**2.3 The emerging of** *equilibrium* **vitrification**

6 Current Frontiers in Cryobiology

referred here as kinetic VF, had been considered as the major method of cryopreservation at that time [Graevsky, 1948a, b; Graevsky & Medvedeva, 1948; Hoagland & Pincus, 1942; Jahnel, 1938; Luyet & Hodapp, 1938; Luyet, 1937; Park *et al.*, 2004; Schaffner, 1942]. Note that some authors contributed the first understanding of the importance of vitrification for biopreservation to a an earlier work of Walter Stiles [Stiles, 1930], as it, for example, is done in [Fahy & Rall, 2007]; we think, the Stiles's notion however was vague and had had a marginal impact. It was Luyet's work, which would make cryopreservation a *science*. From the outset, he recognized that ice damage must be avoided and vitrification could be a method for long-term preservation of cell viability [Luyet, 1937]. In 1938 Luyet and Hodapp achieved survival of frog spermatozoa vitrified by plunging into liquid air [Luyet & Hodapp, 1938], and later several Western European groups reported their experiences with attempts in kinetic vitrification of fowl [Schaffner, 1942], human [Hoagland & Pincus, 1942; Jahnel, 1938; Parkes, 1945], and rabbit spermatozoa [Hoagland & Pincus, 1942] with varying success. While not directly related to the K-VF of sperm, a clear notion of vitrification as the only way of viable stabilization of cells has been expressed by Graevsky in USSR. As he worked with bacteria, it was natural to use a bacterial sample collection loop to freeze the cells in thin pellicles [Graevsky, 1948a, b]. A similar approach was used by Hoagland and Pincus in Germany in 1942 [Hoagland & Pincus, 1942], which seems a very natural approach for very fast K-VF. Yet, in the money-driven 21st century, the term *"Cryoloop"* is a registered as a trademark.

Apparently, those early scientists would have infringed the trademark law now!

USSR in the era of WWII followed by the Cold War.

**2.2 The rise of slow freezing** 

These early efforts of K-VF of sperm did not receive the recognition they deserved, hindered by the low repeatability and poor survival, as well as difficulties in communication due to various "iron walls" that existed between scientists of the Western Allies, Germany and

The breakthrough came from an independent discovery of the protective role of a permeable CPA glycerol by two groups in 1948-49 [Polge *et al.*, 1949; Smirnov, 1949].The high permeability of glycerol to the sperm membrane in conjunction to relatively low toxicity seemed to be the crucial factor; both groups unsuccessfully tried either non-permeable sugars such as glucose (Parkes's group) or very permeable but very toxic lower alcohols such as ethanol or methanol (Smirnov). The high membrane permeability of glycerol and, thus, fast penetration inside the cells allowed to preserve the cells using slow (10-40 OC/min) freezing, and very moderate warming rates by direct thawing on air or in a water bath. It then became the mainstream of the cryopreservation methods, and a vast variety of cell species of different biological taxa have been preserved by slow (also called *equilibrium*) freezing. It revolutionized two very important fields: the cattle industry (with preservation of bovine sperm and later bovine embryos) and cryopreservation of blood components. It is worth noting that 12 years before the discovery of Parkes's and Smirnov's groups, Bernstein and Petropavlovski had reported the protective role of glycerol during the freezing of sperm [Bernstein & Petropavlovski, 1937] to -20OC, but that work had gone largely unnoticed.

With the development of Peter Mazur's equations and the 2-factor hypothesis of cryodamage [Mazur, 1963; Mazur *et al.*, 1972] and work of other cryobiologists on slow (equilibrium) freezing in 1960's, 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 (CPA's) used. On the other hand, Greg Fahy and colleagues [Fahy *et al.*, 1984] reported the vitrification of a whole organ--a rabbit kidney--using high pressure and *extremely high concentrations* of permeable vitrificants. We will call that approach, which for all intents and purposes will be clarified later, *equilibrium* vitrification. The needs of more quick and robust methods of cryopreservation of mammalian embryos had been clear, since Mazur and colleagues and Wilmut had obtained the "frozen mice" by SF in 1972 [Whittingham *et al.*, 1972; Wilmut, 1972].Plus, Fahy's initial report led to the collaboration between him and W. Rall (former Mazur's student, who specialized in freezing embryos) and the first successful vitrification of mouse embryos was reported a year after Fahy's first report [Rall & Fahy, 1985]. The first human baby from a vitrified embryo was reported in 1990 ?? [Gordts *et al.*, 1990]. Since then, vitrification has become an equally spread assisted reproduction technique (ART) as programmed slow freezing of embryos and, especially, oocytes for *in vitro* fertility (IVF) (see [Rezazadeh *et al.*, 2009] for examples and background).

