Preface

Almost a decade has passed since the last textbook on the science of cryobiology and the most common methods of cryopreservation was published [Fuller *et al*, 2007], to which we will refer as *"the previous book"* here and below. When it was published, it became a useful guide for both "seasoned" cryobiologists and those who had just started their journey to this fascinating science.

However, there have been some serious tectonic shifts in cryobiology, which were perhaps not seen on the surface but may have a profound effect on both the future of cryobiology and on the development of new cryopreservation methods. We feel that it is time to revise the previous paradigms and dogmas, discuss the conceptually new cryobiological ideas and introduce the recently emerged practical methods of cryopreservation. The present books, *Current Frontiers in Cryobiology* [Katkov, 2012A] (referred here as *Book 1*) and *Current Frontiers in Cryopreservation* [Katkov, 2012B] (*Book 2*), will serve the purpose. These two books are not a substitute for *the previous book* but are rather complementary, so we highly recommend to all readers who want to know the background on which *the current books* were written to read *the previous book* as well.

Before we describe the current books, let us first briefly compare them to the previous book in retrospective. First of all, there were some very promising directions a decade ago that unfortunately did not meet the expectations. Molecular biology and genetics, particularly in regards to expression of stress proteins and other pathways related to the cell injury, have not introduced any serious breakthroughs except for the use of ROCK inhibitors for cryopreservation of human embryonic and induced pluripotent stem cells. The latter really was a revolutionary discovery, which however, was not made by cryobiologists; it was just *"*picked up*"* by them from the Watanabe's seminal work [Watanabe *et al*, 2007] (see the Chapter by Martin- Ibáñez in *Book 1* for details). In general, however, all those molecular biology tools have helped the solution but have not solved the cryopreservation problems *per se*. One of the backlashes of this new era is that the "traditional" cryobiologists now have little chances of getting a grant from many funding agencies such as NIH, whose panels are dominated by molecular biologists and geneticists, unless the applicant is willing to study those pathways and use of transcriptomics, proteomics, metabolomics, and other *"*omics*"*. Yet, all those very expensive tools have so far added a little to the science of cryobiology, and especially to the practices of cryopreservation. Moreover, it is sad to see the how some new publications *"*rediscover the wheel", repeating many achievements of cryobiologists that had been done one or two decades before but were not referred as full size papers on PubMed, and these novel rediscoveries are often done at a much greater cost. We must agree with the author of *Foreword* of the previous book, who insightfully wrote "*I see now much of the early ground being replowed, often by equally empirical methods, albeit as far greater expenses…The concept of science as a community of colleagues engaged in public service … has been eroded by the cost of research and the emergence of industry as not only a major source of research funding but as the ultimate exploiter of the results, and we have no choice but to play the game"* (Foreword in [Fuller *et al*, 2007] by H. Meryman). However, we hope that might change in the future and that an alliance between cryobiology and molecular and cellular biology will bring *real* practical fruits.

Preface XI

thermodynamics and the definition of the vitreous phase as the state with enormous viscosity, as well as that it is in contradiction with the Stokes-Einstein Law of diffusion. Numerous reports, which state *Tg* of the sample as high as + 60OC while drying was stopped at, say, *Tdr,f* = +20OC are *incorrect*. Such overestimation of *Tg* (which in fact is 20-25 degrees lower than *Tdr,f*) lead to unsubstantiated expectations of long stability at supra-zero, even ambient temperatures at relatively *high* water content of the sample, which has never proven the case in thorough experiments (with some reservation to platelets as rather *"cell debris"* than true cells). All these data, if checked properly, are in fact either artifacts - the results of incorrect gravimetrical measurements or the use of inaccurate methods such as DSC. We are confident that the real viscosity, not those *mysterious* high temperature DSC peaks, should be measured for correct determination of the *biostabilization Tg* (defined as the point at

And as *the last but not the least in our list* is the notion that all those ice-blockers, freeze and shock proteins, and other promising from a decade ago classes of molecules have not so far shown to be used in cryopreservation protocols alone but always in a concert with *ole good* permeable cryoprotectants and impermeable sugars, and other low molecular weight molecules, which have been around for decades. This is especially true in regards to vitrification, specifically of organs: *"the promise of the 21st century medicine"* has remained as far from the completion as it was 25+ years ago with the report on *equilibrium* vitrification of a kidney by Fahy and colleagues. On the other hand, the assisted reproduction cryobiology is rapidly moving toward *kinetic* vitrifcation, the very method of cryopreservation described by the most prominent pioneer of cryopreservation, Father Basil Luyett, more than 7 decades ago. We specifically dedicated our *Chapter 1* both to the memory of this brilliant scientist and to the detailed analysis of the situation, the difference between the two approaches to vitrifcation (*kinetic* vs. *equilibrium*), and to a quite opposite foreseeable future for them.

