echolocation. He demonstrated experimentally that when exposed to temperatures at which water freezes, the animal and human spermatozoa became motionless, only to become active again when the temperature was raised. It took a century for this remarkable finding to be further investigated by Paolo Mantegazza (1831–1910), who documented that human spermatozoa could be frozen at subzero temperatures (−17 °C was the lowest temperature achieved by him), and still be revived upon warming (Sandler, 1973; Bwanga, 1991; Mantegazza, 1866). A true visionary, Mantegazza proposed and predicted the existence of sperm banking for artificial fertilization (Bwanga, 1991; Sztein et al., 2018); it took almost another century for this proposition to become reality (Watson, 1979).

Paul Bert (1833–1886), a French surgeon and physiologist carried out extensive work on the grafting of tissues, limbs and organs in animals (Seghers and Longacre, 1964). The experiments were published as two successive doctoral theses, where he also included his study of the effect of exposing preoperatively the grafts to low temperatures (Bert and De la Greffe, 1863; Bert and De la Vitalité Propre des Tissus, 1866). In general, the lower was the storage temperature, the more successful was the outcome of grafting. At the lowest temperature tested by Bert (−18 °C) (Bert and De la Vitalité Propre des Tissus, 1866), no damage to the tissue was seen. He described hundreds of animal experiments, also attempting to make sense of the knowledge accumulated, and argued for applying grafting to human patients. Unfortunately, the account of his work was buried in two doctoral theses with poor dissemination among peers, while Bert himself abandoned soon afterwards the scientific career to become a politician.

At the beginning of the 20th century, considerable activity was reported in Europe and USA regarding the implantation of blood vessels, which triggered an interest in preservation of arterial or venous ex-vivo segments needed for grafting. The foremost representative of this era was Alexis Carrel (1873–1944), one of the few genuine accomplished surgeon-scientists of all times, who modernized vascular surgery and set the groundwork for future human organ transplantations. In 1912, he was the first surgeon to be awarded a Nobel Prize in medicine. His experiments with animal vascular tissues (Carrel, 1907; Carrel, 1908) showed that grafts stored in a refrigerating unit, at temperatures between 0 °C and 4 °C for up to 35 days, led to optimal postoperative outcomes. Rather surprisingly, Carrel also reported (Carrel, 1908) that the grafts frozen for a few days prior to surgery did not perform well in the long-term, probably because of the damage caused by the growing ice crystals. Balanced salt solutions, isotonic to blood plasma, were employed preferentially as preservation media. In a noteworthy experiment (Carrel, 1912a), he removed a segment of popliteal artery from the leg of a human patient and preserved it for 24 days in an isotonic solution at ∼0 °C before transplanting it into the abdominal aorta of a dog. The animal lived in good health for over 4 years when it died from an unrelated cause. He evaluated a large number of freezing media, concluding that isotonic solutions and petrolatum (Vaseline) were the most successful regarding the surgical outcome, within storage periods as long as 1 year at temperatures between −3 °C and 15 °C, usually employing the range 0 °C–1 °C.

Carrel developed a hypothesis regarding the preservation of tissues between collection and implantation stages (Carrel, 1910; Carrel, 1912b). He postulated the existence of two forms of life, latent and active. In turn, latent life involved two different conditions. The “unmanifested actual life” is the condition where metabolism is reduced almost to cessation, but not completely, a stage called by Carrel “general” death; although it can occur suddenly, the general death is a temporary condition that slowly advances to total breakdown of cellular protoplasm and “elemental” death. He defined the other condition, the “potential” life as the suspension of all vital processes, e.g., metabolism, growth, motility etc. This condition could prevent general death and allow preservation of explanted tissues “outside of the body for an indefinite period of time” (Carrel, 1912b). Going further, Carrel emphasized that the other form of life, the active life, can be maintained for a tissue isolated from a living organism, as demonstrated in 1910 by KeshishianHarrison’s (2004), if normal nutrition was provided through artificial means. Carrel’s experiment (Carrel, 1912b) with an isolated chicken’s heart confirmed that crucial finding. His message will remain forever with his successors: the vascular tissue for transplantation must be alive if we aim at a satisfactory outcome.

In current terms, latent life takes effect when an organism enters a reversible ametabolic state called cryptobiosis (Keilin, 1959; Wright, 2001). It is a state of suspended animation that can be caused by dessication (anhydrobiosis), low temperatures (cryobiosis), lack of oxygen (anoxybiosis), high salt concentration (osmobiosis), or high levels of metabolic toxins (chemobiosis). Research on cryptobiosis began centuries ago with the experimental observations on cryobiosis (Power, 1664) and anhydrobiosis (Needham, 1743; Baker, 1764; Mercier Dupaty, 1788) of nematodes. Employing microscopic techniques, van Leewenhoeck (Wright, 2001; Van Leewenhoeck, 1678; Hoole, 1807) carried out investigations on the anhydrobiosis of protists, bacteria, and rotifers. It appears that when formulating his ideas, Carrel was aware of much of that previous work.