For detailed state of the art of vitrification of reproduction cells, see several Chapters of this Book and Book 2, as well an excellent book by Tucker and Lieberman [Tucker & Liebermann, 2007]. Several Chapters in that book will be referred throughout this Chapter as well. Particularly, an interesting history and even possible natural occurrence of E-VF in nature is described in the Chapter 1A of that book by Fahy and Rall ("*Certain Alaskan beetles dehydrate sufficiently to generate concentrations of up to 10 mol/L of endogenous glycerol,26 which is enough to vitrify aqueous solutions under laboratory conditions*") [Fahy & Rall, 2007]. Note, however that this particular Chapter 1A is substantially biased against K-VF in favor of E-VF, which we will address throughout the following sub-chapters, and toward the founder of the method, Father Luyet, including some far from diplomatic language escapades. That part will be addressed at the end of the Chapter.

#### **2.4 Vitrification** *of the majority* **of reproductive cells is moving from** *equilibrium* **to**  *kinetic approach*

While slow freezing showed its limitations for certain cell types (e.g. oocytes), a new era started when Rall and Fahy vitrified mouse embryos [Rall & Fahy, 1985] using essentially the same high concentrations of vitrificants vitrified by Fahy *et al.* used in its original report [Fahy *et al.*, 1984]. However, such high concentrations (40-60 % v/v) of VFA's such as

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 9

eagles) failed completely, which actually would prompt us to an even more interesting hypothesis of *"The Universal Preservation Protocol"* and prediction of the *"Race for the Pace"*.

**3.1 All five basic methods of long-term biostabilization cell requires vitrification of the** 

We have defined *5 major ways* of cell stabilization that all lead to low- or high-temperature VF of intracellular milieu as we outlined in [Katkov *et al.*, 2006], which are shown on a schematic phase diagram (**Fig. 1**) adapted from [Devireddy & Thirumala, 2011] with some

Equilibrium (slow) freezing (points A-B' in green) allows to freeze-out the bulk of both extracellular and intracellular water (which escapes from the cell as the extracellular liquid phase becomes more and more concentrated) to ice. Finally, the cells are vitrified in the inter-ice "channels" that are surrounded by ice but always make a connected network (due to barometric restrictions) and surrounded by ice. Yet, the glass transition temperature in those channels is still low so the cells must be stored in LN2 at -196 OC, in nitrogen vapor, or in industrials freezers at -130OC and for a limited time at higher temperature than the *Tg* of water (around -136OC), for example in more accessible -80OC freezers. This is the mainstream conventional cryopreservation, which in the majority of cases requires the use

Ice-free *equilibrium* vitrification (E-VF) of cell suspensions, tissues, and organs at very low temperatures and moderate to high rates of freezing (points E-F in red). This method requires the use of high concentrations of vitrificants, which elevates the viscosity of the milieu and prevents the ice formation during cooling and de-vitrification (sometimes called re-crystallization, which is not exactly the same) during warming. Some researchers [Fahy & Rall, 2007] refer to this method as *"vitrification proper",* and in its "pure form" (see below) has had very limited success in preserving animal oocytes, embryos, some tissues and *one*