On the other hand, there are new directions (or the old ones, replowed with deeper and more thorough plowing techniques) on the horizon of cryobiology. Among them, we can mention the attractiveness of cryopreservation of *adherent* cells (often in monolayers) not only for the benefit of the cells *per s*e (by avoiding anoikis triggered apoptosis, etc), but mainly for the convenience of the rapidly emerging field of cell based high throughput and high content analyses, where cells can be frozen, stored, shipped, and *ready-to-go* after thawing directly in multi-well plates (see Chapter by

The other serious breakthrough that was missed by many authors a decade ago is the kinetic vitrifcation of sperm and the emergence of what we call *"Race for the Pace"*, a set of new devices for ultra-fast cooling of samples such as Open Pulled Straws, Microdroplets, Vitrification on the Solid Surface, VitMaster (slush cooling), Cryogenic Oscillating Heat Pipes, Quartz Capillaries, and some others (see Chapter by Cipri *et al* in *Book 1*). We think that many of these devices are rather transient to a new generation

which viscosity reaches 10 x 1013.6 Pa x sec).

Martín-Ibáñez in *Book 1*).

The slogan *"Let Us Learn from Mother Nature",* while being attractive *per se* (it is actually imbedded in the title of our first Chapter by Katkov *et al* in *Book 1*), must be taken with a grain of salt. Yes, Mother Nature has liquid crystals in biological membranes, but LCD TV screens were invented by man. Yes, there are rotifers and other "molecular motors" in cells, but the wheel was discovered and built by the human race. And finally, there are TV, radio, internal combustion engines, and many other devices and apparatuses that have no close analogy in wildlife. Similarly, while some robust creatures are well adapted to survive for short time at up to -20OC, there is no place on Earth that cools down below the glass transition temperature of water (- 136OC), and there is no place on Earth where liquid nitrogen is present. Ergo, practically no one natural biosystem can adapt to such low temperatures just by natural selection, it needs human help to be stabilized for infinite time at -196OC. Thus, while learning something from the natural phenomena, it is our strong opinion that we should not rely on them too much: the money for supplying an Antarctica deep lake drilling or a Mars expedition can be spent much more efficiently and *usefully for humankind* if channeled to the development of a new controllable freezer for cryopreservation of large tissues and organs, similar for instance, to the described in our *Book 1* by Butler and Pegg.

The next large area where the progress has been quite slower than it was expected a decade ago is lyophilization and desiccation of cells of vertebrates. So far, there is no compelling evidence that would convince us that there is a method of freeze-drying or desiccation that has produced *viable* mammalian cells that can be stored for sufficient amount of time (> 2 years) at temperature above +4OC despite the fact that the opposite was claimed many times in the last 50 years. We briefly explored this aspect in our *Chapter 1* of *Book 1*. We think that this field has remained to be trapped in a set of scientific misconceptions, such as the possibility of drying the sample to the glass transition *Tg* that is above the final temperature or drying *Tdr,f*, or the related misleading concept of the possibility of substantial movement of water in a sample below its glass transition. We think that such statements violate the laws of thermodynamics and the definition of the vitreous phase as the state with enormous viscosity, as well as that it is in contradiction with the Stokes-Einstein Law of diffusion. Numerous reports, which state *Tg* of the sample as high as + 60OC while drying was stopped at, say, *Tdr,f* = +20OC are *incorrect*. Such overestimation of *Tg* (which in fact is 20-25 degrees lower than *Tdr,f*) lead to unsubstantiated expectations of long stability at supra-zero, even ambient temperatures at relatively *high* water content of the sample, which has never proven the case in thorough experiments (with some reservation to platelets as rather *"cell debris"* than true cells). All these data, if checked properly, are in fact either artifacts - the results of incorrect gravimetrical measurements or the use of inaccurate methods such as DSC. We are confident that the real viscosity, not those *mysterious* high temperature DSC peaks, should be measured for correct determination of the *biostabilization Tg* (defined as the point at which viscosity reaches 10 x 1013.6 Pa x sec).

