Discussion of use of Xenon as a cryoprotectant

1. AApppplliieedd CCrryyoobbiioollooggyy –– SScciieennttiiffiicc SSyymmppoossiiuumm oonn CCrryyoonniiccss
PPoossssiibbllee MMeecchhaanniissmmss
ooff CCrryyoopprrootteeccttiivvee EEffffeecctt ooff XXeennoonn
 IIggoorr VV.. AArrttyyuuhhoovv ((pprreesseennttiinngg aauutthhoorr))
 AAlleexxaannddeerr YYuu.. PPuullvveerr
 AAlleexx GG.. PPeerreegguuddoovv
 VVaassssiilliiii II.. AArrttyyuukkhhoovv
CCoonnttaacctt:: IIggoorr..AArrttyyuuhhoovv@@bbiiooaaggiinngg..rruu
OOccttoobbeerr 44--55tthh,, DDrreessddeenn,, GGeerrmmaannyy

2. AAbbssttrraacctt
Use of xenon (and, possibly, other inert gases) for cryopreservation has been proposed as early as in
1960s. Our recent experiments (discussed in another report at this conference*) show a certain promise of
this approach as well. Nevertheless, mechanisms of xenon action are still poorly understood. There can be
several possible (mutually non-exclusive) explanations:
1.Accumulation of hydrophobic Xe atoms in the cellular lipid membranes providing
membranoprotective effect, or in hydrophobic "pockets" of proteins, thus stabilizing them and preventing
them from denaturation.
2.Formation of crystals of Xe clathrate hydrate rather than that of ice, possibly with finer grain size or
less damaging shape (ice crystals are needle-like, while Xe hydrate crystals are grain-like).
3.Accumulation of Xe atoms in the interfacial layer between liquid water and ice slowing the growth of
the latter (ice-blocking effect).
4.Vitrification of Xe water solution.
In the study** we focus on the first three items from this list. We have conducted a large scale
molecular dynamics modeling of processes in aqueous Xe solution near and below freezing point. Formation
of Xe crystallohydrates and the effect of Xe on the growth of ice crystals are investigated. Some promising
implications of our study results for cryoprotection are discussed.
____________________________
* A. Pulver, A. Tselikovsky, N. Pulver, I. Artyuhov, V. I. Artyukhov, A. Peregudov. Application of
clathrate forming gases for vitrification of organs and whole organisms.
** V. I. Artyukhov, A. Yu. Pulver, A. Peregudov, I. Artyuhov. Can xenon in water inhibit ice growth?
Molecular dynamics of phase transitions in water-Xe system. J. Chem. Phys. 141, 034503 (2014),
doi: 10.1063/1.4887069

3. Robert W. Prehoda, Suspended animation: the research possibility that may allow
man to conquer the limiting chains of time, Chilton Book Co., 1969.

4. PPaatteenntt bbyy PP.. SShhcchheerrbbaakkoovv aanndd VV.. TTeellppuuhhoovv
«Biological object (heart, kidney, etc) should be
cooled in water up to 0° C at simultaneous
saturation with the mixture of xenon, krypton,
argon at the ratio of 2.5:47.5:50 rot.%, then one
should force out water with gaseous mixture
mentioned and at the pressure of 1.5 atm. it is
necessary to decrease temperature up to -43° C,
then one should decrease the pressure of gaseous
medium up to normal level and continue cooling
up to -196° C. The innovation enables to achieve
reliable and prolonged cryoconservation of animal
heart being the constituent of the whole organism
(in situ), in a transferred transplant conserved for 6
h against the time of cold cardioplegia and up to
the moment of restoring contractile activity.
Moreover, it has been possible to achieve
restoration of adequate cardiac activity.»
Shcherbakov P.V., Telpuhov V.I. Method for organs
and tissues cryoconservation in situ.
http://russianpatents.com/patent/226/2268590.html

5. Physiological pprrooppeerrttiieess ooff xxeennoonn
• Absolutely non-toxic;
• Rapidly both penetrates tissues and leaves
them;
• Perfect anaesthetic?;
• Suppresses cellular metabolism?;
• Acts as antihypoxant?;
• Reduces inflammation?.
___________________
? Nature of these abilities is not known yet…

6. IIssssuueess wwiitthh ccoommmmoonn ccrryyoopprrootteeccttaannttss
• Need for enormous concentrations that inevitably are
toxic;
• Need for very high cooling rates in order to achieve
vitrification;
• Problems with perfusion because of high viscosity at
low temperatures and high concentrations;
• Poor penetration both when entering the cell and
when leaving it;
• Need for very high warming rates in order to avoid
crystallisation;
• Problems with non-uniform washout from
organ/organism.

