Historical Overview of Tissue Preservation by Low Temperatures Under Pressure, Eliminating Ice Formation

Introduction: The Significance of Cryopreservation and the Problem of Ice Formation

Cryopreservation is a cornerstone of modern biology and medicine, providing stable and safe storage for a wide range of biological materials, including organisms, organelles, tissues, and cells.[1] This technology is fundamental to biobanking, regenerative medicine, fertility preservation, and advanced research in neuroscience and pulmonology.[2] The ability to preserve organs "on demand" could have a transformative impact on healthcare, comparable to breakthroughs in cancer treatment.[3, 4] The broad application of cryopreservation, from basic research to clinical transplantation and biodiversity conservation, underscores its role as a foundational technology in contemporary biology and medicine. Advancements in this area, especially those targeting existing limitations, promise substantial scientific and societal benefits beyond immediate clinical applications.

Despite significant progress, a major challenge in cryopreservation remains the damage caused by ice crystal formation. Uncontrolled crystallization is the most critical factor limiting the survival of structured tissues and organs.[3] As water freezes into ice I, it expands, exerting mechanical forces that damage cell membranes and structures, ultimately compromising tissue integrity.[5, 6, 7] This process also induces cellular dehydration and osmotic stress, further affecting viability.[1, 5] The expansion of water during freezing at atmospheric pressure is a fundamental cause of mechanical injury to biological systems and is a central focus in developing improved cryopreservation strategies.

The use of high pressure during freezing presents a promising method to address this issue. Under such conditions, ice formation can be suppressed in favor of amorphous, glass-like ice.[8, 9, 10, 11] This state better preserves biological samples by minimizing structural changes.[10, 11] High pressure alters the phase diagram of water,[9, 7, 10, 12] making it possible to achieve vitrification at lower cooling rates and in larger volumes. This approach reduces reliance on ultra-rapid freezing or high concentrations of cryoprotective agents, marking a conceptual shift in cryopreservation practice.

Historical Roots: Early Concepts and the Contribution of Serafim Vasilievich Znamensky

The history of cryopreservation and the use of cold in biological research predates the advent of modern technologies. In its early stages, the focus was primarily on the destructive effects of low temperatures rather than their preservative potential. Between 1819 and 1879, English physician James Arnott described the use of ice and salt mixtures to induce localized tissue destruction—a technique that would later be known as cryosurgery—employed for tumor palliation and anesthesia.[13] Although these methods were not aimed at preserving biological function, they laid important groundwork for understanding how cold interacts with living tissues.

By the late 19th century, efforts to liquefy gases had led to the development of refrigerants such as liquid air, which was first commercially produced by Carl von Linde in 1895.[13] These advances made it possible to reach much lower temperatures, thus opening new possibilities for experimentation. The transition from crude methods of cryoablation to the refined techniques of cryopreservation illustrates a fundamental evolution in scientific understanding. Rather than exploiting the destructive force of cold, researchers began to explore its potential to preserve biological systems—an approach made possible through progress in physics and engineering.

Cryobiology began to emerge as a distinct scientific discipline in the 1940s.[15] A pivotal breakthrough occurred in 1948, when Polge, Parkes, and Smith accidentally discovered that glycerol could protect fowl spermatozoa from freeze damage.[16] Initially referred to as "vitrification," this discovery proved to be transformative for the field of cryopreservation.[17] By 1950, Smith had demonstrated the use of glycerol for preserving human red blood cells, and in 1959, dimethyl sulfoxide (DMSO) was introduced and rapidly adopted as the new standard cryoprotectant.[16] The accidental nature of glycerol’s discovery underscores the importance of serendipity in scientific progress, while its swift implementation—along with the identification of DMSO—helped establish modern approaches to mitigating ice-related cellular damage well before pressure-based techniques became common.

The idea of using pressure during freezing to prevent ice crystal formation was introduced by Hans Moor and Ulrich Riehle in 1968.[8] Nevertheless, it was not until the mid-1980s that the first commercial high-pressure freezing (HPF) device—the Balzers HPM 010—was brought to market. The nearly two-decade delay between theoretical proposal and technological realization highlights the complexity of engineering a system that could apply physical principles in a reliable, reproducible manner. Though the behavior of water under pressure had already been explored, adapting this knowledge to biological systems required overcoming substantial technical barriers.

