Introduction
Cryopreservation of tissues and organs has made tissue and organ transplantation possible and has been regarded as an important move in treating serious diseases and has increased survival rates. Although the cryopreservation of tissues has emerged and has aided in organ and tissue transplantation in various clinical scenarios, WHO (World Health Organization) estimates that less than ten percent of the organ transplantation demands have been met globally. Due to the short storage time offered for storing the donated organs, which is around 4 to 36 hours, many of the organs, even though healthy, are being discarded because of the exceeded preservation time. This is one significant reason for the shortage of organs available for transplantation. Recent times in the field of cryopreservation have evolved with various modern techniques of cryopreservation to increase the storage time of the cryopreserved organs, making them available and eligible for tissue and organ transplantation.
What Is Cryopreservation?
Cryopreservation is also called cryo conservation. Cryopreservation is a process in which biological material like cells, tissues, or organs are preserved by retaining their physiological functions by freezing at a very low temperature of around (80 to 196) degrees Celsius, as the cellular metabolism tends to decrease with the decrease in temperature and hence can be preserved for a longer duration of time.
What Are the Basic Concepts of Cryopreservation?
The basic concept of cryopreservation depends on two factors:
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The nature and complexity of the cryopreserved tissue.
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The time duration required.
What Are the Commonly Used Techniques of Cryopreservation?
1. Conventional Slow Freezing:
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This technique employs gradual cooling of the tissues at a rate of one degree Celsius per minute.
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However, cryopreservation using this conventional technique is quite challenging as slow cooling causes the formation of ice crystals on the outer layer of the cells, causing mechanical injury.
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Also, an osmotic injury will be encountered due to the extrusion of water from the cell, causing increased solute concentrations.
2. Directional Freezing:
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This technique involves maintaining a constant temperature gradient during the process of cooling to reduce the formation of ice crystals.
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Mechanical damage to the cells by the ice crystal is reduced in this process.
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In this technique, a uniform cooling rate of organs and tissues is achieved and can be advised in cases of rapid cooling, too.
3. Vitrification:
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Vitrification refers to the rapid freezing of tissues, skipping the crystallization phase and directly forming an amorphous or non-crystalline phase.
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This is achieved by keeping the cooling rates faster than the critical cooling rate (CCR), which prevents the formation of ice crystals.
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As the technique involves continuous warming (for utilizing the tissues for organ transplants) and cooling, the tissues undergo continuous thermal stress, which can be prevented with the help of cryopreservative agents (CPAs).
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The most commonly used CPAs are glycerol, DMSO (dimethyl sulfoxide), and Trehalose.
What Are the Limitations of the Commonly Used Cryopreservation Techniques?
1. Formation of Ice Crystals:
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The cells undergoing the cooling and rewarming process of cryopreservation often face the challenge of ice crystal formation.
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Slow cooling causes the formation of ice crystals on the outer layer of the cells, causing mechanical injury.
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Slow cooling processes are most commonly associated with ice crystal formation and are difficult to manage even after adopting the best preservation protocols.
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Also, an osmotic injury will be encountered due to the extrusion of water from the cell, causing increased solute concentrations.
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Rapid cooling results in intracellular ice formation due to the inadequate time rendered and the water gets entrapped in the cell, causing mechanical damage to the cells, tissues, and organs.
2. Toxicity Caused Due to Higher Levels of CPAs:
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Dimethyl sulphoxide acts as a potent toxic agent damaging mitochondria, proteins, and other structures.
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As the intracellular gap junctions play an important role in cell function and coordination, the destruction of these structures and the tissues or organs transplanted soon after cryopreservation may fail to function properly in the recipient, leading to transplant failure.
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The conventional vitrification process uses high levels of CPAs of around four to eight molar to prevent ice crystal formation, which can cause lethal effects on the cells and tissues.
3. Heat and Mass Transfer in Complex Structures:
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Due to the complexity of the cellular arrangements, the diffusion of water and CPAs becomes irregular, and uniform concentration cannot be achieved and hence results in toxicity.
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Difficulty in mass and heat transfer is attributed to the macroscopic complexity of the tissues and thermal conductivity.
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This causes thermal stress and damage during the process of cryopreservation.
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Apart from the survival of individual cells, the preservation of intercellular connections is also vital as the tissues transplanted may fail to perform the activities after cryopreservation.
4. Oxidative Damage:
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Under normal conditions, the metabolic processes like oxidative and redox reactions are well balanced.
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During cryopreservation, due to the low temperature, enzyme activity is affected and also leads to deficiency of ATP and excess accumulation of calcium in the cells, leading to oxidative damage and generation of reactive oxygen species (ROS).
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Excessive ROS causes damage to the DNA, oxidation of protein, and lipid peroxidation, which impairs cellular function and negatively affects the vitality of the cell, post-cryopreservation, and organ transplantation.
What Are the Recent Advances Adopted in the Cryopreservation of Tissues?
The evolution of recent advances in the field of cryopreservation is influenced by the limitations of conventional techniques in retaining the quality of cellular functions and longevity of the tissues and organs in the post-transplantation period. The recent techniques are based on the following principles:
1. Reducing the Toxicity Caused Due to CPAs: CPAs being used to prevent ice crystal formation became toxic and caused damage to the cells and organs, which led the research field to discover highly efficient CPAs with lower toxicity issues. Some of them include antifreeze proteins, macromolecular polymers, and nanomaterials. Preparing cocktail proportions by combining various CPAs in specific molar preparations has proved to reduce toxicity. Some of the cocktail preparations include VS41A and natural deep eutectic systems (NADES). Hypothermic machine perfusion (a technique devised to prevent reperfusion injury by loading a cold perfusate into the circulation) using several loading and unloading steps of CPA can reduce toxicity.
2. Discovering Newer Cryopreservation Techniques: To compensate for the ice crystal formation and the mechanical stress caused by it in the cells and tissues, newer techniques, such as:
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Nanoparticle-mediated intracellular trehalose (CPA) delivery.
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Microfluidics-controlled hydrogel devised for cell protection.
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Sand-induced ice nucleation.
It has achieved better results in the process of cryopreservation. Adopting magnetic and laser fields to cater to the nano-warming process to avoid any mechanical stress to the tissues caused either by the formation of ice crystals, thermal stress, and improper perfusion of CPAs due to the complex anatomy of large organs and tissues. Adequate use of additives like antioxidants and caspase inhibitors has proven to increase the vitality of the cells post-cryopreservation. These newer techniques involve adequate infusion of CPAs and other magnetic nanoparticles through a hypothermic machine perfusion process followed by rapid heating in a uniformly mediated radiofrequency of a magnetic field to prevent the process of devitrification (crystallization) and thermal stress.
Conclusion
Cryopreservation of organs and tissues is now a more promising entity for various tissue and organ transplantation. Newer advances in the field, such as combining vitrification and nano-warming techniques, have helped ward off various challenges like the formation of ice crystals, thermal stress, and the toxicity caused by CPAs. These techniques are very useful in cryopreservation of smaller tissues. However, large complex tissues still face certain challenges in retaining vitality due to their more refined structures, and hence, research in this regard will help larger complex organs in the future. Further evolution of research in the field of cryopreservation of vital organs like the kidney, liver, and heart will greatly influence the post-cryopreservation quality and vitality in the post-transplantation period, positively affecting life expectancies.