X Preface

practical fruits.

our *Book 1* by Butler and Pegg.

especially to the practices of cryopreservation. Moreover, it is sad to see the how some new publications *"*rediscover the wheel", repeating many achievements of cryobiologists that had been done one or two decades before but were not referred as full size papers on PubMed, and these novel rediscoveries are often done at a much greater cost. We must agree with the author of *Foreword* of the previous book, who insightfully wrote "*I see now much of the early ground being replowed, often by equally empirical methods, albeit as far greater expenses…The concept of science as a community of colleagues engaged in public service … has been eroded by the cost of research and the emergence of industry as not only a major source of research funding but as the ultimate exploiter of the results, and we have no choice but to play the game"* (Foreword in [Fuller *et al*, 2007] by H. Meryman). However, we hope that might change in the future and that an alliance between cryobiology and molecular and cellular biology will bring *real*

The slogan *"Let Us Learn from Mother Nature",* while being attractive *per se* (it is actually imbedded in the title of our first Chapter by Katkov *et al* in *Book 1*), must be taken with a grain of salt. Yes, Mother Nature has liquid crystals in biological membranes, but LCD TV screens were invented by man. Yes, there are rotifers and other "molecular motors" in cells, but the wheel was discovered and built by the human race. And finally, there are TV, radio, internal combustion engines, and many other devices and apparatuses that have no close analogy in wildlife. Similarly, while some robust creatures are well adapted to survive for short time at up to -20OC, there is no place on Earth that cools down below the glass transition temperature of water (- 136OC), and there is no place on Earth where liquid nitrogen is present. Ergo, practically no one natural biosystem can adapt to such low temperatures just by natural selection, it needs human help to be stabilized for infinite time at -196OC. Thus, while learning something from the natural phenomena, it is our strong opinion that we should not rely on them too much: the money for supplying an Antarctica deep lake drilling or a Mars expedition can be spent much more efficiently and *usefully for humankind* if channeled to the development of a new controllable freezer for cryopreservation of large tissues and organs, similar for instance, to the described in

The next large area where the progress has been quite slower than it was expected a decade ago is lyophilization and desiccation of cells of vertebrates. So far, there is no compelling evidence that would convince us that there is a method of freeze-drying or desiccation that has produced *viable* mammalian cells that can be stored for sufficient amount of time (> 2 years) at temperature above +4OC despite the fact that the opposite was claimed many times in the last 50 years. We briefly explored this aspect in our *Chapter 1* of *Book 1*. We think that this field has remained to be trapped in a set of scientific misconceptions, such as the possibility of drying the sample to the glass transition *Tg* that is above the final temperature or drying *Tdr,f*, or the related misleading concept of the possibility of substantial movement of water in a sample below its glass transition. We think that such statements violate the laws of

And as *the last but not the least in our list* is the notion that all those ice-blockers, freeze and shock proteins, and other promising from a decade ago classes of molecules have not so far shown to be used in cryopreservation protocols alone but always in a concert with *ole good* permeable cryoprotectants and impermeable sugars, and other low molecular weight molecules, which have been around for decades. This is especially true in regards to vitrification, specifically of organs: *"the promise of the 21st century medicine"* has remained as far from the completion as it was 25+ years ago with the report on *equilibrium* vitrification of a kidney by Fahy and colleagues. On the other hand, the assisted reproduction cryobiology is rapidly moving toward *kinetic* vitrifcation, the very method of cryopreservation described by the most prominent pioneer of cryopreservation, Father Basil Luyett, more than 7 decades ago. We specifically dedicated our *Chapter 1* both to the memory of this brilliant scientist and to the detailed analysis of the situation, the difference between the two approaches to vitrifcation (*kinetic* vs. *equilibrium*), and to a quite opposite foreseeable future for them.

On the other hand, there are new directions (or the old ones, replowed with deeper and more thorough plowing techniques) on the horizon of cryobiology. Among them, we can mention the attractiveness of cryopreservation of *adherent* cells (often in monolayers) not only for the benefit of the cells *per s*e (by avoiding anoikis triggered apoptosis, etc), but mainly for the convenience of the rapidly emerging field of cell based high throughput and high content analyses, where cells can be frozen, stored, shipped, and *ready-to-go* after thawing directly in multi-well plates (see Chapter by Martín-Ibáñez in *Book 1*).

