**Epilogue:** *"In Defence of the Genius***"(Editor's Reflection)**

#### *"In Defense of the Genius"*

Dr. Gregory Fahy called in his Chapter #1A [Fahy & Rall, 2007] the Luyet method and his promotion of kinetic VF as *"Lyuet's tyranny"* so my choice of words in this Chapter is just as a *"symmetrical response* "to that stye*,* nothing personal is intended. Greg spent a great deal of time in that and other numerous reviews and lectures describing the Luyet's unsuccessful attempts to implement K-VF in late 1930s -beginning of 1950's. However, we have not spent *so* much time in *this* Chapter on describing the *failure* to realize the promised potential of equilibrium VF and ice blockers for organ vitrification since 1984, even though Dr. Fahy and his colleagues have had in order of magnitude more resources, knowledge, and time than that of Father Luyet had had in 1939-1954. We would call this situation as being *"stuck in the rut"* and *"the promise is not fulfilled"*.

At the same time, we completely agree with a statement that Fahy and Rall made in a subchapter titled "*The ghost of Luyet"*:

*"Here we can only note the irony that, having been launched by breaking free of Luyet's tyranny of ultrarapid cooling, vitrification methods have now essentially turned back closer to Luyet's original idea of cooling as quickly as possible with minimal intracellular exposure to cryoprotectants* [i.e., kinetic vitrifcation VF, *I.I. K.*]*,*, *albeit this time using at least marginally adequate concentrations of intracellular solutes. The ghost of Luyet lives on in the form of this ongoing methodological evolution, and we think he would have been pleased to see how his ideas about vitrification ultimately related to the now widespread use of vitrification as a practical and successful method of cryopreservation long after he, himself, had abandoned this approach."*[Fahy & Rall, 2007]

As a cryobiologist, who has been working in the same field, the Editor (at the Eves of 2012) might announce the following "resolutions":


30 Current Frontiers in Cryobiology

4. We predict that while those speeds are relatively high for the majority of cell species (in range 200,00-1,000,000 OC/min), we have already achieved those critical rates in one well-documented case, namely humans as well as some other species of vertebrate sperm, thanks to the early work in the 1930s and by the Isachenkos in this century. 5. We believe that those high speeds are achievable for *all* species but that needs entirely new cryogenic equipment. Such rates if they are high enough to surpass the highest *Bcr***'**s would be *universally* applicable to *any* type of suspendial and single cells so we will be witnessing the "*Race for the Pace*" very soon, some groups have been already actively

Thermodynamic analysis of the most recent attempts of creating novel systems for kinetic VF such as cryogenic oscillating heat pipes [Jiao et al., 2006; Jiao et al., 2009], nano-droplets [Demirci & Montsesano, 2007], quartz capillaries [Risco et al., 2007], and some others approaches that claim *"ultra-fast"* rates (see a comprehensive review by Criado in this Book), which in our opinion, do not produce the rates fast enough to reach the majority of B*cr*'s without using exogenous permeable (and thus, potentially toxic) vitrificants. Thermodynamical considerations that prove this statement are not in the scope of this Chapter and will be done elsewhere. In fact, the *hyper*-fast rates of cooling and warming will be needed, and there is about of an order or two of magnitude difference between *"ultra-"*

Introduction of such a *"Universal Kinetic Vitrification Protocol*" applicable for *all* cells (at least for those that are in suspension or make a thin attached layer) would shift the whole paradigm in cryopreservation of germplasm (and other types of suspendial cells) and in cryobiology as a science. It will require both new equipment for realization of hyper-fast rates (on which we are working now) and new methods of measurements. For example, it is not clear how *Th*, *Tm*, and *Td* curves on the Fahy diagram would behave at speeds of cooling and warming in order of thousands OC/min, and how that could be measured: they may disappear completely! In any case, it will open not only the possibility of development of a uniform protocol and equipment for all existing and (which especially important) *new* types of cells and species, but it will also bring new, very challenging but exciting horizons for

Dr. Gregory Fahy called in his Chapter #1A [Fahy & Rall, 2007] the Luyet method and his promotion of kinetic VF as *"Lyuet's tyranny"* so my choice of words in this Chapter is just as a *"symmetrical response* "to that stye*,* nothing personal is intended. Greg spent a great deal of time in that and other numerous reviews and lectures describing the Luyet's unsuccessful attempts to implement K-VF in late 1930s -beginning of 1950's. However, we have not spent *so* much time in *this* Chapter on describing the *failure* to realize the promised potential of equilibrium VF and ice blockers for organ vitrification since 1984, even though Dr. Fahy and his colleagues have had in order of magnitude more resources, knowledge, and time than that of Father Luyet had had in 1939-1954. We would call this situation as being *"stuck in the* 

At the same time, we completely agree with a statement that Fahy and Rall made in a sub-

and *"hyper-"* (cf. *ultrasonic* and *supersonic* speed of flight as an example).