*Intracellular* ice-free *kinetic* vitrification of a bulk solution by very fast (abrupt) plunging into a cooling agent such as liquid nitrogen (points G-H in purple). The extremely high rate of cooling (104–106 OC/min) and practically instant warming prevents ice formation inside the cells (the ice still can be formed outside but it has no time to cause any osmotic damage to the cells as K-VF occurs in fractions of a second). As the result, it does not require the use of potentially toxic high concentrations of "CPAs" (vitrificants) or no permeable exogenous vitrificants at all, it is referred to as *"CPA-free vitrification"* by the Isachenkos in regards to sperm. We deliberately include in this method cooling of sperm at much lower rates because the very high *Tg* of the intracellular milieu does not require such high rates. This is one of

Slow freezing to moderately low (around -40 OC -- -60OC) temperatures, which comprises two steps; i) primary drying - sublimation of the bulk of ice at very high vacuum (points A-D, and ii) secondary drying of the 'cake' at elevated (up to +30-40 OC) temperatures (points D-C). This method is called *lyophilization* and it is widely used in food production, microbiology and in the pharmaceutical industry; but so far it has had very limited

**3. Five basic methods of long-term cell biostabilization:** *pro's* **&** *con's* 

of permeable and impermeable cryoprotective agents (CPAs).

applications in the preservation of *animal* cells and higher plants.

organ, as well as some plant specimens.

the major themes of this chapter.

**intracellular milieu** 

corrections and additions.

glycerol, DMSO, and PG are osmotically damaging and chemically toxic so they are intolerable for many cells such as oocytes and spermatozoa, many of which can withstand at best 10-15% DMSO or glycerol. As a result, researchers moved from *equilibrium* VF to much more rapid *kinetic* vitrification that requires much lower concentrations. It is especially clear for CP of oocyte, which cannot tolerate either slow freezing or equilibrium VF apparently due to their cytoskeletal osmotic fragility. To date, many methods and sample carriers have been designed for K-VF of oocytes and embryos, but they all require small sample volumes and precise timing, which makes them vulnerable to technical errors. We will further explore this aspect in a sub-Chapter below.

#### **2.5 Resurrection and rise of** *kinetic* **vitrification of sperm: the Isachenkos' contribution**

The true "second wind" of the *kinetic* VF was brought in with re-discovering of VF of human spermatozoa *without* any exogenous vitrificants (a.k.a., "*cryoprotectants*" even though they play a completely different role than in slow equilibrium freezing) by the Isachenkos and their colleagues. It came with two seminal appears and two presentations in 2002 and 2003, which, as one of the author remembers, stirred a pot and met a lot of resistance and denial from vitrification experts and other prominent "classical" cryobiologists. In 2002, Vladimir and Evegenia Isachenkos and their colleagues reported that human sperm can be vitrified without endogenous vitrificants ('cryoprotectants" as they called it). It worked with the same success or even better than slow freezing [Nawroth *et al.*, 2002], so the Isachenkos showed that it *did* work. Later, Igor Katkov joined the team and tried to explain *why* it actually worked in [Isachenko *et al.*, 2003] and gave a presentation in CRYO-2003 in Coimbra [Katkov *et al.*, 2003]). It was clearly emphasized that at least three factors might have played a crucial role in the successful K-VF of human sperm without exogenous permeating vitrificants: i) small size of the cells, ii) compartmentalization, and iii) high amount and *concentration* of endogenous natural vitrificants such polymers, sugars and nucleotides. We will explore those aspects later in some detail. This quite novel at the time notion is so *"well known"* now that does not even need mentioning the source (e.g., p.649 [Isachenko *et al.*, 2011]); however, it was not so *"obvious"* back in 2003. Here we want to emphasize that despite of skepticism, denial, or even open hostility towards publications and presentations faced by the Isachenkos (and by Katkov as their strong proponent), the method had matured into a *technology*, which proved to be robust and feasible for ART practitioners as well brought food for thoughts to those who works in the realm of basic cryobiology. Most importantly, the results led to the birth of healthy babies and at least one group has repeated the Isachenko method and has obtained good results completely independently- they report their data in Chapter 3 [Moskovtsev *et al.*, 2012]. The authors dedicate a separate Chapter 2 in this Book for summarizing their achievements [Isachenko *et al.*, 2012]. Below, we not only briefly explore progress of the method but also show that even as the staunchest opponent of the method (more precisely, interpretation of the results) as Dr. Fahy has also evolved in his perception of "legitimacy" of *kinetic* vitrification, which we had never doubted at the beginning.