The other serious breakthrough that was missed by many authors a decade ago is the kinetic vitrifcation of sperm and the emergence of what we call *"Race for the Pace"*, a set of new devices for ultra-fast cooling of samples such as Open Pulled Straws, Microdroplets, Vitrification on the Solid Surface, VitMaster (slush cooling), Cryogenic Oscillating Heat Pipes, Quartz Capillaries, and some others (see Chapter by Cipri *et al* in *Book 1*). We think that many of these devices are rather transient to a new generation of hyper-fast coolers and warmers, but yet, the rapid ascent of kinetic vitrification is the phenomenon that has been largely missed and often simply ignored by the *"*classical cryobiologists "at the end of the last century and the beginning of the current one. Our books dedicate a lot of space to those aspects and their future directions.

Preface XIII

difference is the type of the paper as described earlier, yet in many cases, this difference is rather vague: we do not consider chapters in *Book 2* as "second class" at all: *Books 1* and *2* are inseparable. The sections and chapters of the books are as follows**:** 

Section *"Basic Cryobiology and Kinetic Vitrification"* opens *Book 1*. The first, and the two following chapters are dedicated to kinetic vitrification as the re-emerging method of cryopreservation. Chapter 1 by Katkov and colleagues reiterates the idea that basically all methods of long-term stabilization of cells are in fact different ways (the authors identify 5 of them) of vitrifcation of the intracellular milieu. The chapter gives a detailed thermodynamical description and analysis of the methods. The second part of the chapter is dedicated to the kinetic vitrification of human and animal spermatozoa, the concept of the *"Universal Cryopreservation Protocol*" and what the author called *"Race for the Pace"*, though the last one needs a separate chapter and is only mentioned briefly as one of the future directions. The chapter by *Isachenko and colleagues* tells the story of successful vitrification of human and animal spermatozoa, and its emerging as a valuable tool applied to the assisted reproduction technologies. The third chapter, by a Canadian group (Moskovtsev *et al*) is an *independent* report of the success of vitrifcation of human sperm without permeable (and potentially toxic) cryoprotectants (vitrificants) with certain modifications of the Isachenko's method. The chapter by Gao & Zhou is dedicated to the basic cryobiology of osmotic effects, prevention of the osmotic injury, as well as to the equipment for the optimal addition and elution of

Section *"Stem Cells and Cryopreservation in Regenerative Medicine"* in *Book 1* is presented by a review by Martín-Ibáñez on cryopreservation of human *pluripotent* stem cells; it is the cutting edge of the contemporary cryobiological science where major discoveries have been made very recently. Cryopreservation of *adult* rat mesenchymal stem cells by vitrification is the theme of the chapter by Bahadori *et al* in *Book 2*. It is the one of the chapters when the Editor disagrees with the evaluation of the convenience of cryopreservation of stem cells by vitrification in small containers such OPS, but as we said before, we judged the experimental science, not the concept, and the former one was self-evidently good. The chapter by Campbell & Brockbank reports very interesting results on cryopreservation of adherent smooth muscle and endothelial cells, a direction that, as we mentioned before, may bring about some interesting practical applications. The other two chapters in *Book 2* are the one by U. Santos and colleagues, dedicated to cryopreservation of musculoskeletal cells and tissues, and the other application of regenerative medicine - cryopreservation of allograft for knee

Section *"Human Assisted Reproduction Techniques (ART)"* opens with a review by Liebermann on vitrifcation of embryos and oocytes, a fast developing method of ART. Juergen and Michael Tucker have edited an excellent book dedicated to the use of vitrifcation in human ART [Liebermann & Tucker, 2007] that we highly recommend for reading to the specialists in the field. This review in *Book 1* summarizes the latest

osmotically active permeable cryoprotective agents (CPAs).

ligament construction is the theme of the Chapter by Bitar *et al*.

There are also some other differences between our books and *the previous book* published by CRC, which are mainly determined by the very nature of how the Open Access operates. To begin with, our books are closely related but yet are different. *Book 1* contains mainly reviews that were written by the leaders in the field and were solicited by the Editor. In contrast, *Book 2* (in general, with some exceptions) is dedicated mostly to the reports of concrete methods of cryopreservation, and its chapters are often written by young or emerging scientists who want to make their discoveries public as soon as possible. The Editor is well aware of how discouraging and often devastating the reviewing process in "standard' journals can be just because the reviewer(s) did not share innovative ideas proposed by the author, even though the experimental or mathematical aspects of the manuscript raised no questions. The Editor of these books has reviewed all submitted chapters, about a dozen of them has been rejected, and among 41 published chapters, many were revised one, two, or sometimes three times. But that was always regarding the quality of the manuscripts, not the quality of the author's science; if I sometimes disagreed with the author's opinion, I then *"let the cryo people go"* with their perception, not mine or the one of some external reviewer. In the revolutionary spirit of Open Access, let the common reader, not an elitist reviewer, be the judge in the end!