Within this broader historical context, the contributions of Serafim Vasilievich Znamensky (1910–1995) deserve recognition. A distinguished Russian oncologist, Znamensky devoted 48 years to the healthcare system in Norilsk, where he established and led the city’s oncological dispensary.[20] Among his scientific achievements is a paper titled Tissue Preservation by Low Temperatures Under Pressure, Eliminating Ice Formation—a topic that directly reflects the core goals of present-day cryopreservation research.[20] His personal archive, which spans the years 1938 to 1991, includes early work in this area.[20] Znamensky graduated from the medical faculty of the Kharkiv Medical Institute in 1941,[20] and reportedly received a scientific award in Kyiv around 1940. According to accounts, Academician Bogomolets remarked at the time, “You don’t understand, you fool, what you have invented. If you don’t abandon your work, in two years you’ll be a Stalin Prize laureate.” This anecdote highlights both the scientific promise and early recognition of Znamensky’s work during his student years.

The following table presents key milestones in the development of pressure-assisted cryopreservation, offering a clearer view of the timeline and interconnected nature of these discoveries.

Table 1: Key Milestones in Pressure-Assisted Cryopreservation (Historical Context)

Year(s)

Event/Discovery

Key Figures

Significance

1819-1879

Application of cold in cryosurgery

James Arnott

Early use of cold for tissue destruction and anesthesia 13

1877

Liquefaction of gases under high pressure

Louis-Paul Cailletet, Raoul Pictet

Beginning of high-pressure use to achieve low temperatures 13

1895

Commercial production of liquid air

Carl von Linde

Enabled achievement of lower temperatures for experiments 13

1940s

Emergence of scientific cryobiology

Basile Luyet

Beginning of systematic studies on the effects of low temperatures on biological systems 15

1948

Discovery of glycerol's cryoprotective properties

Christopher Polge, Audrey Smith, Alan Parkes

Breakthrough in preventing ice damage, laying the foundation for modern cryopreservation 16

1950

Application of glycerol for human red blood cell cryopreservation

Audrey Smith

Expansion of cryoprotectant use to human cells 15

1959

Recognition of DMSO as a cryoprotectant

James Lovelock, Audrey Smith

Establishment of the "gold standard" for cryoprotectants 15

~1940

Award-winning paper "Tissue Preservation by Low Temperatures Under Pressure, Eliminating Ice Formation"

Serafim Vasilievich Znamensky

Early domestic research directly related to the report's topic, receiving recognition

1968

Proposal for freezing biological samples under high pressure

Hans Moor, Ulrich Riehle

First theoretical concept of high-pressure freezing 8

1983

First human embryo cryopreservation

Monash University Team

Significant success in cryopreserving complex biological structures 3

1985

Introduction of the first commercial HPF apparatus (Balzers HPM 010)

Balzers

Transition from theoretical concept to practical technology

2005

Introduction of isochoric freezing concept

Boris Rubinsky

New thermodynamic approach to cryopreservation 23

Recent developments

Development of HPF systems HPM Live µ and Leica EM ICE

CryoCapCell, Leica Microsystems

Increased reliability and versatility of HPF 25

2023

Founding of BioChoric (isochoric cold storage startup)

Boris Rubinsky

Commercialization of isochoric methods

Physical Principles of Pressure-Assisted Cryopreservation

Understanding the physical principles behind pressure-assisted cryopreservation is essential for developing effective preservation methods. Water, the primary constituent of biological systems, exhibits several unusual properties. Notably, its solid form—ice I—is less dense than its liquid state, meaning that water expands upon freezing. This expansion is a major cause of mechanical damage to cells and tissues during conventional freezing.

The water phase diagram illustrates how the freezing point of ice I changes under varying pressures. As pressure increases, the melting point of ice I decreases, reaching a minimum near −22°C at approximately 2045–2076 bar (207.5 MPa). Beyond this pressure, the melting point begins to rise again. High pressure also depresses the homogeneous nucleation temperature of ice, pushing it down to about −92°C at 2045 bar.[9] These changes expand the range of conditions under which water can remain supercooled—liquid below 0°C without ice formation. The negative slope of the ice I melting curve in the phase diagram is central to pressure-assisted cryopreservation. Rather than simply inhibiting ice formation, pressure actively lowers the freezing point, enabling water to remain in a liquid or vitrified state at temperatures far below zero. This principle defines the operational range of high-pressure freezing (HPF) systems and informs the development of isochoric cryopreservation technologies.