The Isachenko group has recently expanded K-VF method to other mammalian species (dogs) and to an even more distance vertebrate taxon, the fish (see below and also a separate Chapter [Isachenko *et al.*, 2012]). However, our experiments on K-VF of sperm of rodents was not so successful, and attempts of kinetic VF of sperm of the polar bear and raptor birds (falcons and

glycerol, DMSO, and PG are osmotically damaging and chemically toxic so they are intolerable for many cells such as oocytes and spermatozoa, many of which can withstand at best 10-15% DMSO or glycerol. As a result, researchers moved from *equilibrium* VF to much more rapid *kinetic* vitrification that requires much lower concentrations. It is especially clear for CP of oocyte, which cannot tolerate either slow freezing or equilibrium VF apparently due to their cytoskeletal osmotic fragility. To date, many methods and sample carriers have been designed for K-VF of oocytes and embryos, but they all require small sample volumes and precise timing, which makes them vulnerable to technical errors. We will further

The true "second wind" of the *kinetic* VF was brought in with re-discovering of VF of human spermatozoa *without* any exogenous vitrificants (a.k.a., "*cryoprotectants*" even though they play a completely different role than in slow equilibrium freezing) by the Isachenkos and their colleagues. It came with two seminal appears and two presentations in 2002 and 2003, which, as one of the author remembers, stirred a pot and met a lot of resistance and denial from vitrification experts and other prominent "classical" cryobiologists. In 2002, Vladimir and Evegenia Isachenkos and their colleagues reported that human sperm can be vitrified without endogenous vitrificants ('cryoprotectants" as they called it). It worked with the same success or even better than slow freezing [Nawroth *et al.*, 2002], so the Isachenkos showed that it *did* work. Later, Igor Katkov joined the team and tried to explain *why* it actually worked in [Isachenko *et al.*, 2003] and gave a presentation in CRYO-2003 in Coimbra [Katkov *et al.*, 2003]). It was clearly emphasized that at least three factors might have played a crucial role in the successful K-VF of human sperm without exogenous permeating vitrificants: i) small size of the cells, ii) compartmentalization, and iii) high amount and *concentration* of endogenous natural vitrificants such polymers, sugars and nucleotides. We will explore those aspects later in some detail. This quite novel at the time notion is so *"well known"* now that does not even need mentioning the source (e.g., p.649 [Isachenko *et al.*, 2011]); however, it was not so *"obvious"* back in 2003. Here we want to emphasize that despite of skepticism, denial, or even open hostility towards publications and presentations faced by the Isachenkos (and by Katkov as their strong proponent), the method had matured into a *technology*, which proved to be robust and feasible for ART practitioners as well brought food for thoughts to those who works in the realm of basic cryobiology. Most importantly, the results led to the birth of healthy babies and at least one group has repeated the Isachenko method and has obtained good results completely independently- they report their data in Chapter 3 [Moskovtsev *et al.*, 2012]. The authors dedicate a separate Chapter 2 in this Book for summarizing their achievements [Isachenko *et al.*, 2012]. Below, we not only briefly explore progress of the method but also show that even as the staunchest opponent of the method (more precisely, interpretation of the results) as Dr. Fahy has also evolved in his perception of "legitimacy" of *kinetic* vitrification, which we

The Isachenko group has recently expanded K-VF method to other mammalian species (dogs) and to an even more distance vertebrate taxon, the fish (see below and also a separate Chapter [Isachenko *et al.*, 2012]). However, our experiments on K-VF of sperm of rodents was not so successful, and attempts of kinetic VF of sperm of the polar bear and raptor birds (falcons and

**2.5 Resurrection and rise of** *kinetic* **vitrification of sperm: the Isachenkos'** 

explore this aspect in a sub-Chapter below.

had never doubted at the beginning.

**contribution** 

eagles) failed completely, which actually would prompt us to an even more interesting hypothesis of *"The Universal Preservation Protocol"* and prediction of the *"Race for the Pace"*.