Another "democratic" aspect in our books in the times of globalization is that the contributions were made by people from 27 countries from *all* continents (except Antarctica). Editor *greatly* appreciates the *invaluable* contribution of the American, Australian, British, Canadian, and New Zealand scientists to the field, and 10 out of 42 Chapters in our books were contributed by authors from those countries. However, cryobiology has long existed in many other languages and cultures. We found the tone of some, especially "historical" reviews written by prominent cryobiologists that may make an impression that the scientists of *that* linguistic domain have predominantly contributed to cryobiology quite uncomfortable; in other words: *"If it is not published in English - it doesn't exist"* so to speak. In contrast to such biases, the seminal works of Luyett, Smirnov, Jahnel, Boutron, Milovanov, Cassou, Ostashko, Sumida, Kopeika, and many other scientists whose first language of publication and/or mother tongue were not English, but whose *pioneering* contribution to the theory and practice of low temperature stabilization has been recognized over the World, is also highly regarded in our books.

Yet another difference is that the chapters of our books are grouped into topics (Sections) that are *"*subject oriented*"* rather than loosely flocked to the *"*Themes*"* so none would wonder why one chapter on freezing of plants is in one section, while another one ends up in another. The sections are the same for both our *books*, the only difference is the type of the paper as described earlier, yet in many cases, this difference is rather vague: we do not consider chapters in *Book 2* as "second class" at all: *Books 1* and *2* are inseparable. The sections and chapters of the books are as follows**:** 

XII Preface

of hyper-fast coolers and warmers, but yet, the rapid ascent of kinetic vitrification is the phenomenon that has been largely missed and often simply ignored by the *"*classical cryobiologists "at the end of the last century and the beginning of the current one. Our books dedicate a lot of space to those aspects and their future directions.

There are also some other differences between our books and *the previous book* published by CRC, which are mainly determined by the very nature of how the Open Access operates. To begin with, our books are closely related but yet are different. *Book 1* contains mainly reviews that were written by the leaders in the field and were solicited by the Editor. In contrast, *Book 2* (in general, with some exceptions) is dedicated mostly to the reports of concrete methods of cryopreservation, and its chapters are often written by young or emerging scientists who want to make their discoveries public as soon as possible. The Editor is well aware of how discouraging and often devastating the reviewing process in "standard' journals can be just because the reviewer(s) did not share innovative ideas proposed by the author, even though the experimental or mathematical aspects of the manuscript raised no questions. The Editor of these books has reviewed all submitted chapters, about a dozen of them has been rejected, and among 41 published chapters, many were revised one, two, or sometimes three times. But that was always regarding the quality of the manuscripts, not the quality of the author's science; if I sometimes disagreed with the author's opinion, I then *"let the cryo people go"* with their perception, not mine or the one of some external reviewer. In the revolutionary spirit of Open Access, let the common

Another "democratic" aspect in our books in the times of globalization is that the contributions were made by people from 27 countries from *all* continents (except Antarctica). Editor *greatly* appreciates the *invaluable* contribution of the American, Australian, British, Canadian, and New Zealand scientists to the field, and 10 out of 42 Chapters in our books were contributed by authors from those countries. However, cryobiology has long existed in many other languages and cultures. We found the tone of some, especially "historical" reviews written by prominent cryobiologists that may make an impression that the scientists of *that* linguistic domain have predominantly contributed to cryobiology quite uncomfortable; in other words: *"If it is not published in English - it doesn't exist"* so to speak. In contrast to such biases, the seminal works of Luyett, Smirnov, Jahnel, Boutron, Milovanov, Cassou, Ostashko, Sumida, Kopeika, and many other scientists whose first language of publication and/or mother tongue were not English, but whose *pioneering* contribution to the theory and practice of low temperature stabilization has been recognized over the World, is also highly regarded

Yet another difference is that the chapters of our books are grouped into topics (Sections) that are *"*subject oriented*"* rather than loosely flocked to the *"*Themes*"* so none would wonder why one chapter on freezing of plants is in one section, while another one ends up in another. The sections are the same for both our *books*, the only

reader, not an elitist reviewer, be the judge in the end!

in our books.