**Epilogue:** *"In Defence of the Genius***"(Editor's Reflection)** 

working on it now.

basic cryobiology as well.

*"In Defense of the Genius"* 

*rut"* and *"the promise is not fulfilled"*.

chapter titled "*The ghost of Luyet"*:


But that would be a topic of our other story, here we must stop and say just only that: kinetic vitrification of sperm, the early child of Father Luyett and the other *pioneers of the cryobiological frontiers*, is very much alive and on the march! And our own success and failures, honestly described in this Chapter, have only strengthened the position K-VF as a viable (not marginal!) and very promising method of cryopreservation.

#### **Appendix 1. Some peculiar similarities between [Fahy & Rall, 2007] and our earlier papers, which are not cited there**

We invite the readers to compare the physical description of vitrification in that chapter by Fahy and Rall, particularly sub-chapter *"The kinetic basis of vitrification"*, and the first part of the *"Optimal storage below Tg"* with our preceding publications (pp. 71 and 75 in [Katkov & Levine, 2004] and pp. 353-4 "*6. Storage at temperatures higher than Tg of water"* in [Katkov *et al.*, 2006] respectively). The only substantial difference is that "*the most widely used"* WLF equation (25) in our paper [Katkov & Levine, 2004].

$$\eta(T) = \eta(T\_{\mathcal{S}}) 10^{\frac{-C\_1(T-T\_{\mathcal{S}})}{C\_2 + (T-T\_{\mathcal{S}})}} = \eta(T\_{\mathcal{S}})e^{\frac{-2.303C\_1(T-T\_{\mathcal{S}})}{C\_2 + (T-T\_{\mathcal{S}})}}\tag{1}$$

is replaced by the VTF equation in [Fahy & Rall, 2007] as following:

$$
\eta(T) = \eta(T\_{\g})e^{\frac{B}{T - T\_v}}\tag{2}
$$

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 33

A (human native) B (human, slow freezing) C (human, VF 0.5 mL)

D (bull native) E (bull, slow freezing) F (bull, VF 0.5 mL)

**A** and **D** – native sperm, **B** and **E** spermatozoa frozen slowly with glycerol, **C** and **F**- sperm

Approximately 50% of human and bull sperm survived slow freezing,. The vitrified cells are




Fig. 9. Attempts to vitrify human (**A**-**C**) and bovine (**D**-**F**) spermatozoa using "large

not visibly damaged but no motile spermatozoa were observed for both species

volume"(500 μL) method [Isachenko *et al.*, 2011]

would allow us to vitrify larger volumes.

vitrified accordingly the Isachenko protocol.

different topic.

where *η(T)* is the viscosity at temperature *T above* the glass transition temperature *T*g, *η(Tg*) is the viscosity at the glass transition tem temperature, *Tv* is the "Vogel temperature" [Zhai & Salomon, 2011] and *C1*, *C2* and *B* are empirical constants.

If the assumption of a linear relationship between the fractional free volume and temperature holds (free volume theory for WLF), VTF equation can be transformed into the WLF equation so the equations (1) and (2) must be combined as an equality, i.e.:

$$\frac{-2.303\text{C}\_1(T - T\_\text{g})}{\text{C}\_2 + (T - T\_\text{g})} = \frac{B}{T - T\_v} \text{ },\tag{3}$$

which is true at *any T* and *Tg* in the range of being considered. Equalizing the numerators and denominators separately, the relationships between the WLF and VTF constants can be found as following:

$$C\_1 = \frac{B}{2.303(T\_\% - T\_v)}\tag{4}$$

$$C\_2 \equiv T\_{\mathcal{g}} - T\_{v\text{ \textquotedblleft}} \tag{5}$$

which coincides with [Zhai & Salomon, 2011].

Beside this substitution of WLF with VTF, which accordingly to [Zhai & Salomon, 2011] "*has a more profound physical meaning that relates both thermodynamic and kinetic concepts*", there are definite similarities between [Fahy & Rall, 2007] and [Katkov & Levine, 2004; Katkov *et al.*, 2006], which of course might be purely accidental (with a notion that those two papers had been sent to G.F. by I.I.K. well before 2007), so we will follow the spirit of the Open Access, namely *"Let's the readers to decide"*.