The subsequent impressive expanse of research on cryobiology and cryopreservation, and on ensuing applications, were summarized in some excellent reviews (Keilin, 1959; Wright, 2001; Luyet and Gehenio, 1940; Smith, 1958; Bojic et al., 2021; Freitas-Ribeiro et al., 2022; Chen et al., 2023; Arav and Natan, 2024; Khaydukova et al., 2024). It was estimated (Coriell et al., 1964) that by 1940 over 4,000 publications had been already dedicated to the biological effects of subzero temperatures. While significant success was achieved through the development of improved methods based on controlling the freezing/cooling and thawing/warming processes, and by finding better cryoprotective agents, the progress was slower in the field of transplantation of large organs (Bennett, 2025).

Cryopreservation, known also as cryogenic preservation, is an effective method for the long-term storage of cell suspensions, excised tissues, and small or large whole organs. However, its success can be substantially affected by injury to cells and tissues caused by the freezing/cooling and thawing/warming processes. Indeed, as water is omnipresent within biological systems, its solidification into ice crystals within intracellular or extracellular space is fatal to the cell. Therefore, knowledge of water-to-ice phase transition at subzero temperatures and its effect on cytoplasm, cell membrane, and cellular interactions is crucial for finding means to reduce the lethality associated with cryopreservation techniques. Our understanding of the effects of ice’s presence in biological systems, the mechanism of cryodamage, and also the role of cryoprotectants has been summarized in some earlier landmark reviews and in more recent publications as well (Wood et al., 1956; Trump et al., 1965; Mazur et al., 1972; Mazur, 1984; Toner et al., 1990; Karlsson and Toner, 1996; Asghar et al., 2014; Fu et al., 2022; Pegg et al., 2015; Best, 2015; Elliott et al., 2017; Eskandari et al., 2020; Chang and Zhao, 2021; Murray and Gibson, 2022; Landecker, 2024).

A brief description of cryogenic processes based on such publications may be useful in this context. While refrigeration of biologic matter can be employed successfully in certain situations, its major drawback is the limited shelf life of the stored specimens. Cryopreservation at subzero temperatures, e.g., from −130 °C to −196 °C (the boiling point of liquid nitrogen) extends the shelf life to hundreds of years; for instance, a shelf life of a thousand years was estimated theoretically for cells stored in liquid nitrogen (Mazur, 1984). However, the interval from −15 °C to −60 °C during the cooling stage, and passed again during thawing, induces lethal damage to cells due to ice formation. In principle, the damage due to freezing (cryodamage) can be caused by (a) the ice crystals destroying mechanically the cells by perforating and shredding their structure, or (b) secondary effects due to changes in the concentration of solutes in the liquid phase. Both mechanisms are relevant, and the distribution of their inputs is controlled by factors such as cell type, and cooling rate. For instance, at fast cooling, formation of damaging intracellular ice crystals is favored, while large osmotic forces are generated contributing to the rupture of cell membrane. At slow cooling, extracellular ice formation is favored, and the resulting ice crystals cause physical damage to cells, while cells are also subject to prolonged exposure to high concentrations of harmful solutes, both intracellularly and extracellularly. An ideal cooling rate has to be low enough to avoid intracellular ice but high enough to reduce solute effects. The fact that each cell type has a different cooling rate versus survival profile complicates the process if tissues and full organs are involved. Regarding the thawing/warming rate, although the rapid procedures appear to be preferred, it may lead sometimes to cell survival lower than provided by a slower rate. Ice crystal can increase in size (recrystallization) and induce cellular damage and lysis.

All mentioned events could be partially alleviated by the addition of cryoprotective agents (CPAs), also known as cryoprotectants. They have been introduced in methodology in order to reduce or prevent cryoinjury, mainly through diminishing ice formation by lowering the concentration of solutes. The classic cryoprotectants such as glycerol and glycols (Polge et al., 1949) and dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959) have limitations, yet DMSO is still routinely employed. As they become common components in most cryopreservation protocols, the CPAs lost the initial novelty, and it was suggested that their actual involvement might have been overlooked (Landecker, 2024). In fact, all elements in the trinity freezer/target/cryoprotectant are equally essential. Extensive research is currently carried out to develop more effective CPAs including ice binders, nucleation inhibitors, bio-inspired agents, and substances that may display protective effects even if based on mechanisms not fully elucidated (Murray and Gibson, 2022). CPAs can be either permeating (e.g., DMSO), or non-permeating (e.g., sucrose, trehalose). They are commonly mixed with a vehicle solution, such as RPMI 1640 culture medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, human albumin, or Krebs-Henseleit buffer (KHB) solution. The CPA-vehicle solution mixtures are known as cryomedia.