Section *"Basic Cryobiology and Kinetic Vitrification"* opens *Book 1*. The first, and the two following chapters are dedicated to kinetic vitrification as the re-emerging method of cryopreservation. Chapter 1 by Katkov and colleagues reiterates the idea that basically all methods of long-term stabilization of cells are in fact different ways (the authors identify 5 of them) of vitrifcation of the intracellular milieu. The chapter gives a detailed thermodynamical description and analysis of the methods. The second part of the chapter is dedicated to the kinetic vitrification of human and animal spermatozoa, the concept of the *"Universal Cryopreservation Protocol*" and what the author called *"Race for the Pace"*, though the last one needs a separate chapter and is only mentioned briefly as one of the future directions. The chapter by *Isachenko and colleagues* tells the story of successful vitrification of human and animal spermatozoa, and its emerging as a valuable tool applied to the assisted reproduction technologies. The third chapter, by a Canadian group (Moskovtsev *et al*) is an *independent* report of the success of vitrifcation of human sperm without permeable (and potentially toxic) cryoprotectants (vitrificants) with certain modifications of the Isachenko's method. The chapter by Gao & Zhou is dedicated to the basic cryobiology of osmotic effects, prevention of the osmotic injury, as well as to the equipment for the optimal addition and elution of osmotically active permeable cryoprotective agents (CPAs).

Section *"Stem Cells and Cryopreservation in Regenerative Medicine"* in *Book 1* is presented by a review by Martín-Ibáñez on cryopreservation of human *pluripotent* stem cells; it is the cutting edge of the contemporary cryobiological science where major discoveries have been made very recently. Cryopreservation of *adult* rat mesenchymal stem cells by vitrification is the theme of the chapter by Bahadori *et al* in *Book 2*. It is the one of the chapters when the Editor disagrees with the evaluation of the convenience of cryopreservation of stem cells by vitrification in small containers such OPS, but as we said before, we judged the experimental science, not the concept, and the former one was self-evidently good. The chapter by Campbell & Brockbank reports very interesting results on cryopreservation of adherent smooth muscle and endothelial cells, a direction that, as we mentioned before, may bring about some interesting practical applications. The other two chapters in *Book 2* are the one by U. Santos and colleagues, dedicated to cryopreservation of musculoskeletal cells and tissues, and the other application of regenerative medicine - cryopreservation of allograft for knee ligament construction is the theme of the Chapter by Bitar *et al*.

Section *"Human Assisted Reproduction Techniques (ART)"* opens with a review by Liebermann on vitrifcation of embryos and oocytes, a fast developing method of ART. Juergen and Michael Tucker have edited an excellent book dedicated to the use of vitrifcation in human ART [Liebermann & Tucker, 2007] that we highly recommend for reading to the specialists in the field. This review in *Book 1* summarizes the latest achievements in the area. Another chapter in *Book 1* (Bigelow & Copperman) is also dedicated to cryopreservation of human oocytes, and altogether, both chapters provide a good glance at the comparative advantages of slow freezing vs*.* vitrifcation in cryopreservation of human eggs. As the background in cryopreservation of human spermatozoa is extensively covered by three chapters of the first section in *Book 1*, the third chapter of this section written by Honaramooz discusses the recent advances in cryopreservation of testicular tissues. The chapter in *Book 2* by Criado covers a very "hot" topic of contamination associated with vitrifcation in the so called "open systems", in which there is a direct contact (or a possibility of such) of the vitrified sample with liquid nitrogen. The chapter also provides a comprehensive review of current containers used for vitrifcation in human ART.

Preface XV

**Igor I. Katkov, Ph.D.**

San Diego, California,

Stem Cell Center

and

USA

Head of Cryobiology and Biostabilization

Chief Scientific Officer of CELLTRONIX

Burnham-Sanford Institute for Medical Research

dedicated to cryopreservation of species of spices plants, while the review by Quain *et al* discusses the current advances in cryopreservation of vegetatively propagated tropical crops. Similar subject (vegetatively propagated crops), but with an emphasis on the thermal analysis of the cryopreservation methods using DSC, is covered by Zámečník *et al* in *Book 2*. In the same book, Martinez-Montero and colleagues cover current frontiers in cryopreservation of sugarcane and pineapple, C. Santos reports the results of cryopreservation of cork oak, and Radha *et al* discuss cryopreservation of a medicinal Indian plant of *Icacinaceae* species. The Chapter by Burritt covers an interesting topic of action of proline as a "natural" multi-functional cryoprotectant that is accumulated in higher plants under stress, and can be considered as an attractive

