**3.2.2 On reasoning of creating** *"xerobanks"* **of dried genetic material**

Secondly, as the nucleus of somatic cells can be kept intact after desiccation, it (theoretically) can be used for cloning by somatic-cell intracellular nuclear transfer (SCNT). So, those two aspects, ICSI and SCNT raise the question whether the xerobanks of both gametes and somatic cells should be created for human, model (laboratory), agricultural and wildlife species. We personally believe (though it might change with the time) that except for xerobanks of sperm of laboratory animals, such as transgenic mice and rats, for which both ICSI and SCNT have been well established [Katkov, 2008], the other types of xerobanks are not a necessity, and people should focus their resources and money (which are often scarce in this field) on the methods that have been proven to produce viable cells ( i.e. on cryopreservation). In situations where the cold chain is not as easily available (for example, for the preservation of a genome of species that are on the verge of extinction), drying could be considered as the last resort, but for now, it should not be considered as an alternative to cryobanks. That might change where ICSI and SCNT become routine for many species, but so far we should concentrate on CP. And again, it is gametes, embryos and other reproductive cells that should be preserved first to save genetic material of endangered species even after their death [Maksudov *et al.*, 2009] while, for example, the CP of stem and other somatic cells should be kept as the last resort when the reproductive cells are unavailable. Note, that some other authors of this Book are much more optimistic on that matter of both drying (e.g., the Chapter by Joseph Saragusty ([Saragusty, 2012] sub-chapters 2.3 and 4.2), and cryobanks of stem cells for restoration of species ( [Saragusty, 2012], subchapter 4.3).

#### **3.3 Slow freezing: Still the mainstream of cryopreservation but…**

As we mentioned above, the discovery of "enigmatic glycerol" [Polge *et al.*, 1949; Smirnov, 1949] led to the explosion of methods of cryopreservation and types of species cryopreserved and development of the first cryobanks that marked the 1950's. It revolutionized first the cattle industry, than blood transfusion and many others followed. However, while many of them being successful, the method *per se* remained semi-empirical. However, it has changed with introduction of the 2-factor hypothesis and the equations for the equilibrium slow freezing (minimal intracellular ice formation) by Peter Mazur [Mazur, 1963; Mazur *et al.*, 1972]. Using this truly fundamental approach, Mazur and colleagues in USA and Ian Wilmut in UK were be able to cryopreserve the mouse embryo [Whittingham *et al.*, 1972; Wilmut, 1972]. Since then, slow freezing has been the mainstream of modern cryobiology, and while VF is an

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 13

Fig. 1. Five ways of vitrification: A corrected and supplemented phase diagram adapted

2. Points **E-F** (**red**): *equilibrium vitrification* (often referred as VF per se). The very viscous solution of the permeable vitrificant (solute) prevents the formation and/or growth of both intracellular and *extracellular* ice the sample can vitrify without the ice phase practically at *any rate* of cooling and warming (the **E-**F is locate at higher concentration than the line of freezing (heterogeneous ice nucleation) shows in a sketchy form in magneto color crosses the *Tg* line at lower concentrations and only two phases, amorphous and liquid, exist on the

3. Points **G-H** (**purple**): *kinetic vitrification* that occurs *intracellularly* at a much lower concentration of the vitrificant or even without permeable VFA. This however, requires much higher rates of cooling and warming so the damaging ice crystals cannot be formed during rapid cooling and re-crystallization (de-vitrification) will be blocked and, and thus,

4. Points **A-D-C** (**orange**): *freeze-drying (lyophilization)* (not **A**-**D**, as originally is stated in [Devireddy & Thirumala, 2011]). **A-D** (orange) represents freezing and sublimation of ice (primary drying) followed by elevation of temperature of drying above OC (secondary

5. Points **A-C** (**orange**): *desiccation (xeropreservation)* is either vacuum or air/humidity chamber drying where the temperature of drying is always above OC so no freezing phase is present. Note that the temperature drying *Tdr* is *always* above the glass transition temperature of the sample *Tg* (blue curve) on definition (otherwise, evaporation will not occur due to extremely high viscosity), while for stable storage, the temperature of storage *Tst* must be *below* Tg, so the conditions of stable drying are following *Tst < Tg < Tdr* (final). Many papers on drying of biological reports *Tg* above *Tdr*, which as incorrect, see [Katkov and Levine, *Tg*] for details and possible explanation of such "paradox". It can explain instability of samples at long storage

See also other set of phase diagrams in the Fig. 4 and explanation in the text.

1. Points **A-B'** (**green**): *slow equilibrium freezing*, often called *cryopreservation per se*. Note, that the solute concentration is dynamically changing during freezing of extracellular ice so the

**Light Blue** line represents the glass temperature *Tg* curve of the sample.

original authors' line **A-B** (**orange**) is substituted by **A-B'** (**green**).

will not damage the cells during very fast warming.