Vitrification, or the formation of glass, involves solidifying water into a non-crystalline, amorphous state that avoids the formation of damaging ice crystals. Because amorphous ice preserves cellular ultrastructure, it is ideal for biological preservation.[8] Achieving vitrification at atmospheric pressure requires extremely high cooling rates—often hundreds of thousands of degrees Celsius per second. However, under high pressure, these requirements are significantly reduced to a few thousand °C/s,[8] making it possible to vitrify larger samples ranging from 100–200 micrometers up to 0.6 mm in thickness. The combination of lower nucleation and melting points under pressure extends the time and temperature "window" for vitrification, addressing one of the main limitations of traditional cryopreservation: the inability to preserve thick samples without ice damage.

Cryoprotective agents (CPAs) play an essential complementary role. These substances lower water’s freezing point, increase solute concentration, and reduce ice formation. CPAs also inhibit the growth of extracellular ice crystals and reduce the release of latent heat during crystallization.[27] Effective CPAs must be capable of penetrating cells and should have low toxicity. Common examples include ethylene glycol, glycerol, and dimethyl sulfoxide (DMSO). While pressure-assisted cryopreservation reduces the need for extreme cooling rates or high CPA concentrations,[23] chemical protection remains important. CPAs help manage osmotic stress and further suppress crystallization, suggesting that the best preservation outcomes arise from combining physical strategies (such as pressure and cooling rate control) with chemical ones.[1]

In recent years, a thermodynamically distinct method—isochoric cryopreservation—has gained interest. Traditional cryopreservation typically occurs under isobaric (constant pressure) conditions, usually at atmospheric pressure.[23] In contrast, isochoric systems operate at constant volume, where pressure varies passively in response to volume changes caused by ice formation. In these systems, the expansion of ice Ih increases internal pressure, establishing a new thermodynamic equilibrium. This method offers a stable environment for maintaining supercooled liquid or ice–liquid mixtures at sub-zero temperatures.[23] By shifting control from externally applied to internally generated pressure, isochoric cryopreservation presents new opportunities for preserving organs and complex tissues, offering both stability and scalability.

Modern Methods and Technologies

Modern pressure-assisted cryopreservation methods represent the pinnacle of engineering and a deep understanding of water physics.

High-Pressure Freezing (HPF)

HPF is a key method for immobilizing biological samples, especially for analysis with electron microscopy (EM). Its principle is based on rapidly freezing samples under high pressure, usually in the range of 2076-2100 bar, with a very high cooling rate exceeding 2000 K/s. These conditions prevent ice crystal formation, instead promoting the formation of amorphous ice.

Modern equipment, such as the HPM Live µ (developed by CryoCapCell, based on the historical BalTec HPM010) and Leica EM ICE, demonstrates high reliability and versatility in achieving these critical parameters. These apparatuses are capable of synchronizing pressure application and cooling of the sample within milliseconds.18 The development of HPF equipment capable of applying 2100 bar pressure and achieving cooling rates over 2000 K/s within milliseconds 10 represents the pinnacle of engineering achievements in cryopreservation. This level of precision and speed is necessary to overcome the kinetic barriers to vitrification in biological samples, making it a highly specialized, expert technique.10

Preparation of samples for HPF involves placing them between small metal carriers (usually aluminum or gold-coated copper), which not only transmit pressure but also aid in heat extraction.10 It is crucial to avoid air bubbles in the sample, as they can reduce the pressure plateau and cause deformation.9 Often, cryoprotectants or fillers, such as hexadecane or bovine serum albumin, are used to ensure full contact and further protection.9 After vitrification, samples can be prepared for further analysis using various EM techniques, including freeze substitution (FS) and cryo-EM. FS involves gradually raising the temperature to replace amorphous ice with a solvent containing chemical additives, which allows for the preservation of a stabilized structure.8

Isochoric Cryopreservation

Isochoric cryopreservation represents an approach based on preserving biological material in a liquid phase at sub-zero temperatures under constant volume conditions. In this system, pressure is passively generated by the freezing of a portion of the water, which allows ice and liquid water to coexist in thermodynamic equilibrium down to -21°C.

This method includes several variants: isochoric freezing (preservation in the liquid portion of the ice-liquid equilibrium), isochoric supercooling (increasing the metastability of supercooled water), and isochoric vitrification.23 Continuous monitoring of temperature and pressure is mandatory 32, as a change in pressure within the isochoric chamber can signal the onset of freezing.23 A typical experimental protocol involves preparing a cooling bath (e.g., a mixture of water and 50/50 ethylene glycol to achieve temperatures down to -30°C), carefully filling the isochoric vessel with the solution without air bubbles to avoid measurement errors, and maintaining a low initial pressure (around 1 bar).33 Repeating experiments is critically important for data validation.33 The passive generation of pressure in isochoric systems offers a distinct advantage for scaling compared to active hydraulic HPF systems. This intrinsic self-regulation, where freezing itself generates protective pressure, simplifies mechanical complexity for larger volumes, making it a promising avenue for organ preservation at scale, as evidenced by research on human-sized liver preservation.