Vitrification is an alternative method for preservation that allows cooling the cells to cryogenic temperatures without the formation of ice crystals (Pegg et al., 2015; Rall and Fahy, 1985; Fahy et al., 2015). The non-freezing aqueous solutions hosting the biological targets solidifies by fast cooling, leading to enormous viscosities (∼10¹⁵ cP), when the medium turns into a glassy or vitreous material devoid of ice. In this context, the terms ‘freezing’ and ‘thawing’ should be replaced by, respectively, ‘cooling’ and ‘warming’ (or ‘rewarming’). While involving the use of high concentrations of CPAs, vitrification is generally regarded as a successful technique that may display some advantages when compared to other cryopreservation procedures. However, the trade-off between the toxicity and concentration of CPAs remains a challenge. An accurate account of vitrification’s fundamentals and methodology is available (Fahy et al., 2015).

Tissues and organs are systems comprising densely packed cells and extracellular matrix (ECM) components, all assembled with a specific architectonics in order to generate integrated functional entities. To maintain the ECM organization and intercellular relationships following a preservation procedure is crucial. The application to multicellular systems of cryopreservation techniques specific to single-cell systems provides in general disappointing results, especially for organs. Upon freezing, extracellular ice crystals are produced that can damage indirectly the cells and directly the host as such, in which case they may also lead to functional damage. Multicellular systems contain a collection of cell types with variable survival responses to freezing, yet all these types are subjected to the same cooling and thawing processes. Therefore, larger concentrations of CPAs are needed to broaden the survival range. In such systems, the mechanisms of cryodamage become more complex at molecular and ultrastructural levels. Other factors that contribute to cryodamage in organs include non-uniform freezing or thawing rates, anomalous heat and mass transport processes, uneven expansion and contraction leading to fracture, and uneven distribution of CPAs.

We should mention that in organs that requires their own intact vasculature for securing a successful transplantation, the cryodamage ensues through the rupture of capillaries regardless of how many cells survived. The ice forms intraluminally, and rupture occurs when its volume overcomes the limits maintained through the original strength and elasticity of the wall. That may be relevant for vessels larger than capillaries.

Normothermic and subnormothermic preservation techniques involve normal temperatures, more precisely near the physiologic temperature (36 °C–38 °C), or in the range 20 °C–33 °C, respectively. The process of cryobiosis is not involved in these procedures, as they do not aim at suspending the metabolism in order to avoid degradation of the preserved biological material, and therefore are practicable only for very short periods of storage. For an improved viability of stored cells, methods based on encapsulation within biocompatible gels have been developed, however the storage time remains limited. For the normothermic and subnormothermic preservation of organs, machine perfusion techniques, pioneered by Carrel and Lindbergh (Carrel and Lindbergh, 1935), have been further significantly refined and are used occasionally in the transplantation of kidney, liver or heart. The principle of perfusion-assisted preservation is to duplicate the original physiological milieu of the organ by combining oxygen-carrier substances, nutrients, antithrombotic drugs, and other agents.

A significant number of cryopreservation techniques are now available, and developmental work is in continual progress aimed at improvements. It is important to mention that the implementation of these methods is associated with unavoidable technical and economical drawbacks, such as dependence on continuous electricity supply, and requirement for large working spaces to accommodate refrigerators, freezers, back-up freezers, and auxiliary equipment. The procedures employing liquid nitrogen need regular refreshments, such generating significant additional costs.

Based on the available literature (Freitas-Ribeiro et al., 2022; Chen et al., 2023; Arav and Natan, 2024; Khaydukova et al., 2024; Taylor et al., 2019; Criswell et al., 2023; Ozgur et al., 2023), we have summarized below the current storage techniques involving low temperatures.

Hypothermic method does not involve subzero temperatures, and is commonly known as ‘cold storage.’ It is entirely based on refrigeration (1 °C–8 °C) and is used for short-term storage, i.e., for a few days, but preferably no longer than a few hours. It is generally applicable when other techniques are unsuccessful for particular biological targets. Hypothermia is based on the slowing effect that a low temperature has on the rate of chemical and biochemical processes, a fundamental natural law. It is routinely used in laboratories for in vitro experiments involving cells or tissues that are freshly available for proximal use. It cannot provide a basis for long-term storage and banking of biological material for transplantation, which requires much lower temperatures and longer durations. However, when combined with perfusion, it can be used to preserve organs such as kidneys or heart.