[Suzuki, 2006] that are often claimed to have *Tg* +60-70 OC.

from [Devireddy & Thirumala, 2011].

right side of the x-axis.

drying) **D-C**.

emerging method that will one day replace SF for many types of cells it has not been done yet: right now SF is an imperative for the majority of cell types.

With the development of Peter Mazur's equations and the 2-factor hypothesis of cryodamage and work of other cryobiologists on slow (equilibrium) freezing in 1960s, it became clear that a particular cell would need its own optimal cryopreservation protocol, which would largely depend on the cell cryobiological and physiological parameters as well as on the type of cryoprotective agents (CPAs) used. Particularly, equilibrium freezing of embryos would require very slow pace of cooling (0.3-1 OC/min) so the whole cryopreservation process would take several hours. In contrast, for small oblate (flat) ellipsoids such as the red blood cells (RBCs) with an excellent surface-to area ratio, which would allow them to lose water very quickly, the optimal freezing rate of cooling would be in the range of several thousand OC/min. Thus, if we consider an intermediate cooling rate, say 10 OC/min, it would kill oocytes at a very fast rate due to the intracellular ice formation (IIF). But the same cooling rate is too slow for RBC's so they will be dead, due to excessive shrinkage and prolonged degradation ("solute effects"). Yet, for lymphocytes, which are intermediate between oocytes and RBC's that rate would be optimal.

Addition of a CPA shifts the survival curve toward the lower rate and higher survival, which indicates that the CPA protects mostly during suboptimal cooling (see Fig. 2) acting as an osmotic buffer that prevents excessive shrinkage and other "solute effects"[Lovelock, 1953; Mazur, 1970, 1984; Mazur & Koshimoto, 2002]. The effect of protective action of the CPA is much more pronounced for larger bone marrow cells while small erythrocytes perfectly survive the absence of CPA if cooled fast enough. The optimal concentration, however, is in the same magnitude of 1- 2 M. Note that the mechanism of cryoprotective action of CPAs such as glycerol or DMSO at slow sub-optimal cooling rates is absolutely different and works at much lower concentrations than their role as vitrificants ("thickeners") that elevate the viscosity during vitrification (VF). From that standpoint, they should NOT be called "CPAs" but rather "VFAs" in case of VF.

Thus, the optimal ("*maximum maximorum*") concentration of the CPA (more precisely, the combination of concentration of CPA *and* rate of cooling) are unique for a particular type of cells. These two concepts are illustrated in Figures 2 and 3. The bottom line is that SF often needs elaborate multi-step protocols, whichrequires special equipment, and it can do exceptionally well, especially if combined with other "tricks" that are specific to the particular species of cells. A good, recent example is the CP of human pluripotent (embryonic and induced alike) stem cells (hESc's and iPSC's respectively). Introduction of i) multi-step freezing, ii) ROCK inhibitors in combination with full cell dissociation, and iii) freezing pluripotent SC's in adherent stage as they are prone to *anoikis* (cell death after cell are detached from extracellular matrix, [Wagh *et al.*, 2011]) have dramatically increased survival and functionality of human pluripotent cells after SF ([Katkov *et al.*, 2011; Li *et al.*, 2008; Martin-Ibanez *et al.*, 2009; Mollamohammadi *et al.*, 2009; Stubban *et al.*, 2007; Ware & Baran, 2007], see Chapter by Martin-Ibanez [Martin-Ibanez, 2012] in this Book). It now highly supersedes various vitrification techniques proposed from time to time [Beier *et al.*, 2011; Reubinoff *et al.*, 2001; Zhou *et al.*, 2004] despite what is claimed otherwise by the authors.

However, the strengths of SF freezing can be its weaknesses as well: it indeed needs *elaborative* protocols that have to be developed for each new species of cells separately. Secondly, it is difficult to implement for CP of large chunks if tissues, and especially if we are talking about CP of a whole organ. Yet, the methods and equipment are being developed, see a chapter by Butler in our Book [Butler & Pegg, 2012].

emerging method that will one day replace SF for many types of cells it has not been done yet:

With the development of Peter Mazur's equations and the 2-factor hypothesis of cryodamage and work of other cryobiologists on slow (equilibrium) freezing in 1960s, it became clear that a particular cell would need its own optimal cryopreservation protocol, which would largely depend on the cell cryobiological and physiological parameters as well as on the type of cryoprotective agents (CPAs) used. Particularly, equilibrium freezing of embryos would require very slow pace of cooling (0.3-1 OC/min) so the whole cryopreservation process would take several hours. In contrast, for small oblate (flat) ellipsoids such as the red blood cells (RBCs) with an excellent surface-to area ratio, which would allow them to lose water very quickly, the optimal freezing rate of cooling would be in the range of several thousand OC/min. Thus, if we consider an intermediate cooling rate, say 10 OC/min, it would kill oocytes at a very fast rate due to the intracellular ice formation (IIF). But the same cooling rate is too slow for RBC's so they will be dead, due to excessive shrinkage and prolonged degradation ("solute effects"). Yet, for lymphocytes, which are