Self-Pressurized Rapid Freezing (SPRF)

Self-Pressurized Rapid Freezing (SPRF) is a novel technology, developed by Leica Microsystems, for example, that utilizes the tendency of water inside a sealed specimen carrier to expand upon cooling, thereby generating pressure intrinsically rather than through an external hydraulic system.18 This approach allows for high pressures (up to 2010 bar) to be achieved through the formation of crystalline ice and low-density ice within the sealed container.18 SPRF represents a fusion of HPF and isochoric system principles. The internal generation of pressure through water expansion upon cooling allows for the benefits of high pressure (as in HPF) without the complexity of an external hydraulic system, potentially offering a more compact and versatile solution for certain applications. This demonstrates the ongoing evolution in cryopreservation technology aimed at optimizing pressure generation.

Table 2: Comparison of Pressure-Assisted Cryopreservation Methods

Method

Principle

Pressure Range

Cooling Rate

Typical Sample Size/Type

Primary Application

Key Advantages

Key Disadvantages

High-Pressure Freezing (HPF)

Rapid cooling under high external pressure

~2076-2100 bar (207.5-210 MPa) 9

>2000 K/s 10

Thin samples (100-200 µm, up to 0.6 mm) ; cell pellets, monolayers, biopsies 10

Sample preparation for electron microscopy 10

Preserves near-native ultrastructure, prevents ice crystals, faster fixation, greater sample depth 8

Complex, requires expertise, cryoprotectant and contrast issues, high equipment cost 8

Isochoric Freezing

Constant volume confinement, passive pressure generation

Variable, often milder (<40 MPa for medical, up to 200 MPa for food) 23

Not primary factor, aims for stable liquid-ice equilibrium

Nematodes, pancreatic islets, rat hearts, porcine livers, food products 23; potentially human organs

Organ/tissue preservation, food preservation

Passive pressure generation, extended viability, reduced ice damage, bacterial control 23

Barotoxicity at high pressures, engineering challenges for large chambers, limited biological validation 23

Isochoric Supercooling

Constant volume confinement, enhanced supercooled liquid metastability

Variable, often milder (e.g., -3°C) 23

Aims for stable supercooled state

Cardiac microtissues, endothelial cells, HeLa cells, porcine/rodent/human livers 23

Extended hypothermic storage without freezing 23

Significantly longer nucleation induction times, resilience to perturbations, ice-free preservation 23

Barotoxicity, limited biological validation 23

Isochoric Vitrification

Achieving amorphous ice state under isochoric conditions, potentially reduced cooling/CPA requirements

Not explicitly stated, in isochoric context 23

Potentially reduced compared to isobaric vitrification 23

Hawaiian stony coral (centimeter-scale) 23

Vitrification of larger or more complex samples 23

Reduced CPA/cooling rate requirements for vitrification 23

Complex theoretical description, possibility of undetected ice formation, limited biological validation 23

Self-Pressurized Rapid Freezing (SPRF)

Internal pressure generation from water expansion in sealed carrier upon cooling

Achieves up to 2010 bar 18

Rapid freezing

C. Elegans, spermatozoa, E. coli, yeast cells, HeLa cells, feline oocytes 23

Cryofixation for microscopy, reversible cryopreservation 23

Internal pressure generation, avoids external hydraulic system complexity 18

Requires sealed specimen carrier, possibility of low-density ice formation 18

Applications and Successes in Tissue and Organ Preservation

Pressure-assisted cryopreservation methods demonstrate significant successes across various fields, from fundamental research to potential clinical applications.

HPF Applications in Electron Microscopy

High-Pressure Freezing (HPF) is widely used for preparing biological samples for electron microscopy (EM). This method ensures homogeneous preservation and minimal ice crystal artifacts, which is critical for obtaining high-quality images. HPF has been successfully applied to various sample types, including human cell pellets, cell monolayers, mouse brain and liver biopsies, and Arabidopsis thaliana roots and seedlings.10

One of the key advantages of HPF is its ability to increase the depth of freezing without forming damaging ice crystals, up to 100-200 µm, and in some cases up to 0.6 mm. This significantly surpasses the 10-40 µm limitation typical of atmospheric pressure freezing methods.8 HPF's ability to preserve ultrastructure in a near-native state 10 and at greater depths 8 has revolutionized electron microscopy. This enables more accurate structural and functional analyses of biological materials, directly impacting our understanding of cellular and molecular processes by providing high-resolution, artifact-free images.