#### **Appendix 2. On the recent paper by the Isachenkos on "***vitrification in large volumes"*

Recently, the Isachenkos group published a report on vitrification of 500 μL of human sperm vitrified with 0.25 M sucrose Recently, the Isachenkos group published a report on vitrification of 500 μL of human sperm vitrified with 0.25 M sucrose [Isachenko *et al.*, 2011]. Unfortunately, our two other teams that have co-authored this Chapter were not able to repeat the method: both human and bovine spermatozoa sperm survived vey poorly (single alive spermatozoa were observed) after vitrification in 0.5 mL straws. Interestingly enough, morphology of the sperm was practically intact (**Fig. 9**). This, together with the failure of the method even for 25 μL droplets (another Isachenkos modification) to vitrify spermatozoa of polar bear and 4 raptor bird species described in the major text of this Chapter, indicates that the Isachenko method of *"cryoprotectant"*-free cryopreservation" works satisfactory for some species of sperm [Merino *et al.*, 2011a; Merino *et al.*, 2011b; Sanchez *et al.*, 2011] but not for the others, and it is completely inapplicable to big and watery "oocytes and embryos. And without further clarification, the method of kinetic VF in *"large volumes"* have not been able to be repeated independently even for human and bovine sperm. Some of the questions that have been raised from other co-authors of the present Chapter in regards to that paper are:

where *η(T)* is the viscosity at temperature *T above* the glass transition temperature *T*g, *η(Tg*) is the viscosity at the glass transition tem temperature, *Tv* is the "Vogel temperature" [Zhai &

If the assumption of a linear relationship between the fractional free volume and temperature holds (free volume theory for WLF), VTF equation can be transformed into the

*g*

*CT T B C TT TT*

which is true at *any T* and *Tg* in the range of being considered. Equalizing the numerators and denominators separately, the relationships between the WLF and VTF constants can be

> <sup>1</sup> 2.303( ) *<sup>g</sup> <sup>v</sup> <sup>B</sup> <sup>C</sup>*

Beside this substitution of WLF with VTF, which accordingly to [Zhai & Salomon, 2011] "*has a more profound physical meaning that relates both thermodynamic and kinetic concepts*", there are definite similarities between [Fahy & Rall, 2007] and [Katkov & Levine, 2004; Katkov *et al.*, 2006], which of course might be purely accidental (with a notion that those two papers had been sent to G.F. by I.I.K. well before 2007), so we will follow the spirit of the Open Access,

**Appendix 2. On the recent paper by the Isachenkos on "***vitrification in large volumes"* Recently, the Isachenkos group published a report on vitrification of 500 μL of human sperm vitrified with 0.25 M sucrose Recently, the Isachenkos group published a report on vitrification of 500 μL of human sperm vitrified with 0.25 M sucrose [Isachenko *et al.*, 2011]. Unfortunately, our two other teams that have co-authored this Chapter were not able to repeat the method: both human and bovine spermatozoa sperm survived vey poorly (single alive spermatozoa were observed) after vitrification in 0.5 mL straws. Interestingly enough, morphology of the sperm was practically intact (**Fig. 9**). This, together with the failure of the method even for 25 μL droplets (another Isachenkos modification) to vitrify spermatozoa of polar bear and 4 raptor bird species described in the major text of this Chapter, indicates that the Isachenko method of *"cryoprotectant"*-free cryopreservation" works satisfactory for some species of sperm [Merino *et al.*, 2011a; Merino *et al.*, 2011b; Sanchez *et al.*, 2011] but not for the others, and it is completely inapplicable to big and watery "oocytes and embryos. And without further clarification, the method of kinetic VF in *"large volumes"* have not been able to be repeated independently even for human and bovine sperm. Some of the questions that have been raised from other co-authors of the

*g v*

, (3)

*T T* (4)

*C TT* <sup>2</sup> *<sup>g</sup> <sup>v</sup>* , (5)

WLF equation so the equations (1) and (2) must be combined as an equality, i.e.:

2

1

2.303 ( ) ( )

Salomon, 2011] and *C1*, *C2* and *B* are empirical constants.

which coincides with [Zhai & Salomon, 2011].

present Chapter in regards to that paper are:

namely *"Let's the readers to decide"*.

found as following:

A (human native) B (human, slow freezing) C (human, VF 0.5 mL)

D (bull native) E (bull, slow freezing) F (bull, VF 0.5 mL)

Fig. 9. Attempts to vitrify human (**A**-**C**) and bovine (**D**-**F**) spermatozoa using "large volume"(500 μL) method [Isachenko *et al.*, 2011]

**A** and **D** – native sperm, **B** and **E** spermatozoa frozen slowly with glycerol, **C** and **F**- sperm vitrified accordingly the Isachenko protocol.

Approximately 50% of human and bull sperm survived slow freezing,. The vitrified cells are not visibly damaged but no motile spermatozoa were observed for both species


Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 35

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possibility of kinetic VF of sperm) that had been recognized for many year prior to that publication in 2003. Note that the authors confused vitrificants with CPAs: intracellular proteins do not help slow freezing.


Thus, there is a disparity in experimental verification of the method between several groups that have been contributed to this Chapter, which we feel should be clarified.
