Preface

Cryopreservation is the storage of biological material at ultra-low temperatures, preferably that of liquid nitrogen, which arrests all metabolic activities. This technique is widely applied to all organisms ranging from microorganisms through plants to animals and human organs. Theoretically, no genetic changes should occur during cryopreservation, thus permitting indefinite cell preservation. **Cryopreservation** or **cryoconservation** is a process where organelles, cells, tissues, extracellular matrices, organs, or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures ranging from −80 to −210°C using programmable freezers, liquid nitrogen, and liquid nitrogen slush and vapour. A successful cryopreservation procedure therefore entails ensuring the normal functionality of the organism. In recent times, basic cryopreservation has had applications in research and clinical, medical, and agricultural fields.

Typically, freezing or cooling is lethal to most living organisms' cellular, mechanical, and metabolic functionality. Hence the typical procedures involved in cryopreservation include selecting appropriate tissue, conditioning the tissue, determining cooling rate, storing the tissue, thawing the tissue, and subsequently restoring tissue functions. During cryopreservation, several dynamics come into play; these include cell type, size, water content, temperature, and metabolic state. There have been several limitations to the effective application of cryopreservation techniques and storage, although substantial successes have been reported for both animal and plant cells. Cells have to be protected from damage, especially by ice crystals during freezing. Cryoprotectants, also called Cryo Protective Agents (CPAs), are widely used; however, these may be toxic to the cells depending on the type of cell and the extent of exposure. Mode of freezing, thawing, regenerating, and rejuvenating also may render the process successful or otherwise. Viability of cells may be compromised at any step of the process. Naturally, tissues subjected to cryotemperatures contain fluids such as intracellular and extracellular fluids, plasma, interstitial fluid, and transcellular fluid, which make up 10 to 75 percent of tissues. During cooling, these fluids may form ice crystals, which may nucleate to larger crystals and ultimately damage cells. Hence the use of CPAs may render stability and protection to cell membranes, and where proven useful, preserve functionality of tissue as subjected to cryopreservation.

The main objective of this book is to bring to bear factors that affect cells during cryopreservation. Divided into four sections, this book contains eight chapters providing discussions, overviews, and reviews of cryo-techniques.

The first section is on "Recceing Oligomers, Polymers, and Other Cryoprotective Agents (CPAs)." Successful cooling is dependent on the control of extracellular ice formation, protective intracellular dehydration, and the colligative and dehydration properties of the CPA. Hence CPAs ensure a freeze-avoidance mechanism that enables hydrated tissues to survive when exposed to cryogenic temperatures. There are, however, unanswered questions about the precise mechanism of action of CPAs. In their function to protect living cells against damage during cooling,

**II**

**Chapter 7 139**

and Heating **159**

**Chapter 8 161**

*by Yuting Shao, Chao Chen, Qi Zhou, Jun Yang, Xiao Lv, Mingyue Lin* 

Methods of Thermal Analysis as a Tool to Develop Cryopreservation

Dynamics of Water Content in Plant Tissues During Cooling

Cryopreservation in Ophthalmology

Protocols of Vegetatively Propagated Crops

*by Stacy D. Hammond, Miloš Faltus and Jiří Zámečník*

*and Yanlong Bi*

**Section 4**

they act by lowering the freezing point, modifying the crystalline surface of ice, and averting solute accumulation at a given level of dehydration, all of which counteract injury. The use of CPAs is an essential aspect in all cryopreservation protocols, from slow freezing to vitrification. There are several types including non-permeable (or external) and permeable (internal) CPAs, depending on their ability to permeate biological membranes. Some examples of non-permeable CPAs are glucose, sucrose, or polyvinylpyrrolidone (PVP). Dimethyl sulfoxide (DMSO), 1,2-propanediol, glycerol, and ethylene glycol are examples of permeable CPAs.

In the first chapter, "The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis" Dr. Buff et al. highlight that a routinely used CPA is cytotoxic and apparently mutagenic as utilized in slow-freezing solutions. The authors thus propose new non-penetrating CPAs: chitooligosaccharides (COS). These chitosan oligomers molecules are biocompatible, antioxidant, and bacteriostatic. In their chapter, the authors test the probability of reducing penetrating CPAs during slow-freezing procedures. COS are thus proposed as extracellular CPAs that reduce the use of dimethyl sulfoxide (Me2SO). The authors question the biocompatibility of COS on mouse embryos through the analysis of the cells' development. They evaluate the molecules in slow-freezing solutions with a reduced quantity of Me2SO, and use differential scanning calorimetry (DSC) to evaluate the crystallization and melting processes, the amount of crystallized water, the equilibrium temperature, and consequently the impact of different CPAs. In this study, the authors indicated that COS could promote a decrease in the use of penetrating CPAs while ensuring successful vitrification. However, there remains the need for further investigation of the influence of COS on the organization of ice crystals in cryopreserved samples and the ability of COS to increase the glass-forming tendency of the remaining solution.

