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Combined approach to the development of protocol for vitrification of bulky biological objects

*Pulver А., *Artyuhov I.V, **Artyukhov V.I., ***Tselikovsky A.V, *Shamaev N.V., *Pulver N.A, *Peregudov A.

*Institute of Biology of Aging, Moscow, 125284, Russia. E-mail: pulver@bioaging.ru
**Department of Materials Science and NanoEngineering, Rice University, Houston, TX, USA
***N.N. Burdenko Voronezh State Medical Academy, Voronezh, Russia

ABSTRACT
Any means of cryopreservation generally relies upon vitrification, in which cells survive in glass between ice
crystals. Proposals of a simultaneous vitrification of entire samples without the formation of ice even predate the
discovery of the first cryoprotectants, in 1940s. However, the first practical implementation of vitrification (murine
preimplantation embryos) was carried out only in 1985, by Gregory Fahy and William Rall. The present report
provides a brief overview of different glass types resulting from liquids in a living system converting into the glassy
state at low temperatures, as well as the potential benefits of vitrification for long-term large-sized sized biological
objects preservation. We specify the main outstanding obstacles for the implementation of bulky biological objects
vitrification, as well as ways proposed by various researchers to solve some of them.

One of those obstacles is the toxicity of extremely high cryoprotectant concentration (up to 60 % v/w) re-
quired for vitrification. That’s why we turned our attention to xenon (Xe). The main prospective benefit of using Xe

as a cryoprotectant is its absolute nontoxicity on tissue and cellular level, while even the most lowtoxic cryopro-
tectants (such as glycerol, ethylene glycol etc.) are potentially dangerous due to osmotic effects.

In order to assess the cryoprotective potential of Xe we have conducted two series of experiments – with

yeast (S. cerevisidae) and mammalian (CHO-K1 and NIH-3T3) cell cultures in custom-built bronze miniature hy-
perbaric chambers.

Yeast survival in presence of Xe at all tested pressures appeared to be much better than in control experi-
ments and approximately equal or slightly better than with 5% DMSO or glycerol; in experiments with joint appli-
cation of Xe and DMSO or glycerol results have been even better. But in case of mammalian cells convincing results

have been achieved only in experiments with the fastest cooling achievable.

Molecular dynamics simulations of Xe clathrate hydrate and ice crystal growth indicate vitrification of wa-
ter inside cells under combined application of Xe and DMSO/glycerol as a possible explanation of the results ob-
served (details are described in our report “Possible Mechanisms of Cryoprotective Effect of Xenon”).

Based on this hypothesis we offer approaches to both cooling (combined use of traditional cryoprotectants
and clathrate forming gases for vitrification, patent applied), and to the subsequent heating (uniform heating of
complex biological objects by means of electric and magnetic fields’ phased emitters, guided by MRI thermometry,
patent pending – discussed in yet another corresponding report at this conference), providing the theoretical

background for the successful cryopreservation of bulky biological objects. And eventually – up to individual or-
gans and whole organisms.

In addition, we offer some suggestions for improving the efficiency of gas perfusion for chilling procedure,
and application of frequency and width-pulse electromagnetic field modulation within the initial stages of cooling.
Before describing the essence of our propositions, it wouldn't be inappropriate to recall some
matters of common cryobiological knowledge.
Any means of cryopreservation generally relies upon vitrification, in which cells survive in glass
between ice crystals. Even under slow programmable freezing cells are surrounded by unfrozen liquid
between growing ice crystals [Mazur 1984].
Vitrification is a process whereby fluid becomes a solid during cooling (more precisely, exhibits

most of the properties of solids) without any substantial change in molecular arrangement or ther-
modynamic state variables. Hence, no crystallization with the consequent heat release and volume in-
crease. Proposals of a simultaneous vitrification of entire samples without the formation of ice [Luyet

1937] even predate the discovery of the first cryoprotectants, [Polge, Smith et al. 1949] in 1940s.

However, the first practical implementation of vitrification (murine preimplantation embryos) was
carried out only in 1985, by Gregory Fahy and William Rall [Rall, Fahy 1985].
The main condition for the onset of vitrification during cooling is an increase of liquid viscosity

up to about 1013 Poise. On the molecular level it is expressed in loss of rotational and translational de-
grees of freedom, leaving only bond vibration within a fixed molecular structure, which leads to a de-
crease in the specific heat and thermal expansion coefficient [Wowk 2010].