Addition of a CPA shifts the survival curve toward the lower rate and higher survival, which indicates that the CPA protects mostly during suboptimal cooling (see Fig. 2) acting as an osmotic buffer that prevents excessive shrinkage and other "solute effects"[Lovelock, 1953; Mazur, 1970, 1984; Mazur & Koshimoto, 2002]. The effect of protective action of the CPA is much more pronounced for larger bone marrow cells while small erythrocytes perfectly survive the absence of CPA if cooled fast enough. The optimal concentration, however, is in the same magnitude of 1- 2 M. Note that the mechanism of cryoprotective action of CPAs such as glycerol or DMSO at slow sub-optimal cooling rates is absolutely different and works at much lower concentrations than their role as vitrificants ("thickeners") that elevate the viscosity during vitrification (VF). From that standpoint, they

Thus, the optimal ("*maximum maximorum*") concentration of the CPA (more precisely, the combination of concentration of CPA *and* rate of cooling) are unique for a particular type of cells. These two concepts are illustrated in Figures 2 and 3. The bottom line is that SF often needs elaborate multi-step protocols, whichrequires special equipment, and it can do exceptionally well, especially if combined with other "tricks" that are specific to the particular species of cells. A good, recent example is the CP of human pluripotent (embryonic and induced alike) stem cells (hESc's and iPSC's respectively). Introduction of i) multi-step freezing, ii) ROCK inhibitors in combination with full cell dissociation, and iii) freezing pluripotent SC's in adherent stage as they are prone to *anoikis* (cell death after cell are detached from extracellular matrix, [Wagh *et al.*, 2011]) have dramatically increased survival and functionality of human pluripotent cells after SF ([Katkov *et al.*, 2011; Li *et al.*, 2008; Martin-Ibanez *et al.*, 2009; Mollamohammadi *et al.*, 2009; Stubban *et al.*, 2007; Ware & Baran, 2007], see Chapter by Martin-Ibanez [Martin-Ibanez, 2012] in this Book). It now highly supersedes various vitrification techniques proposed from time to time [Beier *et al.*, 2011; Reubinoff *et al.*,

However, the strengths of SF freezing can be its weaknesses as well: it indeed needs *elaborative* protocols that have to be developed for each new species of cells separately. Secondly, it is difficult to implement for CP of large chunks if tissues, and especially if we are talking about CP of a whole organ. Yet, the methods and equipment are being

right now SF is an imperative for the majority of cell types.

intermediate between oocytes and RBC's that rate would be optimal.

should NOT be called "CPAs" but rather "VFAs" in case of VF.

2001; Zhou *et al.*, 2004] despite what is claimed otherwise by the authors.

developed, see a chapter by Butler in our Book [Butler & Pegg, 2012].

Fig. 1. Five ways of vitrification: A corrected and supplemented phase diagram adapted from [Devireddy & Thirumala, 2011].

**Light Blue** line represents the glass temperature *Tg* curve of the sample.

1. Points **A-B'** (**green**): *slow equilibrium freezing*, often called *cryopreservation per se*. Note, that the solute concentration is dynamically changing during freezing of extracellular ice so the original authors' line **A-B** (**orange**) is substituted by **A-B'** (**green**).

2. Points **E-F** (**red**): *equilibrium vitrification* (often referred as VF per se). The very viscous solution of the permeable vitrificant (solute) prevents the formation and/or growth of both intracellular and *extracellular* ice the sample can vitrify without the ice phase practically at *any rate* of cooling and warming (the **E-**F is locate at higher concentration than the line of freezing (heterogeneous ice nucleation) shows in a sketchy form in magneto color crosses the *Tg* line at lower concentrations and only two phases, amorphous and liquid, exist on the right side of the x-axis.

3. Points **G-H** (**purple**): *kinetic vitrification* that occurs *intracellularly* at a much lower concentration of the vitrificant or even without permeable VFA. This however, requires much higher rates of cooling and warming so the damaging ice crystals cannot be formed during rapid cooling and re-crystallization (de-vitrification) will be blocked and, and thus, will not damage the cells during very fast warming.

See also other set of phase diagrams in the Fig. 4 and explanation in the text. 4. Points **A-D-C** (**orange**): *freeze-drying (lyophilization)* (not **A**-**D**, as originally is stated in [Devireddy & Thirumala, 2011]). **A-D** (orange) represents freezing and sublimation of ice (primary drying) followed by elevation of temperature of drying above OC (secondary drying) **D-C**.