Successes of Isochoric Cryopreservation

Isochoric cryopreservation has shown significant successes in both medical and agricultural applications.

Medical Applications:

• Cells and Small Organisms: Research on C. Elegans nematodes demonstrated high survival rates (>95%) at pressures up to 40 MPa.23 Also, successful preservation of rodent pancreatic islets at -3°C/34 MPa without supplementary cryoprotectants was achieved, with 97% viability after 24 hours.23
• Tissues and Microtissues: The method allowed for the preservation of three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues, with recovery of autonomous beating and responsiveness to isoproterenol after 24-72 hours at -3°C.23 Also, preservation of a vascular micro-physiological system with human coronary endothelial cells was achieved, which fully recovered after four hours of reperfusion.23
• Organs: Studies on whole rat hearts showed preservation of ventricular function comparable to controls at -4°C/41 MPa for one hour.23 Supercooling of porcine livers to -2°C for 24-48 hours in University of Wisconsin (UW) solution was demonstrated with no signs of structural degradation.23
• Vitrification: Successful cryopreservation and revival of centimeter-scale multi-polyp fragments of Hawaiian stony coral were achieved using isochoric vitrification.23
• Blood: Early research indicated that red blood cells could tolerate pressures up to ~30 MPa, and blood storage at 34 MPa for seven days resulted in no hemolysis.23
  
  The successful preservation of complex biological entities, such as whole rat hearts, porcine livers, and hiPSC-derived cardiac microtissues, using isochoric methods 23 signifies a critical step towards establishing clinical organ banks. This extends beyond single-cell cryopreservation and addresses the long-standing challenge of preserving large, complex tissues with retained function, which directly impacts transplant medicine.

Food Industry Applications:

Isochoric freezing at -1.5°C/15 MPa effectively inhibited microbial growth and preserved the fresh-like physicochemical and bioactive properties of milk for up to five weeks. This method is also significantly more energy-efficient than conventional freezing.

Scalability:

Vitrification of 0.5 to 3-liter volumes of cryoprotective agents (CPAs) in cryobags, as well as a porcine liver (~1 liter), has been demonstrated.32 This indicates the potential for scaling isochoric methods for the preservation of large biological entities.

Other Cryopreservation Successes (including traditional methods)

Beyond pressure-assisted methods, cryopreservation in general has achieved significant successes. Standard protocols have been established for cryopreserving various cell types, such as red blood cells and reproductive cells, and some tissues, including ovaries and and skin.25 Various tissues and organs have been successfully cryopreserved and transplanted, including rat hearts, rat hindlimbs, rat and sheep ovaries, rodent and porcine livers, human amputated fingers, rabbit kidneys, rabbit jugular veins, human mucosal tissues, human skin, ddy mouse femora, pancreatic islets, human corneas, human adipose tissue, and human brain.33 While the report focuses on pressure-assisted methods, acknowledging broader successes in cryopreservation 25 provides important context. This shows that the field is advancing on multiple fronts, and pressure-assisted methods are a specialized, highly effective subset addressing specific limitations (ice formation in complex structures) that traditional methods struggle with.

Table 3: Tissues and Organs Successfully Preserved Using Pressure-Assisted Methods

Biological Material

Preservation Method

Key Parameters (Pressure, Temperature, Duration)

Result/Viability

Human cell pellets, cell monolayers, mouse brain and liver biopsies, and Arabidopsis thaliana roots and seedlings

HPF

2076-2100 bar, >2000 K/s

Homogeneous preservation, minimal ice crystal artifacts 10

Nematode C. Elegans

Isochoric Freezing / SPRF

Up to 40 MPa (-2°C / 20 MPa to -6°C / 65 MPa)

High survival (>95%) up to ~40 MPa; cryofixation 23

Canine kidney epithelial cells

Isochoric Freezing

96.5 MPa (-10°C) to 205 MPa (-20°C)

Deleterious effects at higher pressures 23

Whole rat hearts

Isochoric Freezing

-4°C / 41 MPa (1 hour)

Recovery of ventricular function comparable to control; barotoxicity at 60-78 MPa 23

Rodent pancreatic islets

Isochoric Freezing

-3°C / 34 MPa

Significant survival up to 3 days (97% viability after 24 hours) 23

HiPSC-derived cardiac microtissues

Isochoric Supercooling

-3°C (24, 48, 72 hours)