Lyophilization and preservation in dry state. Known also as freeze-drying, this method is based on a process of dehydration at low temperature, where both anhydrobiosis and cryobiosis may be involved. The technique consists of three stages, including (a) freezing (between −50 °C and −80 °C), (b) primary drying achieved by the sublimation of ice under controlled vacuum, and (c) secondary drying achieved by removing the residual (non-freezable) water at raised temperatures. The resulting desiccated porous products can be stored for years at room temperature, and their original properties can be reinstated on demand by rehydration with an appropriate amount of water while controlling the osmotic stress.

High-subzero preservation comprises strategies requiring intermediate subzero temperatures, developed as alternatives both to hypothermic and cryogenic methods. Inspired from the stress-tolerance strategies adopted by some species, e.g., frog, crocodile, squirrel, or dolphin (Taylor et al., 2019), this category includes the techniques listed below, using the current terminology.

Supercooling (−4 °C to −6 °C)

A range of chemical compounds has been developed to serve as cryostasis agents (‘revival cocktails’) for enhancing the outcomes of the high-subzero methods.

Deep-subzero preservation employs temperatures in the range −80 °C to −196 °C, and includes both traditional cryopreservation and vitrification techniques, well known and used extensively.

Furthermore, innovative vitrification techniques have been developed lately, including:

Isochoric vitrification (−80 °C to −196 °C), maintaining a constant volume that prevents ice formation

The schematic overview of the aforementioned methods, along with their advantages and drawbacks, is presented in Figure 1.

Recent advances in biopreservation are increasingly focused on extending storage duration while preserving vascular structure and endothelial function. Traditional hypothermic storage at 4 °C remains constrained by ischemic injury and progressive endothelial dysfunction, prompting the exploration of alternative temperature ranges and biologically targeted preservation strategies (Bordet et al., 2024; Hwang et al., 2021). High-subzero preservation approaches, including isochoric preservation, represent a promising direction by enabling lower-temperature storage while suppressing ice formation through constant-volume thermodynamics (Rubinsk et al., 2005; Năstase et al., 2023). These methods reduce mechanical and osmotic stress and may be particularly advantageous for vascular tissues, which are highly susceptible to ice-induced damage. In parallel, progress in vitrification has highlighted the critical importance of rapid and uniform rewarming; volumetric heating techniques such as nanowarming have been developed to mitigate thermal gradients and structural injury during warming (Manuchehrab et al., 2017). Beyond thermal control, new preservation solutions and transport systems are being designed to support multi-day storage while reducing ischemic and reperfusion-associated injury. These formulations increasingly emphasize endothelial protection, oxidative stress reduction, and metabolic suppression rather than osmotic balance alone. Technologies such as XT-ViVo® and TimeSeal® exemplify this trend and may help streamline vascular tissue logistics as validation data continue to accumulate (Heberle et al., 2025; Dong et al., 2025; Hassan et al., 2025; Khaki et al., 2025; Muss et al., 2025; Kim et al., 2025). However, despite preliminary data presented at kidney transplant conferences, no data have been published in the current literature to date (Heberle et al., 2025; Dong et al., 2025; Hassan et al., 2025; Khaki et al., 2025; Muss et al., 2025; Kim et al., 2025). Collectively, these emerging strategies signal a shift toward vascular biopreservation paradigms that integrate optimized temperature management with functional endothelial preservation, supporting longer storage times without compromising post-transplant performance.

A large variety of preservation procedures, ranging from normothermic to deep-subzero temperatures, have been used for the storage of human vascular tissue intended for surgery or research. The procedures present different and specific advantages or disadvantages when compared to each other. Normothermic and hypothermic preservation procedures reduce substantially the costs, but are of limited use and only for short durations. Despite certain advantages (e.g., it avoids the use of liquid nitrogen, assures long duration storage without supervision, and reduces storage and shipping costs), lyophilization could not find a steady niche in the preservation of vascular grafts. Vitrification appears to be effective in reducing substantially the cryodamage, but currently is not frequently applied, one of the likely reasons being the costs involved. The traditional subzero-temperature procedures seem to be the preferred alternative for storing vascular tissue, in spite of obvious disadvantages such as need for liquid nitrogen, and high costs for equipment and shipping. If performed following strictly established protocols, and if associated with immunological monitoring, the conventional approaches to subzero cryopreservation methodology may reduce cryodamage to safe levels and can increase duration of storage, such contributing to successful clinical outcomes.

Whereas the majority of topical publications have reported favorable results irrespective of the preservation procedure, to recommend a particular technique is rather irrelevant.