Cryoprotectants lower the critical cooling rate (which is not less than 107 °С/min [Armitage
1991] for pure water) required for vitrification inversely proportional to their concentration within
the cooling solution, reducing it up to quite available speeds.

Cryopreservation of individual cell suspensions requires nothing more than a slow programma-
ble freezing (SPF), and the vitrification protocols for whimsical preimplantation embryos [Isachenko,

Alabart et al. 2003] are even more easy to apply. However, when dealing with macroscopic objects,
researchers are faced with a number of additional obstacles, namely [Fahy, Wowk et al, 2009; Mazur,
2010; Hopkins, Badeau et al, 2012]:

a. The toxicity of extremely high cryoprotectant concentration (up to 60% v/w) required for vit-
rification.

b. Uneven cryoprotectants distribution in organ parenchyma.
c. Irregularity of cooling (temperature gradients).
d. Insufficient cooling rate.
e. Cracking during storage below glass transition temperature (Tg).
f. Occurrence of nucleation sites at about-Tg temperatures.
g. Insufficiently slow and uneven warming with temperature gradients and local overheating.
h. Devitrification during warming due to insufficient heating rate with ice recrystallization.
i. Reperfusion syndrome at the end of rewarming.
Ice formation during warming happens faster than during cooling because ice nucleation occurs

at lower temperatures than ice growth. This nucleation leads to extensive ice growth at warmer tem-
peratures. That’s why the ‘‘critical warming rates” (minimum warming rates to avoid “devitrification”,

or significant ice formation during warming from a vitrified state) are typically two or more orders of
magnitude greater than critical cooling rates [Hopkins, Badeau et al. 2012].
The same stringent and even contradictory conditions are imposed on storage temperature and

transport of macroscopic samples, because masses of vitrifiable tissues larger than a few cubic centi-
meters almost invariably develop large-scale fractures. Storing them at liquid nitrogen temperature

leads to cracking due to shear stress relaxation, while keeping them close to the glass transition tem-
perature (down to 15 degrees) results in the formation of nanoscale ice crystals due to lateral diffu-
sion of water molecules, which increases the critical warming rate upon subsequent heating.

A detailed review of the entire spectrum of research requires a separate report, and goes beyond

the scope of this work. The more so because no major success has been achieved yet – excluding suc-
cessful vitrification of blood vessels (1996), peripheral nerves, pancreatic islets and so on. Although

speaking of nerves and blood vessels, we consider it to be rather a tissue engineering than cryobiolo-
gy.

The most promising advances in this field were made by “21st Century Medicine”, a company led

by Gregory Fahy and Brian Wowk. In 2005 they have reported on a rabbit kidney that survived vitrifi-
cation and subsequent transplantation with immediate contralateral nephrectomy, successfully func-
tioning for 9 days [Fahy, Wowk et al., 2009]. However, due to the above-mentioned unresolved prob-
lems, this experiment remains anecdotal so far.

We know three possible ways of macroscopic biological objects accelerated warming: the die-
lectric warming [Wusteman, Robinson et al. 2004], vascular perfusion with inert fluids that remain

liquid at cryogenic temperatures [Federowicz, Harris et al. 1999], and gas perfusion [Schimmel,
Wajcner et al. 1964; Bickis & Henderson 1966; Hamilton, Holst et al. 1973; Van Sickle & Jones 2014].
Dielectric warming results in very uneven heating.
Cooling solutions to cryogenic temperatures directly by cryoprotectants is impossible, due to
progredient solute viscosity and peripheral vascular resistance elevation. As a result, the perfusion
rate decreases. From a certain moment, instead of passing through the microvasculature, refrigerants

begin to "shunt" through the major vascular arcades. Respectively the heat exchange is complemen-
tarily weakened and cooling becomes irregular. With a further increase viscosity perfusion either

stops or ruptures in the parenchyma occur. Liquid perfluorocarbon-based coolants are also unable to
solve the problem of sufficient cooling rate for the same reasons, albeit at lower temperatures.
Gaseous coolants, despite their negligible heat capacity, have two to three orders of magnitude
lower viscosity, and freely pass through the vascular bed even at cryogenic temperatures. The most

popular in this area is helium, due to its low condensing temperature and the highest diffusion mobili-
ty.