Section *"Equipment and Assays"* is the last but definitely not the least important section of these two books, as the entire progress of cryobiology and cryopreservation depends on the development of devices and containers for cryopreservation, and proper and adequate assays of cryopreserved cells after resuscitation. The first chapter in *Book 1,* by Butler & Pegg, covers the precision in and control of cryopreservation, the pivotal components in the modern cryopreservation technologies. While this chapter covers mostly slow (equilibrium) programmed freezing, it is supplemented by a review by Cipri *et al*, which discusses some novel equipment and carriers, particularly for vitrification. To some extent it complements the last sub-section of our chapter 1, but we have to emphasize that none of the devices described in the Cipri's chapter can achieve the very rapid rates of cooling as many of the inventors claim. For example, the notion that *VitMaster* can achieve as high as 135,000 OC/min is largely overestimated even for very small samples, as slush freezing does not completely eliminate the Leidenfrost effect. In regard to assays, Partyka and colleagues review the methods of assessment of viability of cryopreserved sperm, many of which can be adapted to other types of cells as well. Finally, Pérez Campos *et al* present some interesting ideas on using X-ray diffraction for the assessment of quality of

In conclusion, *Books 1* and *2* cover a vast variety of topics regarding the current development of both fundamental cryobiology and practical aspects of cryopreservation, and we hope they will help the researches to grasp the background, state of the art, and future of this captivating and very important field of Life Sciences.

CPA candidate for cryopreservation in general.

cryopreserved tissues in tissue banks in *Book 2***.** 

The section *"Farm / Pet/ Laboratory Animal ART"* is generically related to the previous section, but with an emphasis on animal reproductive cells and tissues. The first chapter in *Book 1* by Rodriguez-Martinez covers the cryopreservation of porcine (pig) gametes, embryos and genital tissues. It is followed by a chapter on cryopreservation of embryos of model animals, written by Tsang & Chow. *Book 2* contains a series of reports and mini-reviews on cryopreservation of boar (Kaeoket) and rat (Yamashiro & Sato) spermatozoa, cryopreservation of genetic diversity (sperm, oocytes, embryos, somatic cells) of rabbit species by Jolly *et al*, cryopreservation of ovarian tissues of large domestic animals (cow, pig and sheep) and non-primates (macaque) by Milenkovic and colleagues, and cryopreservation of reproductive cells of domestic animals (Neto *et al*). While there is a certain overlap in the coverage among those chapters, we feel that such diversity enriches the *Book 2* as different points of view are considered.

Section titled *"Cryopreservation of Wildlife Genome"*, particularly of terrestrial vertebrate species, is comprehensively covered by Saragusty and is supplemented by a review on cryopreservation of genome of wild *Felidae* by Paz in *Book 1*.

Section *"Cryopreservation of Aquatic Species"* lacks a general review but several aspects are covered in a variety of chapters. Zilli & Vilella (*Book 1*) discuss the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state of proteins of seawater fish spermatozoa, with a similar topic, but at a different angle and with their own recent experimental results, covered by Li and colleagues in *Book 2*. This book also contains several experimental reports on cryopreservation of sperm of freshwater species, such as European pikeperch and catfish (Bokor *et al*), a variety of Malaysian freshwater species (Chew), brown trout and koi carp (Bozkurt *et al*) and African giant catfish (Omitogun *et al*).

The section titled *"Cryopreservation in Plants"* is the most *populous* and is represented by four chapters in *Book 1* and five chapters in *Book 2*. Two extensive reviews by Kaczmarczyk *et al* and by Kami cover general aspects of plant cryopreservation. Again, while those two chapters overlap in many aspects, they are rather complementary. The third chapter in *Book 1,* written by Babu and colleagues, is dedicated to cryopreservation of species of spices plants, while the review by Quain *et al* discusses the current advances in cryopreservation of vegetatively propagated tropical crops. Similar subject (vegetatively propagated crops), but with an emphasis on the thermal analysis of the cryopreservation methods using DSC, is covered by Zámečník *et al* in *Book 2*. In the same book, Martinez-Montero and colleagues cover current frontiers in cryopreservation of sugarcane and pineapple, C. Santos reports the results of cryopreservation of cork oak, and Radha *et al* discuss cryopreservation of a medicinal Indian plant of *Icacinaceae* species. The Chapter by Burritt covers an interesting topic of action of proline as a "natural" multi-functional cryoprotectant that is accumulated in higher plants under stress, and can be considered as an attractive CPA candidate for cryopreservation in general.