5. Points **A-C** (**orange**): *desiccation (xeropreservation)* is either vacuum or air/humidity chamber drying where the temperature of drying is always above OC so no freezing phase is present. Note that the temperature drying *Tdr* is *always* above the glass transition temperature of the sample *Tg* (blue curve) on definition (otherwise, evaporation will not occur due to extremely high viscosity), while for stable storage, the temperature of storage *Tst* must be *below* Tg, so the conditions of stable drying are following *Tst < Tg < Tdr* (final). Many papers on drying of biological reports *Tg* above *Tdr*, which as incorrect, see [Katkov and Levine, *Tg*] for details and possible explanation of such "paradox". It can explain instability of samples at long storage [Suzuki, 2006] that are often claimed to have *Tg* +60-70 OC.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 15

Fig. 3. The role of a cryoprotective agent (CPA) at slow freezing: Survival of cells of different size (marrow cells, the left panel >> erythrocytes on the right) as function of CPA

concentration and cooling rate. Adapted from [Mazur, 1970].

Fig. 2. The two-factor hypothesis of the cryoinjury by Peter Mazur: survival of cells of different size (oocytes >> lymphocytes > hamster cells >> erythrocytes) as function of the cooling rate. **Top**: Mazur's original graph, adapted from [Mazur *et al.,* 1972; Mazur *et al.,* 2008]. Bottom: Updated for stem cells (large size), yeast moderate) and sperm (slow) in [Cipri *et al.,* 2010]. Note that "slow (sub-optimal) and "fast" (supra-optimal) freezing in this case largely depends on the cells size: e.g., the rate of cooling 10 OC/min is very fast for oocytes (lethal IIF), very slow for erythrocytes ('damage due to the "solute effects") and close to the optimal for lymphocytes.

Fig. 2. The two-factor hypothesis of the cryoinjury by Peter Mazur: survival of cells of different size (oocytes >> lymphocytes > hamster cells >> erythrocytes) as function of the cooling rate.

Bottom: Updated for stem cells (large size), yeast moderate) and sperm (slow) in [Cipri *et al.,* 2010]. Note that "slow (sub-optimal) and "fast" (supra-optimal) freezing in this case largely depends on the cells size: e.g., the rate of cooling 10 OC/min is very fast for oocytes (lethal IIF), very slow for erythrocytes ('damage due to the "solute effects") and close to the optimal for

**Top**: Mazur's original graph, adapted from [Mazur *et al.,* 1972; Mazur *et al.,* 2008].

lymphocytes.

Fig. 3. The role of a cryoprotective agent (CPA) at slow freezing: Survival of cells of different size (marrow cells, the left panel >> erythrocytes on the right) as function of CPA concentration and cooling rate. Adapted from [Mazur, 1970].

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 17

it has as much in common with cryobiology as astrology with astronomy or alchemistry with chemistry. Not surprisingly, cryonicists are banned from publication in all scientific cryobiological journals and from the membership in the cryo-societies as their activities have nothing to do with real scientific premises. Yet, they skillfully wrap their messages, post some valid statements, and add some useful websites, for example on physics of glass transition (apparently, they have good physicists among their "disciples") to make cryonics seem like a *legitimate* science And of course, they cite Fahy's and Brian Wowk's work wherever they can. They actually admit that they buy those ice-blockers from the *"21 CM"*. They are very active in Wikipedia so we can see all biographies of prominent cryonicists, and even much less prominent and rather obscure ones like a former bookkeeper Danila Medvedev in Russia [Wikipedia, 2011c], which the company *"KrioRus"* proudly announces how many bodies and other parts of humans (including some brains, which they call *"Neurovitrification*"*!*), dogs, cats and birds they "vitrified" [Wikipedia, 2011b]. Of course, you can find in Wiki also the biographies of Greg Fahy, Brian Wok, and a detailed description of the *"21 CM"* company. None of these scientists has ever claimed any of the cryonics beliefs openly (they value their scientific carriers as well as an ability to apply for NIH money, for example, which considers cryonics as a pseudo-science), and we don't imply that those cryobiologists and the *current* "21 CM" management are "hidden cryonicists". Moreover, the Company's website clearly distances itself from cryonics (http://www.21cm.com/cryobiology.html). However, the fact that cryonics companies and organizations heavily rely on E-VF and ice-blockers as the major method of preservation and future *resurrection*, their connection, both *"ideological*, (e.g., hiring a *very* controversial Ukrainian scientist and former 21 CM employee Yuri Pichugin) and financial (being presumably valued customers of the *"21 CM"* by buying those ice-blockers) is self-evident (http://www.cryonics.org/century.html, http://en.wikipedia.org/wiki/Cryonics,