Recovery of autonomous beating, maintained responsiveness to isoproterenol 23

Vascular micro-physiological system with human endothelial cells

Isochoric Supercooling

-3°C (24, 48, 72, 96 hours)

Full recovery of barrier function 23

HeLa cells

Isochoric Cryomicroscope / SPRF

-5°C (24, 48, 72 hours)

Largely uncorrupted; vitrification 23

Porcine livers

Isochoric Supercooling / Vitrification

-2°C (24, 48 hours); ~1 liter

No signs of structural degradation; 1-liter scale vitrification 23

Rodent livers

Isobaric Supercooling

-6°C

Successful supercooling 23

Human livers

Isobaric Supercooling

-4°C

Preservation 23

Hawaiian stony coral

Isochoric Vitrification

Not specified

Successful cryopreservation and revival (centimeter-sized) 23

Red blood cells

High Pressure Isobaric

Up to ~30 MPa; 34 MPa (7 days)

Pressure tolerance; no hemolysis 23

Rabbit kidney

High Pressure Isobaric

100 MPa (pre-cooling)

Reversible cryopreservation 23

Yeast cells (S. cerevisiae)

SPRF

Not specified

Vitrification and reversible cryopreservation 23

Feline oocytes

SPRF

Not specified

Reversible cryopreservation 23

Raw milk

Low-pressure isochoric freezing

-1.5°C / 15 MPa (up to 5 weeks)

Inhibition of microbial growth, preservation of fresh physicochemical properties

Advantages and Disadvantages of Pressure-Assisted Cryopreservation Methods

The application of pressure in cryopreservation offers significant advantages but also comes with certain drawbacks that need to be considered.

Advantages

The primary advantage of pressure-assisted cryopreservation methods is the prevention or significant reduction of ice crystal formation. This is achieved by shifting the phase diagram of water and inducing the formation of amorphous ice, which is critically important for preserving the integrity of biological structures.

As a result of this process, preservation of the native state and ultrastructure of samples is achieved. Materials are kept as close as possible to their natural state, which significantly enhances the accuracy of structural and functional analyses.8

Pressure also leads to reduced cooling rate and cryoprotectant (CPA) concentration requirements. Vitrification can be achieved at significantly lower cooling rates (thousands of K/s instead of hundreds of thousands of K/s) and potentially with less CPA concentration needed.8 This simplifies the process and reduces potential CPA toxicity.

Pressure-assisted methods allow for increased sample thickness that can be successfully vitrified, up to 0.6 mm, significantly surpassing atmospheric pressure limitations (10-40 µm).

For isochoric systems, enhanced supercooling stability is characteristic. Isochoric conditions can significantly enhance the metastability of supercooled water, making systems resilient to physical, thermal, and chemical perturbations and allowing for extended storage (hours to weeks).23 Additionally,

passive pressurization in isochoric systems, where pressure is generated by the growth of ice Ih, simplifies the process compared to active mechanical compression.

In the food industry, pressure methods allow for bacterial load management and chemical infusion. The combination of low temperatures and mild high pressures can significantly inactivate non-pathogenic E. coli, Listeria monocytogenes, and Salmonella Typhimurium, and promote deeper penetration of chemical additives and nutrients into food tissues.23

Finally, compared to traditional chemical fixation, pressure-assisted cryopreservation provides faster fixation and simultaneous stabilization of all cellular components, which significantly outperforms the slow action of chemical fixatives.8 The advantages of cryopreservation with the use of pressure extend beyond simply preventing ice formation; they include improved structural preservation, increased sample depth, reduced reliance on high cooling rates/CPA concentrations, and even new applications in food safety. This suite of benefits indicates that pressure is not just a "fix" for the problem of ice damage but a powerful tool that fundamentally improves cryopreservation across multiple parameters.

Disadvantages

Despite numerous advantages, pressure-assisted cryopreservation methods have several significant drawbacks.

One of the most critical is barotoxicity, or pressure-induced damage. High pressures can be detrimental to living biological materials. Studies have shown that survival rates for cells and tissues significantly decrease at higher pressures (e.g., 65 MPa for nematodes, 60-78 MPa for rat hearts).23 The extent of damage depends on the pressure level, duration of exposure, and the specific biological model.23 The threshold for irreversible damage for most medical applications is around 40 MPa for prolonged storage.23

High-Pressure Freezing (HPF) is a complex and expert technique, requiring significant protocol optimization for each sample type.8 This limits its widespread application without specialized knowledge.

While cryoprotectants protect against ice damage, they can affect sample osmolarity and exhibit toxicity, requiring careful selection and testing. CPA toxicity is a significant limitation, especially for tissues and organs, as it can lead to cell death during incubation and cryopreservation.