XIV Preface

considered.

achievements in the area. Another chapter in *Book 1* (Bigelow & Copperman) is also dedicated to cryopreservation of human oocytes, and altogether, both chapters provide a good glance at the comparative advantages of slow freezing vs*.* vitrifcation in cryopreservation of human eggs. As the background in cryopreservation of human spermatozoa is extensively covered by three chapters of the first section in *Book 1*, the third chapter of this section written by Honaramooz discusses the recent advances in cryopreservation of testicular tissues. The chapter in *Book 2* by Criado covers a very "hot" topic of contamination associated with vitrifcation in the so called "open systems", in which there is a direct contact (or a possibility of such) of the vitrified sample with liquid nitrogen. The chapter also provides a comprehensive review of

The section *"Farm / Pet/ Laboratory Animal ART"* is generically related to the previous section, but with an emphasis on animal reproductive cells and tissues. The first chapter in *Book 1* by Rodriguez-Martinez covers the cryopreservation of porcine (pig) gametes, embryos and genital tissues. It is followed by a chapter on cryopreservation of embryos of model animals, written by Tsang & Chow. *Book 2* contains a series of reports and mini-reviews on cryopreservation of boar (Kaeoket) and rat (Yamashiro & Sato) spermatozoa, cryopreservation of genetic diversity (sperm, oocytes, embryos, somatic cells) of rabbit species by Jolly *et al*, cryopreservation of ovarian tissues of large domestic animals (cow, pig and sheep) and non-primates (macaque) by Milenkovic and colleagues, and cryopreservation of reproductive cells of domestic animals (Neto *et al*). While there is a certain overlap in the coverage among those chapters, we feel that such diversity enriches the *Book 2* as different points of view are

Section titled *"Cryopreservation of Wildlife Genome"*, particularly of terrestrial vertebrate species, is comprehensively covered by Saragusty and is supplemented by a review on

Section *"Cryopreservation of Aquatic Species"* lacks a general review but several aspects are covered in a variety of chapters. Zilli & Vilella (*Book 1*) discuss the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state of proteins of seawater fish spermatozoa, with a similar topic, but at a different angle and with their own recent experimental results, covered by Li and colleagues in *Book 2*. This book also contains several experimental reports on cryopreservation of sperm of freshwater species, such as European pikeperch and catfish (Bokor *et al*), a variety of Malaysian freshwater species (Chew), brown trout

The section titled *"Cryopreservation in Plants"* is the most *populous* and is represented by four chapters in *Book 1* and five chapters in *Book 2*. Two extensive reviews by Kaczmarczyk *et al* and by Kami cover general aspects of plant cryopreservation. Again, while those two chapters overlap in many aspects, they are rather complementary. The third chapter in *Book 1,* written by Babu and colleagues, is

current containers used for vitrifcation in human ART.

cryopreservation of genome of wild *Felidae* by Paz in *Book 1*.

and koi carp (Bozkurt *et al*) and African giant catfish (Omitogun *et al*).

Section *"Equipment and Assays"* is the last but definitely not the least important section of these two books, as the entire progress of cryobiology and cryopreservation depends on the development of devices and containers for cryopreservation, and proper and adequate assays of cryopreserved cells after resuscitation. The first chapter in *Book 1,* by Butler & Pegg, covers the precision in and control of cryopreservation, the pivotal components in the modern cryopreservation technologies. While this chapter covers mostly slow (equilibrium) programmed freezing, it is supplemented by a review by Cipri *et al*, which discusses some novel equipment and carriers, particularly for vitrification. To some extent it complements the last sub-section of our chapter 1, but we have to emphasize that none of the devices described in the Cipri's chapter can achieve the very rapid rates of cooling as many of the inventors claim. For example, the notion that *VitMaster* can achieve as high as 135,000 OC/min is largely overestimated even for very small samples, as slush freezing does not completely eliminate the Leidenfrost effect. In regard to assays, Partyka and colleagues review the methods of assessment of viability of cryopreserved sperm, many of which can be adapted to other types of cells as well. Finally, Pérez Campos *et al* present some interesting ideas on using X-ray diffraction for the assessment of quality of cryopreserved tissues in tissue banks in *Book 2***.** 

In conclusion, *Books 1* and *2* cover a vast variety of topics regarding the current development of both fundamental cryobiology and practical aspects of cryopreservation, and we hope they will help the researches to grasp the background, state of the art, and future of this captivating and very important field of Life Sciences.

> **Igor I. Katkov, Ph.D.** Head of Cryobiology and Biostabilization Stem Cell Center Burnham-Sanford Institute for Medical Research and Chief Scientific Officer of CELLTRONIX San Diego, California, USA

#### **References**