**3.5 Modern methods of vitrification of reproductive, stem and other germplasm cells** 

The fate of the second direction of vitrification, which was initiated with the paper by Rall and Fahy [Rall & Fahy, 1985] on E-VF of embryos, was completely different: it definitely has not been stuck in the rut but rather quite opposite. The use of vitrification for cryopreservation of reproductive cells and tissues has boomed over the last 20+ years since that seminal paper was published. However, the modern methods of VF of oocytes, embryos, sperm, ovarian and testicular tissue are in fact the varieties of *kinetic* vitrification. Elaborative multi-step protocols of the addition of VFA's before VF and elution of them after warming have been developed to decrease the toxic and osmotic effects of vitrification. Some of those methods are covered by other Chapters or our Books 1 and 2 and by the above-mentioned excellent book by Tucker and Lieberman [Tucker & Liebermann, 2007]. Up to now, a vast variety of carriers has been developed as well. They are summarized in an excellent review by Saragusty and Arav [Saragusty & Arav, 2011] and is reproduced on Fig. 4. While there is still a debate over what is better for a particular cell type or species, slow freezing or kinetic vitrification, the latter one is gaining ground, particularly for VF of oocytes, thanks to ART scientists and practitioners such as Kuwayama, Vajta, Sheldon, Liebermann, Tucker and many others. Note however, that sometime that "cold war" may

**are in the realm of** *kinetic* **vitrification, but still many questions remain** 

http://cryonics.org /yuri.html).

#### **3.4 Equilibrium vitrification and** *"magic"* **ice blockers: True 21st century medicine or**  *"Fahy's tyranny"* **and the spearhead of cryonics pseudo-science?**

On the other hand, Greg Fahy and colleagues [Fahy *et al.*, 1984] reported vitrification of the whole organ (i.e. kidney), and later report E-VF of mouse embryo by Bill Rall and Greg Fahy [Rall & Fahy, 1985]. Since, the fate of VF of these two types of cells and fields split dramatically: E-VF of the whole organ has been essentially *stuck in the rut*, with *very few progress*, that has been reported mostly by the Fahy's group *per se* [Fahy *et al.*, 2009] despite of 25 years of research and substantial amount of financial support that the author received from many sources including taxpayers money. For example, accordingly the Fahy's company with a promising name *"21st Century Medicine"(21 CM)*, posted on their Wikipedia site "*In 2004 21CM received a \$900,000 grant from the U.S. National Institutes of Health (NIH) to develop solutions and processes to improve human heart transplantations"* [Wikipedia, 2011a]. Since 1+ million dollars (including previous Phase I) in funding and eight years after that announcement, we have not found any progress report or reliable publication on that topic from the company's scientists in scientific peer-reviewed journal. The vitrification of a heart (even an animal one) is not even close to realization apparently.

The company and its scientific team heavily rely on so called *"ice-blockers"*, chemical substances that block the propagation of ice in big samples cooled very slowly, thus helping vitrification. The company has made progress in the development a pipeline of such reagents. However, they are used mostly as "helpers" to lower the osmotic and chemical toxicity of the enormous concentrations of "common" vitrificants that are necessary for equilibrium (slow vitrification). Whether that approach will ever meet real progress in the remaining 88 years of the *21st century medicine*, needs to be seen.

Nonetheless, Dr. Fahy has been very proactive in promoting *equilibrium* vitrification and denying *kinetic* one whenever and wherever it is possible. He basically ignores and calls it *"quasi vitrification"*(e.g., in [Fahy & Rall, 2007]), and in doing so he contradicts himself within three pages of his own review [Fahy & Rall, 2007]! In Fig. 1A.1, he placed the start of citations on vitrification of cells and organs. Of course, he starts counting from his publication 1984 totally ignoring the *earlier* work of Luyet, Boutron, Farrant and other scientists, the very work that Fahy is discussing is a couple of sub-chapters later. Yet, it was he, *"the world's foremost expert in cryopreservation by vitrification"* (http://en.wikipedia.org/ wiki/Twenty-First\_Century\_Medicine), who "truly" vitrified cells *first*. We will come back to this attitude a bit later when we compare E-VF and K-VF. Now, we only say that while his chapter in that book is #1A*,* the majority of the next 19 Chapters in fact describe various *kinetic* vitrification techniques with small size and fast cooling and warming, a typical pattern of K-VF. Few people even mentioned the term ice-blockers, fewer used it in reproduction practice, mainly as "helpers" (see our explanation above).