Engineering challenges for industrial and clinical scaling of isochoric systems remain a major hurdle. High-pressure conditions necessitate thick-walled metallic vessels that are difficult to manufacture, transport, and cool at industrial scales, posing barriers to widespread adoption.

In the case of HPF, some resins used after vitrification can reduce contrast, requiring additional post-staining for improved visualization.10

Challenges for specific sample types also exist. For example, plant material with large vacuoles and air gaps is difficult to cryopreserve with HPF due to issues with proper pressure build-up, which requires additional preparation steps like degassing.10

The cost and accessibility of equipment for specialized HPF and related methods can be very high, making them prohibitive for many research and clinical institutions.8

Despite advances in physical understanding, biological validation of isochoric methods, especially for supercooling and vitrification, is still limited to a few studies, indicating the need for further biological research.23

There are unexplored fundamental knowledge gaps regarding the mechanisms of barotoxicity, its dependence on duration and rate parameters, and its scaling with biological complexity.23

In some cases of isochoric vitrification, ice formation can occur without detection by pressure monitoring, especially if the solution contracts sufficiently to create a vapor cavity that is then filled by ice.23

Finally, hypothermic machine perfusion (HMP), although improving outcomes, carries risks of excessive vascular endothelial damage, inadequate oxygen solubility, and organ edema. The significant advantages of cryopreservation with the use of pressure are often balanced by notable disadvantages, particularly barotoxicity and the engineering complexity of scaling. This indicates that while pressure offers a powerful solution to the problem of ice formation, it introduces new challenges related to biological tolerance and practical implementation, which requires careful optimization for each specific application.

Table 4: Advantages and Disadvantages of Pressure-Assisted Cryopreservation

Category

Specific Point

Explanation/Mechanism

Advantages

Prevention/Reduction of Ice Crystal Formation

Shifts water phase diagram, induces amorphous ice formation

Preservation of Native State and Ultrastructure

Samples are as close as possible to natural state, enhances analysis accuracy 8

Reduced Cooling Rate and CPA Concentration Requirements

Vitrification at thousands of K/s instead of hundreds of thousands of K/s; potentially reduces needed cryoprotectant concentration 8

Increased Sample Thickness

Ability to vitrify samples up to 0.6 mm thick, significantly surpasses atmospheric pressure limits

Enhanced Supercooling Stability (Isochoric Systems)

Increases metastability of supercooled water, resilience to perturbations, extended storage 23

Passive Pressurization (Isochoric Systems)

Pressure generated passively by ice Ih growth, simplifies process

Bacterial Load Management and Chemical Infusion (Food Applications)

Inactivates pathogens, promotes deeper additive penetration 23

Faster Fixation and Simultaneous Stabilization (HPF)

Advantage over chemical fixation 8

Disadvantages

Barotoxicity (Pressure-Induced Damage)

Harmful effects of high pressure on living materials; depends on pressure level, duration, and biological model type 23

Complexity and Expertise Required (HPF)

Highly specialized technique, requiring significant optimization 8

Cryoprotectant Issues

Toxicity, osmolarity effects, need for careful selection

Engineering Challenges for Industrial/Clinical Scaling (Isochoric Systems)

Requires thick-walled metallic vessels, complex to manufacture, transport, and cool

Contrast Issues (HPF)

Some resins reduce contrast, require post-staining 10

Challenges for Specific Sample Types

Plant material (large vacuoles, air gaps) 10

Cost and Accessibility of Equipment

Specialized HPF equipment is expensive and not always available 8

Limited Biological Validation (Isochoric Systems)

More biological studies needed, especially for supercooling and vitrification 23

Knowledge Gaps (Barotoxicity)

Mechanisms, dependencies, scaling with biological complexity remain unexplored 23

Undetectable Ice Formation (Isochoric Vitrification)

Possibility of ice formation without detection by pressure monitoring 23

Vascular Endothelial Damage and Edema (HMP)

Risks associated with hypothermic machine perfusion

Future Directions and Challenges

The future of pressure-assisted cryopreservation of tissues and organs promises significant breakthroughs but faces a number of complex challenges requiring interdisciplinary efforts.