Further to this, Dr. Mark Scott et al. provide a chapter on "Cryoprotection of Platelets by Grafted Polymers." Platelets are lifesaving cells that are difficult to store at 4°C due to the formation of cold storage lesions (CSLs). The deployment of methoxypolyethylene glycol (mPEG), a biocompatible polymer, to the membrane of platelets reduces CSLs. In their study, the authors demonstrate that the covalently grafting platelets to mPEG, termed as PEGylation, serves as a potent CPA allowing platelets to be stored at 4°C, or frozen at −20°C, while maintaining normal hemostatic function and counts. Also, there is the prevention of the formation of overt morphological changes resulting from the CSLs. Ultimately the successful cold storage of platelets improves transfusion safety as it reduces the threat of microbial growth in a blood product to be used for patients. As already discussed, the prolonged used of DMSO is toxic to cells. The exciting news is that the use of frozen platelets does not require DMSO inclusion. Hence this requires that studies are conducted for protocol validation and to clinically ensure that PEGylation can facilitate the cold storage of platelets.

The following chapter by Noha A. Al-Otaibi considers "Cryomedia Formula: Cellular Molecular Perspective." The importance of CPAs cannot be over emphasised since the growing market of cell therapy medicinal products (CTMPs) and biopharmaceuticals demands efficient cryopreservation and CPAs. The review presented here considers topics such as conventional cryomedia compositions and protection mechanisms, quality assessment methods of cryopreserved cells, CPAs' protection action, CPA toxicity and detrimental effects, other biochemical effects, and modulating CPAs' damages via additive agents. In the conclusion, the author

**V**

states, that "Understanding of the protective mechanisms of cryomedia ingredients along with identifying powerful protective compounds to enhance cryomedia

The next section of the book, "Procedures for Cryopreserving Gametes," sheds light on the cryopreservation of sperm. In 1954, there was a report of using cryopreservation in humans where previously frozen sperms were inseminated resulting in pregnancies. Following this, in 1957, fowl sperms were cryopreserved by a team of scientists in the United Kingdom directed by Christopher Polge. One may be wondering about the need to use cryopreservation in human reproduction; will we be playing God? The answer is no, because this is simply a means of assisting natural processes. There may be times that it is clinically safe for the reproductive cell of an individual to be preserved for posterity, especially when undergoing, for example, chemotherapy, which may be detrimental to ones' reproductive abilities in the future. This is just one example, but there are several others. In the first chapter in this section, "Cryobiology and Cryopreservation of Sperm," Ali Erdem Öztürk et al. discuss the hypothesis of cryoinjury during cryopreservation and the associated challenges, especially damage caused by CPAs. As usual, DMSO plays a key role here where damage to DNA and mitochondria as well as formation of reducing oxygen species (ROS) are inevitable. This chapter raises issues that should be further investigated. The chapter is also closely related to the preceding chapter in which the authors discuss the use of less damaging CPAs including oligomers and polymers. The current chapter comprehensively addresses the biology, necrosis, and apoptosis of sperm cells during freezing with data supported by electron microscope images. The discussion provided is a guide to future research and helps to identify critical gaps that must be filled to ensure the full potential of using

performance is highly demanded."

cryopreservation in enriching species' reproducibility.

functioning properties of corneal endothelial cells.

as health status of offspring.

In the next chapter, "Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine," the authors present the imperative function of cryopreservation in assisted reproduction techniques. The need for improved techniques and functionality of sperms, including motility after thawing, is still a challenge although this system has been around for several years. Though new methods including lyophilization have been proposed, they need to be validated, and hence future research needs to investigate and optimize safety methods as well

There is the obvious clinical demand that during surgery where organs have to be isolated and transplanted, the isolated organ should be safely transported, stored, and be functional for a successful use. Technically, where distance and time are not a challenge, this can be effectively carried out, otherwise organs go to waste. It is necessary that demand and supply systems are established with the aim of stabilizing the biological tissues and preventing metabolic and biochemical

processes that render organs nonfunctional. The next section of the book, "Cryobiology Aiding to Organ Transplant," focuses on procedures that have to be improved so that sub-zero temperatures can assist in saving lives. In the first chapter of this section, "Cryopreservation in Ophthalmology," Dr. Shao et al. review how cryopreservation can be deployed to facilitate amniotic membrane and cornea preservation in ophthalmology. There is more emphasis on CPAs in this chapter where glycerol-cryopreserved cornea tissues can be effectively and bio-safely used. The need for equipment, however, complicates the high cost of technical support required. Transportation is still a bottleneck that has to be dealt with to maintain

states, that "Understanding of the protective mechanisms of cryomedia ingredients along with identifying powerful protective compounds to enhance cryomedia performance is highly demanded."