Who has been truly benefitting from Fahy's and his colleagues work? The people that have been engaged in a pseudo-scientific activity called *'cryonics'* (http://en.wikipedia.org/ wiki/Cryonics) They freeze deceased people, or sometimes even just their heads or brains (as did Saul Kent (http://en.wikipedia.org/wiki/Saul\_Kent), the founder of the "21st CM" (http://www.biomarkerinc.com/saul\_kent\_page.html) in the hope that one day the dead will be *"resurrected"* (?!), or even that the brain can be somehow *'translocated'* into a new body. This is at least science fiction and naive beliefs (a type of *"transhumanism"*) and at most a charlatanic snake oil scheme aimed "*to skim off big bucks"* from the human tragedy so

On the other hand, Greg Fahy and colleagues [Fahy *et al.*, 1984] reported vitrification of the whole organ (i.e. kidney), and later report E-VF of mouse embryo by Bill Rall and Greg Fahy [Rall & Fahy, 1985]. Since, the fate of VF of these two types of cells and fields split dramatically: E-VF of the whole organ has been essentially *stuck in the rut*, with *very few progress*, that has been reported mostly by the Fahy's group *per se* [Fahy *et al.*, 2009] despite of 25 years of research and substantial amount of financial support that the author received from many sources including taxpayers money. For example, accordingly the Fahy's company with a promising name *"21st Century Medicine"(21 CM)*, posted on their Wikipedia site "*In 2004 21CM received a \$900,000 grant from the U.S. National Institutes of Health (NIH) to develop solutions and processes to improve human heart transplantations"* [Wikipedia, 2011a]. Since 1+ million dollars (including previous Phase I) in funding and eight years after that announcement, we have not found any progress report or reliable publication on that topic from the company's scientists in scientific peer-reviewed journal. The vitrification of a heart

The company and its scientific team heavily rely on so called *"ice-blockers"*, chemical substances that block the propagation of ice in big samples cooled very slowly, thus helping vitrification. The company has made progress in the development a pipeline of such reagents. However, they are used mostly as "helpers" to lower the osmotic and chemical toxicity of the enormous concentrations of "common" vitrificants that are necessary for equilibrium (slow vitrification). Whether that approach will ever meet real progress in the

Nonetheless, Dr. Fahy has been very proactive in promoting *equilibrium* vitrification and denying *kinetic* one whenever and wherever it is possible. He basically ignores and calls it *"quasi vitrification"*(e.g., in [Fahy & Rall, 2007]), and in doing so he contradicts himself within three pages of his own review [Fahy & Rall, 2007]! In Fig. 1A.1, he placed the start of citations on vitrification of cells and organs. Of course, he starts counting from his publication 1984 totally ignoring the *earlier* work of Luyet, Boutron, Farrant and other scientists, the very work that Fahy is discussing is a couple of sub-chapters later. Yet, it was he, *"the world's foremost expert in cryopreservation by vitrification"* (http://en.wikipedia.org/ wiki/Twenty-First\_Century\_Medicine), who "truly" vitrified cells *first*. We will come back to this attitude a bit later when we compare E-VF and K-VF. Now, we only say that while his chapter in that book is #1A*,* the majority of the next 19 Chapters in fact describe various *kinetic* vitrification techniques with small size and fast cooling and warming, a typical pattern of K-VF. Few people even mentioned the term ice-blockers, fewer used it in

Who has been truly benefitting from Fahy's and his colleagues work? The people that have been engaged in a pseudo-scientific activity called *'cryonics'* (http://en.wikipedia.org/ wiki/Cryonics) They freeze deceased people, or sometimes even just their heads or brains (as did Saul Kent (http://en.wikipedia.org/wiki/Saul\_Kent), the founder of the "21st CM" (http://www.biomarkerinc.com/saul\_kent\_page.html) in the hope that one day the dead will be *"resurrected"* (?!), or even that the brain can be somehow *'translocated'* into a new body. This is at least science fiction and naive beliefs (a type of *"transhumanism"*) and at most a charlatanic snake oil scheme aimed "*to skim off big bucks"* from the human tragedy so

**3.4 Equilibrium vitrification and** *"magic"* **ice blockers: True 21st century medicine or** 

*"Fahy's tyranny"* **and the spearhead of cryonics pseudo-science?** 

(even an animal one) is not even close to realization apparently.

remaining 88 years of the *21st century medicine*, needs to be seen.

reproduction practice, mainly as "helpers" (see our explanation above).