Scaling for Clinical Application (Human Organs)

A key goal is to overcome current volume limitations and achieve successful vitrification and rewarming of whole human organs, such as hearts, kidneys, and livers, ranging from 0.5 to 3 liters. Current challenges include efficient heat and mass transfer, preventing cracking during cooling and rewarming, and ensuring uniform cryoprotectant penetration, which leads to failures at scale.37 The consistent focus on "human organ scale" as a future goal, despite current limitations in heat transfer and rewarming 37, underscores that whole human organ cryopreservation remains the "holy grail" of the field. This means that while significant progress has been made with cells and small tissues, the complexity of large, vascularized organs presents unique, multifaceted challenges requiring integrated solutions beyond just pressure.

Development of New Cryoprotectants

The toxicity of existing cryoprotectants, such as DMSO and glycerol, is a major limitation, especially for tissues and organs. New CPAs with reduced toxicity are needed that can safely penetrate all cell layers within an organ without damage, which is not possible with current toxic CPAs. The toxicity of current cryoprotectants is a major bottleneck, particularly for tissues and organs. This indicates that even with advancements in pressure technology, the development of novel, less toxic CPAs is a critically important, independent research priority for achieving successful cryopreservation at a clinical scale. The chemical aspect remains a limiting factor.

Addressing Barotoxicity and Fundamental Knowledge Gaps

Despite the advantages, barotoxicity remains a significant limitation.23 A deeper understanding of the mechanisms of barotoxicity, its dependence on exposure duration and rate, and its scaling with biological complexity is needed.23 The observation that different biological models have varying sensitivities and that damage is time- and pressure-dependent 23 highlights that barotoxicity is a complex, multi-parametric problem. Addressing it requires fundamental research into biological responses to pressure, beyond just the physical effects on water.

Industrial Translation of Isochoric Chambers

Despite scientific successes, industrial translation of isochoric freezing has not yet been achieved due to the engineering requirements for "industrial scale" isochoric chambers.23 These chambers must be thick-walled metallic vessels that are challenging to manufacture, transport, and cool, hindering their widespread industrial adoption. The gap between scientific success in the lab and industrial translation of isochoric cryopreservation 23 indicates significant engineering and manufacturing challenges associated with high-pressure vessels. This means that scaling these promising methods requires not only biological and physical breakthroughs but also substantial investment and innovation in materials science and the design of large-scale pressure vessels.

Integration with Other Technologies

The future of organ cryopreservation likely lies in a multimodal approach, integrating pressure-assisted methods with other advanced technologies such as machine perfusion (MP) and nanowarming.37 MP allows for graft viability assessment, functional repair, and reduction of ischemia-reperfusion injury.39 This indicates that no single technique will be a complete solution; rather, a combination of physical, chemical, and engineering strategies will be needed to overcome the multifaceted challenges of long-term organ preservation.

Expanding Application Areas

Exploring the potential of isochoric freezing for transporting organoids to the space station and generating livers in zero gravity demonstrates the versatility of this technique and its potential to address unique challenges beyond conventional clinical or food applications. This points to broader scientific impact and future directions that push the boundaries of biological preservation.

Conclusion

The historical overview of tissue preservation by low temperatures under pressure, eliminating ice formation, reveals a long journey from early, intuitive applications of cold to modern high-tech methods. From the pioneering work of James Arnott in cryosurgery and the accidental discovery of glycerol's cryoprotective properties to Moor and Riehle's conceptual proposals for pressure-assisted freezing, the field has constantly evolved. The contribution of the Russian oncologist Serafim Vasilievich Znamensky, whose work directly addressed tissue preservation under pressure and received recognition during his student years, highlights the early conceptualization of this problem across different scientific schools.

Modern methods, such as High-Pressure Freezing (HPF) and various forms of isochoric cryopreservation, have achieved significant successes in preventing ice crystal formation, preserving native ultrastructure, and expanding the storage capabilities of various biological materials. HPF has become an indispensable tool in electron microscopy, allowing for the examination of samples with unprecedented detail. Isochoric methods demonstrate the potential for preserving larger and more complex biological structures, including whole organs, opening new horizons for transplantology and biobanking.

However, despite these achievements, significant challenges remain. Barotoxicity, or pressure-induced damage, is a serious limitation requiring a deep understanding of biological mechanisms and their dependence on pressure parameters. The complexities of scaling for large organs, the need for developing new, less toxic cryoprotectants, and the considerable engineering difficulties in creating industrial isochoric chambers also present substantial hurdles.

Nevertheless, the prospects for pressure-assisted cryopreservation remain highly promising. Integration with other advanced technologies, such as machine perfusion and nanowarming, as well as the exploration of novel application areas, including space biology, point to the transformative potential of this field. Further interdisciplinary research, combining physics, engineering, chemistry, and biology, will be essential to overcome the remaining barriers and realize the full potential of tissue preservation by low temperatures under pressure.

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