7. TThhrreeee qquueessttiioonnss
1. Does xenon really act as a cryoprotectant?
2. If 'Yes', then what are the mechanisms?
3. How can it be applied in practice, especially
for cryopreservation of bulk biological objects?

8. Possible ((mmuuttuuaallllyy nnoonn--eexxcclluussiivvee))
eexxppllaannaattiioonnss
1.Accumulation in cell membranes preventing liquid crystal to gel
phase transition and thus maintaining membrane elasticity,
or/and in protein molecules' hydrophobic "pockets" preventing
denaturation.
2.Formation of xenon hydrate crystals that compete with ice for
water molecules, and are for some reason less destructive for
cells and tissues. For example they can be finer and/or have less
damaging shape, e.g. granular instead of needle-like that of ice.
3.Ice-blocking effect due to accumulation in the ice-water
interface zone.
4.Vitrification of xenon-water solution. Its conditions may differ
from that of pristine water.

9. XXeennoonn iinn cceelllluullaarr mmeemmbbrraanneess
From: R.D. Booker, A.K. Sum. Biophysical changes induced by xenon on phospholipid
bilayers. Biochimica et Biophysica Acta 1828 (2013) 1347–1356

10. Possible ((mmuuttuuaallllyy nnoonn--eexxcclluussiivvee))
eexxppllaannaattiioonnss
1.Accumulation of xenon in cell membranes, preventing liquid
crystal to gel phase transition and thus maintaining membrane
elasticity or/and in protein molecules' hydrophobic "pockets"
thus preventing them from denaturation. - probable!
2.Formation of xenon hydrate crystals which compete with ice ones for water
molecules and are for some reason less destructive for cells and tissues. For
example they can be finer and/or have less damaging shape, e.g. granular
instead of needle-like that of ice.
3.Ice-blocking effect of xenon due to it's accumulation in the ice-water
interface zone.
4.Vitrification of xenon-water solution. It's conditions may differ from that of
pristine water.

11. Vasilii I. Artyukhov et al., Can xenon in water inhibit ice growth? Molecular
dynamics of phase transitions in water–Xe system. JCP 141, 034503 (2014)

12. SSoommee ccoommppuuttaattiioonnaall ddeettaaiillss
Modelling software: GROMACS*
Time step 2 fs
Number of water molecules 4000
Number of xenon atoms 40, 80, 120, 160, 200
* D. van der Spoel et al, J. Comput. Chem. 26, 1701–1718 (2005).

13. SSoolluubbiilliittyy ooff xxeennoonn iinn wwaatteerr
T,°C
Solubility
0.01
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
-25 -20 -15 -10 -5 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Extrapolation
Semi-empirical dependence ln s = A + B/T + C ln T

14. TTeemmppeerraattuurree//CCoonncceennttrraattiioonn
ccoommbbiinnaattiioonnss ssttuuddiieedd::
DDuurraattiioonn ooff ssiimmuullaattiioonn rruunnss ((nnss))
T,°K 280 270 265 260 255 250
1% 80 80 50 100 100 >100
2% 50 50 50 100 100 >100
3% 30 30 50 100 100 >100
4% 30 30 100 100 100 >100
5% 30 50 100 100 100 >100
*Homogenous crystallization observed

15. Radial Distribution FFuunnccttiioonn XXee--OO aatt 226600°°KK

16. Solvation sshheellll aarroouunndd XXee aattoomm iinn wwaatteerr
Source: D. Paschek, J. Chem. Phys. 120, 6674 (2004).

17. CCrryyssttaalllloohhyyddrraattee ooff xxeennoonn

18. HHoommooggeennoouuss nnuucclleeaattiioonn
aatt tthhee 55%% mmooll.. XXee//mmooll.. HH22OO ssyysstteemm aatt 227700°°KK

19. Two microcrystals ooff xxeennoonn ccllaatthhrraattee hhyyddrraattee

20. Possible ((mmuuttuuaallllyy nnoonn--eexxcclluussiivvee))
eexxppllaannaattiioonnss
1.Accumulation of xenon in cell membranes, preventing liquid crystal to gel
phase transition and thus maintaining membrane elasticity or/and in protein
molecules' hydrophobic "pockets" thus preventing them from denaturation. -
probable!
2.Formation of xenon hydrate crystals which compete with ice
ones for water molecules and are for some reason less
destructive for cells and tissues. For example they can be finer
and/or have less damaging shape, e.g. granular instead of
needle-like that of ice. - probable!
3.Ice-blocking effect of xenon due to it's accumulation in the ice-water
interface zone.
4.Vitrification of xenon-water solution. It's conditions may differ from that of
pristine water.