it has as much in common with cryobiology as astrology with astronomy or alchemistry with chemistry. Not surprisingly, cryonicists are banned from publication in all scientific cryobiological journals and from the membership in the cryo-societies as their activities have nothing to do with real scientific premises. Yet, they skillfully wrap their messages, post some valid statements, and add some useful websites, for example on physics of glass transition (apparently, they have good physicists among their "disciples") to make cryonics seem like a *legitimate* science And of course, they cite Fahy's and Brian Wowk's work wherever they can. They actually admit that they buy those ice-blockers from the *"21 CM"*. They are very active in Wikipedia so we can see all biographies of prominent cryonicists, and even much less prominent and rather obscure ones like a former bookkeeper Danila Medvedev in Russia [Wikipedia, 2011c], which the company *"KrioRus"* proudly announces how many bodies and other parts of humans (including some brains, which they call *"Neurovitrification*"*!*), dogs, cats and birds they "vitrified" [Wikipedia, 2011b]. Of course, you can find in Wiki also the biographies of Greg Fahy, Brian Wok, and a detailed description of the *"21 CM"* company. None of these scientists has ever claimed any of the cryonics beliefs openly (they value their scientific carriers as well as an ability to apply for NIH money, for example, which considers cryonics as a pseudo-science), and we don't imply that those cryobiologists and the *current* "21 CM" management are "hidden cryonicists". Moreover, the Company's website clearly distances itself from cryonics (http://www.21cm.com/cryobiology.html). However, the fact that cryonics companies and organizations heavily rely on E-VF and ice-blockers as the major method of preservation and future *resurrection*, their connection, both *"ideological*, (e.g., hiring a *very* controversial Ukrainian scientist and former 21 CM employee Yuri Pichugin) and financial (being presumably valued customers of the *"21 CM"* by buying those ice-blockers) is self-evident (http://www.cryonics.org/century.html, http://en.wikipedia.org/wiki/Cryonics, http://cryonics.org /yuri.html).

#### **3.5 Modern methods of vitrification of reproductive, stem and other germplasm cells are in the realm of** *kinetic* **vitrification, but still many questions remain**

The fate of the second direction of vitrification, which was initiated with the paper by Rall and Fahy [Rall & Fahy, 1985] on E-VF of embryos, was completely different: it definitely has not been stuck in the rut but rather quite opposite. The use of vitrification for cryopreservation of reproductive cells and tissues has boomed over the last 20+ years since that seminal paper was published. However, the modern methods of VF of oocytes, embryos, sperm, ovarian and testicular tissue are in fact the varieties of *kinetic* vitrification. Elaborative multi-step protocols of the addition of VFA's before VF and elution of them after warming have been developed to decrease the toxic and osmotic effects of vitrification. Some of those methods are covered by other Chapters or our Books 1 and 2 and by the above-mentioned excellent book by Tucker and Lieberman [Tucker & Liebermann, 2007]. Up to now, a vast variety of carriers has been developed as well. They are summarized in an excellent review by Saragusty and Arav [Saragusty & Arav, 2011] and is reproduced on Fig. 4.

While there is still a debate over what is better for a particular cell type or species, slow freezing or kinetic vitrification, the latter one is gaining ground, particularly for VF of oocytes, thanks to ART scientists and practitioners such as Kuwayama, Vajta, Sheldon, Liebermann, Tucker and many others. Note however, that sometime that "cold war" may

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 19

spatula; **Fifth raw**: nylon mesh - arrow points at the nylon mesh, plastic blade, Vitri-Inga. **B**: tubing carriers. **First raw** (top to the bottom)**:** 0.25-mL mini-straw, 0.25 ml mini-straw, Open-pulled straw (OPS), Superfine OPS (SOPS), Flexipet-denuding pipette (170 μm end hole); **Second raw:** CryoTip (opena and loaded), high-security vitrification device; **Third raw:** pipette tip, **Fourth raw**: sealed pulled straw (left), (Cryopette (top right), Rapid-I

**4. Equilibrium** *vs***. kinetic vitrifcation; Evolution of the "Fahy's" phase** 

largely depends on the currently achievable rates of cooling and warming.

This sub-chapter discusses in detail the phase diagram ("*Fahy-Rall*" vitrification diagram). We will also discuss using this diagram the two basic and reciprocal ways of achieving VF, which can be done: i) by cooling and warming at relatively moderate rates but very high concentrations of exogenous (and often toxic) vitrification agents/enhancers (VFAs), which is defined as *equilibrium* VF and ii) by increasing the rate of cooling with a few or not at all exogenous VFAs present, which we refer as *kinetic* VF. We will also emphasize that the border of "*non-achievable*" and "*achievable*" VF that was once set up by Fahy is arbitrary and

**Fig. 5A** depicts the *original* diagram published by *Fahy* et al in **1984** [Fahy *et al.*, 1984]. The diagram is divided in 4 distinctive zones. **Zone IV** is the *equilibrium VF*, when it occurs at any practical rates of cooling and warming as it lies to the right of the junction of *Tm* ( i.e., no ice forming during cooling) and *Td* (no de-vitrifcation during warming). It is basically the zone where the line **E-F** on **Fig. 1** is drawn but with the notion that *Tg* in the Fahy's diagrams (apparently, for glycerol) lies substantially lower than in Devireddy's diagram (*Tg* of a fully dehydrated sample is well above 0 OC while *Tg* of glycerol is in range of -90OC and below [Pouplin *et al.*, 1999]. For *Tg*'s of some popular vitrificants see Table 1 in [Katkov & Levine, 2004]. **Zone III** is the zone when vitrification occurs. The left border is the junction of *Th* (showed in dotted line as it is hardly to estimate *Th* of very viscous samples) and glass transition curve *Tg* and it occurs at concentrated *Cv*'s, the minimal concentrations where *equilibrium* vitrification during cooling occurs at practically any speed. However, such concentration still may produce de-vitrification during re-warming as the devitrification curve *Td* crosses the melting (equilibrium warming) curve at the critical concentration of

devitrification *Cdv*. Thus, this Zone III is the zone where warming must be done fast.

and *Cdv* so they move to the left into the area of the lower concentrations.