The next section of the book, "Procedures for Cryopreserving Gametes," sheds light on the cryopreservation of sperm. In 1954, there was a report of using cryopreservation in humans where previously frozen sperms were inseminated resulting in pregnancies. Following this, in 1957, fowl sperms were cryopreserved by a team of scientists in the United Kingdom directed by Christopher Polge. One may be wondering about the need to use cryopreservation in human reproduction; will we be playing God? The answer is no, because this is simply a means of assisting natural processes. There may be times that it is clinically safe for the reproductive cell of an individual to be preserved for posterity, especially when undergoing, for example, chemotherapy, which may be detrimental to ones' reproductive abilities in the future. This is just one example, but there are several others. In the first chapter in this section, "Cryobiology and Cryopreservation of Sperm," Ali Erdem Öztürk et al. discuss the hypothesis of cryoinjury during cryopreservation and the associated challenges, especially damage caused by CPAs. As usual, DMSO plays a key role here where damage to DNA and mitochondria as well as formation of reducing oxygen species (ROS) are inevitable. This chapter raises issues that should be further investigated. The chapter is also closely related to the preceding chapter in which the authors discuss the use of less damaging CPAs including oligomers and polymers. The current chapter comprehensively addresses the biology, necrosis, and apoptosis of sperm cells during freezing with data supported by electron microscope images. The discussion provided is a guide to future research and helps to identify critical gaps that must be filled to ensure the full potential of using cryopreservation in enriching species' reproducibility.

In the next chapter, "Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine," the authors present the imperative function of cryopreservation in assisted reproduction techniques. The need for improved techniques and functionality of sperms, including motility after thawing, is still a challenge although this system has been around for several years. Though new methods including lyophilization have been proposed, they need to be validated, and hence future research needs to investigate and optimize safety methods as well as health status of offspring.

There is the obvious clinical demand that during surgery where organs have to be isolated and transplanted, the isolated organ should be safely transported, stored, and be functional for a successful use. Technically, where distance and time are not a challenge, this can be effectively carried out, otherwise organs go to waste. It is necessary that demand and supply systems are established with the aim of stabilizing the biological tissues and preventing metabolic and biochemical processes that render organs nonfunctional. The next section of the book, "Cryobiology Aiding to Organ Transplant," focuses on procedures that have to be improved so that sub-zero temperatures can assist in saving lives. In the first chapter of this section, "Cryopreservation in Ophthalmology," Dr. Shao et al. review how cryopreservation can be deployed to facilitate amniotic membrane and cornea preservation in ophthalmology. There is more emphasis on CPAs in this chapter where glycerol-cryopreserved cornea tissues can be effectively and bio-safely used. The need for equipment, however, complicates the high cost of technical support required. Transportation is still a bottleneck that has to be dealt with to maintain functioning properties of corneal endothelial cells.

**IV**

solution.

facilitate the cold storage of platelets.

they act by lowering the freezing point, modifying the crystalline surface of ice, and averting solute accumulation at a given level of dehydration, all of which counteract injury. The use of CPAs is an essential aspect in all cryopreservation protocols, from slow freezing to vitrification. There are several types including non-permeable (or external) and permeable (internal) CPAs, depending on their ability to permeate biological membranes. Some examples of non-permeable CPAs are glucose, sucrose, or polyvinylpyrrolidone (PVP). Dimethyl sulfoxide (DMSO), 1,2-propanediol, glycerol, and ethylene glycol are examples of permeable CPAs.

In the first chapter, "The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis" Dr. Buff et al. highlight that a routinely used CPA is cytotoxic and apparently mutagenic as utilized in slow-freezing solutions. The authors thus propose new non-penetrating CPAs: chitooligosaccharides (COS). These chitosan oligomers molecules are