21. SSyysstteemmss wwiitthh iiccee
(a) pure water at 275°K,
(b) 1% mol. Xe/mol. H2O solution at 274°K,
(c) 4% mol. Xe/mol. H2O solution at 271°K.
(d) Structure of ice–liquid interface: Xe and O atom density profiles for
systems (a) and (c).

22. Possible ((mmuuttuuaallllyy nnoonn--eexxcclluussiivvee))
eexxppllaannaattiioonnss
1.Accumulation of xenon in cell membranes, preventing liquid crystal to gel
phase transition and thus maintaining membrane elasticity or/and in protein
molecules' hydrophobic "pockets" thus preventing them from denaturation. -
probable!
2.Formation of xenon hydrate crystals which compete with ice ones for water
molecules and are for some reason less destructive for cells and tissues. For
example they can be finer and/or have less damaging shape, e.g. granular
instead of needle-like that of ice. - probable!
3.Ice-blocking effect of xenon due to it's accumulation in the ice-water
interface zone. - unlikely...
4.Vitrification of xenon-water solution. It's conditions may differ from that of
pristine water.

23. DDeeppeennddeennccee ooff XXee ddiiffffuussiioonn ccoonnssttaanntt
oonn ccoonncceennttrraattiioonn aanndd tteemmppeerraattuurree

24. Extrapolation ooff DDiiffffuussiioonn ccoonnssttaanntt
ttoo lloowweerr tteemmppeerraattuurreess
2
1.5
1
0.5
0
245 250 255 260 265 270 275 280 285
D, H20 D, 1% Xe D extrapol.
T0 extrapol= 248.7°K; Is it Tg?!

25. Possible ((mmuuttuuaallllyy nnoonn--eexxcclluussiivvee))
eexxppllaannaattiioonnss
1.Accumulation of xenon in cell membranes, preventing liquid crystal to gel
phase transition and thus maintaining membrane elasticity or/and in protein
molecules' hydrophobic "pockets" thus preventing them from denaturation. -
probable!
2.Formation of xenon hydrate crystals which compete with ice ones for water
molecules and are for some reason less destructive for cells and tissues. For
example they can be finer and/or have less damaging shape, e.g. granular
instead of needle-like that of ice. - probable!
3.Ice-blocking effect of xenon due to it's accumulation in the ice-water
interface zone. - unlikely...
4.Vitrification of xenon-water solution. It's conditions may differ
from that of pristine water. - Could be? At ~-25°C?!

26. TTaakkee--hhoommee mmeessssaaggee
• Xenon may serve as a cryoprotectant coming
with several valuable features at once – and
no drawbacks at all.
• Most intriguing: possible (not yet established)
ability to vitrify water as high as -10’s °C
• Can be used in combination with tolerable
concentrations of common cryoprotectors.
• We are open to any form of collaboration in
further investigation of this possibility.

27. SSoommee rreeffeerreenncceess
1. R. W. Prehoda, Suspended animation: the research possibility that may allow man to conquer the limiting chains of time,
Chilton Book Co., 1969
2. S. Sheleg et al., Cardiac Mitochondrial Membrane Stability after Deep Hypothermia using a Xenon Clathrate Cryostasis
Protocol – an Electron Microscopy Study. Int J Clin Exp Pathol (2008) 1, 440-447
3. D. S. Laptev et al., The Use of Inert Gas Xenon for Cryopreservation of Leukocytes. Bulletin of Experimental Biology and
Medicine, Vol. 157, No. 2, June, 2014, pp. 282-284
4. Shcherbakov P.V., Telpuhov V.I. Gas Influenced Immortality. Chemistry and Life 2006;8:34-39 (in Russian).
5. Shcherbakov P.V., Telpuhov V.I. Method for organs and tissues cryoconservation in situ.
http://russianpatents.com/patent/226/2268590.html, http://www.findpatent.ru/patent/226/2268590.html (in Russian)
6. Vasilii I. Artyukhov et al., Can xenon in water inhibit ice growth? Molecular dynamics of phase transitions in water–Xe
system. JCP 141, 034503 (2014)
7. R.D. Booker, A.K. Sum. Biophysical changes induced by xenon on phospholipid bilayers. Biochimica et Biophysica Acta 1828
(2013) 1347–1356
8. Rodin V.V., Isangalin F.Sh., Volkov V.Ya. Structure of water protein solutions in a presence of xenon clathrate. Cryobiology &
Cryo-Medicine. Kiev: Naukova Dumka, 1984, pp. 3-7 (in Russian).
9. Rodin V.V., Novikov I.A., Volkov V.Ya. Investigation of the influence of xenon on the DNA-bound water system using the
method of NMR. Cryobiology 1989;4:35-38 (in Russian)
10. Ryan D. Booker et al., Xenon Hydrate Dissociation Measurements With Model Protein Systems. J. Phys. Chem. B 2011, 115,
10270–10276