**Zone II**, called by Fahy and colleagues at that time (!) *"doubly unstable"* lies at concentration below *Cv*. At those concentrations, both cooling and warming must be done fast to avoid ice formatting and devitrification respectively. That is what we call kinetic vitrification as it deals with the speed of cooling and re-warming rather than with the equilibrium values. It means that the border between that Zone II, where vitrification is achievable with the **Zone I**, where successfully vitrification is impossible at any "reasonable speed" of cooling and warming largely depends on the rate of that cooling and warming: it is reciprocal to the *Cv*

Thus, **there are 2 basic and reciprocal ways of achieving VF**: i) by raising the concentration, and as result, the viscosity of the intra- and extracellular milieu at relatively moderate and even slow rates of cooling but very high concentrations VFAs,

(right-bottom); **Fifth raw**: JY Straws.

**diagram** 

See [Saragusty & Arav, 2011] for more details and references.

erupt and evolve into a "hot war" when it comes to which set of VF media, the protocol, and the carrier are better. Thus, while being faster and simpler than slow freezing (though much farther from automation and "full proof"), vitrification at this moment has been struggling basically with the same problem as the SF has been plagued with: each type of cells, the carrier, and VF media need own protocol, and very often a VF media that work for open carries are too diluted for closed carriers, while using open carriers raises concern of contamination etc. The bottom line is that *kinetic* vitrification as it is now, offers a vast variety of the methods, that have to be checked and adjusted when a new type of cells of/or new species of animals are in consideration.

As we can see later, our experience with vitrifcation led us to conclude that it might change soon, but before moving further, we have to look in more detail at the distinction and principal differences between equilibrium and kinetic vitrification from the standpoint of thermodynamics. In other words, we have to look at the supplemental phase diagram, or as we call it here, the "Fahy's diagram" as it was first published and explained in detail from the cryobiological audience by Fahy and colleagues [Fahy *et al.*, 1984].

A B

Fig. 4. Vitrification carrier systems [Saragusty & Arav, 2011]. **A:** surface carriers. **First raw:** electron microscope grid, minimum drop size; Cryotop; **Second raw:** Cryoloop, Hemi-straw; **Third raw:** Cryoleaf, fiber plug, **Fourth raw**: direct cover VF, VF

spatula; **Fifth raw**: nylon mesh - arrow points at the nylon mesh, plastic blade, Vitri-Inga. **B**: tubing carriers. **First raw** (top to the bottom)**:** 0.25-mL mini-straw, 0.25 ml mini-straw, Open-pulled straw (OPS), Superfine OPS (SOPS), Flexipet-denuding pipette (170 μm end hole); **Second raw:** CryoTip (opena and loaded), high-security vitrification device; **Third raw:** pipette tip, **Fourth raw**: sealed pulled straw (left), (Cryopette (top right), Rapid-I (right-bottom); **Fifth raw**: JY Straws.

See [Saragusty & Arav, 2011] for more details and references.

18 Current Frontiers in Cryobiology

erupt and evolve into a "hot war" when it comes to which set of VF media, the protocol, and the carrier are better. Thus, while being faster and simpler than slow freezing (though much farther from automation and "full proof"), vitrification at this moment has been struggling basically with the same problem as the SF has been plagued with: each type of cells, the carrier, and VF media need own protocol, and very often a VF media that work for open carries are too diluted for closed carriers, while using open carriers raises concern of contamination etc. The bottom line is that *kinetic* vitrification as it is now, offers a vast variety of the methods, that have to be checked and adjusted when a new type of cells of/or

As we can see later, our experience with vitrifcation led us to conclude that it might change soon, but before moving further, we have to look in more detail at the distinction and principal differences between equilibrium and kinetic vitrification from the standpoint of thermodynamics. In other words, we have to look at the supplemental phase diagram, or as we call it here, the "Fahy's diagram" as it was first published and explained in detail from

the cryobiological audience by Fahy and colleagues [Fahy *et al.*, 1984].

A B

**A:** surface carriers. **First raw:** electron microscope grid, minimum drop size; Cryotop; **Second raw:** Cryoloop, Hemi-straw; **Third raw:** Cryoleaf, fiber plug, **Fourth raw**: direct cover VF, VF

Fig. 4. Vitrification carrier systems [Saragusty & Arav, 2011].

new species of animals are in consideration.