Further to this, Dr. Mark Scott et al. provide a chapter on "Cryoprotection of Platelets by Grafted Polymers." Platelets are lifesaving cells that are difficult to store at 4°C due to the formation of cold storage lesions (CSLs). The deployment of methoxypolyethylene glycol (mPEG), a biocompatible polymer, to the membrane of platelets reduces CSLs. In their study, the authors demonstrate that the covalently grafting platelets to mPEG, termed as PEGylation, serves as a potent CPA allowing

platelets to be stored at 4°C, or frozen at −20°C, while maintaining normal hemostatic function and counts. Also, there is the prevention of the formation of overt morphological changes resulting from the CSLs. Ultimately the successful cold storage of platelets improves transfusion safety as it reduces the threat of microbial growth in a blood product to be used for patients. As already discussed, the prolonged used of DMSO is toxic to cells. The exciting news is that the use of frozen platelets does not require DMSO inclusion. Hence this requires that studies are conducted for protocol validation and to clinically ensure that PEGylation can

The following chapter by Noha A. Al-Otaibi considers "Cryomedia Formula: Cellular Molecular Perspective." The importance of CPAs cannot be over

emphasised since the growing market of cell therapy medicinal products (CTMPs) and biopharmaceuticals demands efficient cryopreservation and CPAs. The review presented here considers topics such as conventional cryomedia compositions and protection mechanisms, quality assessment methods of cryopreserved cells, CPAs' protection action, CPA toxicity and detrimental effects, other biochemical effects, and modulating CPAs' damages via additive agents. In the conclusion, the author

biocompatible, antioxidant, and bacteriostatic. In their chapter, the authors test the probability of reducing penetrating CPAs during slow-freezing procedures. COS are thus proposed as extracellular CPAs that reduce the use of dimethyl sulfoxide (Me2SO). The authors question the biocompatibility of COS on mouse embryos through the analysis of the cells' development. They evaluate the molecules in slow-freezing solutions with a reduced quantity of Me2SO, and use differential scanning calorimetry (DSC) to evaluate the crystallization and melting processes, the amount of crystallized water, the equilibrium temperature, and consequently the impact of different CPAs. In this study, the authors indicated that COS could promote a decrease in the use of penetrating CPAs while ensuring successful vitrification. However, there remains the need for further investigation of the influence of COS on the organization of ice crystals in cryopreserved samples and the ability of COS to increase the glass-forming tendency of the remaining

In the next chapter, "Current Advancements in Pancreatic Islet Cryopreservation Techniques," Dr. Rodriguez et al. outline the limitations of effective storage during donor-recipient cross-matching after islet isolation. The authors characterize aspects of the islet cryopreservation method under the following topics: history of islet cryopreservation and characteristics of the islet cryopreservation process such as cryoprotection and thawing. They present advances in islet CPA technology with a focus on permeating and non-permeating reagents, as well as the use of alginatebased microencapsulation technology. In conclusion, the authors are hopeful that a standard cryopreservation protocol, islet banking, would be more feasible and that eventually transportation "would no longer be throttled by the donor-recipient mismatch."

The final section of the book is titled "Dynamics of Water Content in Plant Tissues during Cooling and Heating." Water is a critical component of organisms and serves as the medium for metabolic activities occurring inter and intracellularly. Water thus gets in the way when cells need to be cooled, prompting the questions, how much water should a cell lose and how should this be done to maintain viability and genetic integrity of the organism? In the chapter, "Methods of Thermal Analysis as a Tool to Develop Cryopreservation Protocols of Vegetatively Propagated Crops," Dr. Hammond et al. place emphasis on the challenge of reducing water content of vegetative plant tissues to facilitate successful cryotreatment. It is critical that glass transition is formed during cooling because if ice crystals are formed, the cells are damaged. The application of DSC, temperature modulation DSC, and quasi-isothermal modulation DSC (QITTMDSC) are used here to monitor heat flow types in measuring the quantity of freezable water. The authors conclude by highlighting the significance of apprehending the dynamics of water content in plant tissues when cooling and heating as part of the development of a dependable cryopreservation protocol. Although the study recommends the standard DSC method for day-to-day work with known thermal properties of the sample and non-overlapping thermal events, QITTMDSC is endorsed for exact measurement of heat capacity in equilibrated conditions, which can help in identifying the state of matter.

This book is the contribution of more than thirty-five authors from eighteen institutions in seven countries. A rich source of information is provided on current developments and reviews of previous research as well as existing standard operating procedures. The book brings to the fore that although cryopreservation has progressed very well, there is much more to be explored, particularly in the case of CPAs. I am personally interested in having CPAs that will not compromise the biology of cells before they are exposed to sub-zero temperatures and will allow tissues to function normally after thawing. In a situation where there are no restrictions to the quantity of CPAs that can be used, the subjection of cells to cryopreservation will be effortlessly conducted. Though the question still persists: "Is cryopreservation just an illusion for some biological systems?" Let us work towards answering this question as we pursue cryobiology and cryopreservation.

Section 1

Recceing Oligomers,

Polymers and Other

Cryoprotective Agents

**1**

## Section 1