Of course, the gas perfusion has its own shortcomings:
 low heat capacity forces the use of high pressure to increase the heat transfer, which is
fraught with barotrauma;
 gas embolism - particularly during rewarming, when the gaseous coolant is replaced by a
liquid;
 «drying up» of endothelium and the adjacent tissue areas.

In view of the foregoing, for vitrification of isolated organs (and eventually up to intact organ-
isms), suitable for transplantation after storage and rewarming, we plan the following:

1. Application of combined use of traditional cryoprotectants with clathrate forming gases as a
means for vitrification. We turned our attention to xenon [Prehoda 1969; Rodin, Isangalin et al.

1984; Shcherbakov, Tel’pukhov et al. 2004; Sheleg, Hixon et al. 2008] because of its absolute non-
toxicity on tissue and cellular level, while even the most low-toxic cryoprotectants (such as glycer-
ol, ethylene glycol etc.) are potentially dangerous due to osmotic effects.

In order to evaluate the cryoprotective potential of xenon we have conducted two series of exper-
iments – with baker’s yeast (S. cerevisiae) and mammalian cell cultures (CHO-K1 and NIH-3T3) in

custom-built bronze miniature hyperbaric chambers.

Yeast survival at all xenon pressures tested (3 to 7 at) after 2 hours of pressure chamber expo-
sure at -20°C with subsequent chamber immersion in liquid nitrogen (68,5 plus or minus 6%)

turned out to be better than in control (35 plus or minus 6%) and comparable or even higher to
that of 5% DMSO or glycerol application (50 plus or minus 25%). Joint application of xenon +
DMSO or glycerol showed 71 plus or minus 15% of cell survival.
However, experiments with mammalian cells at xenon partial pressures of 2.5 to 12 at upon slow
programmable freezing (SPF) showed complete cells dissolution upon conventional rewarming.
Even after designing a sophisticated decompression protocol we could not achieve survival upon

thawing. This made us disappointedly conclude that xenon has no cryoprotective properties apply-
ing SPF. Nevertheless, 2,8 plus or minus 2,3% of control group cells frozen by the abovementioned

“yeast” protocol of cells survived and, moreover, fastest achievable cooling speed (direct placing
of pressurized hyperbaric chamber into liquid nitrogen, which we considered an absolute death
sentence) in the other control experiment improved cells survival up to 22,5 plus or minus 13,4%.

Large scale molecular dynamics simulations of xenon clathrate hydrate and ice crystal growth (de-
tails are recently described in our report "Possible Mechanisms of Cryoprotective Effect of Xenon")

indicate vitrification of water inside cells, especially under combined application of xenon and
DMSO, as a possible explanation of these results.

The main thing for us is that the hydrophobic solubility of xenon in low temperature water in-
creases with cooling faster than the thermal stability of clathrate hydrate [Artyukhov, Pulver et al.

2014].
As a result, it should turn out that traditional cryoprotectants, by suppressing xenon crystalline

hydrate formation on cooling stage, would allow the solution to be saturated by xenon to a signifi-
cant increase in liquid viscosity. This will enable vitrification using a much lesser amount of chem-
ical cryoprotectants, and thus yield large decrease in toxicity. We have submitted a patent applica-
tion.

Sounds so simple, even primitive. Why didn't anybody think of this before?..
2. Second critical step is to develop a methodology for a fundamentally new approach to a uniform
heating of complex biological objects by means of electric and magnetic fields’ phased emitters,

guided by MRI thermometry – also patent pending and discussed in yet another corresponding re-
port at this conference.

3. Creation of an improved carrier solution for cryoprotectants with an emphasis on organ condition-
ing, prevention of chilling injury, neutralization of reactive oxygen species, activation of apoptosis

cascades and overall reperfusion syndrome during rewarming.
4. Slight improvement of gas perfusion methodology. Increasing the heat exchange efficiency should
be achieved through a conjunction of elevated persufflation pressure with modulation of sonic and
ultrasonic waves.

5. Application of frequency and width-pulse electromagnetic field modulation within the initial stag-
es of cooling, not confining to a simple 50 Hertz oscillation used in Cells Alive System low-
temperature freezer of Abiko сorporation [Owada, Saito 2010].

6. Determination of the best storage temperature for objects, vitrified by our method of (taking into
account the alleged stabilizing effect of gas cryoprotectants).
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