Organ preservation at low temperature: a physical and biological problem

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biological problem

J. Aussedat, P. Boutron, P. Coquilhat, J. Descotes, Gérôme Faure, M. Ferrari, Laurence Kay, J. Mazuer, P. Monod, J. Odin, et al.

To cite this version:

J. Aussedat, P. Boutron, P. Coquilhat, J. Descotes, Gérôme Faure, et al.. Organ preservation at low

temperature: a physical and biological problem. Journal de Physique I, EDP Sciences, 1993, 3 (2),

pp.515-531. �10.1051/jp1:1993147�. �jpa-00246738�

Classification

Physics

Abstracts

87.90 64.70 65.90

Organ preservation at low temperature

_{: a}

physical and biological problem

J.

Aussedat(~),

P.

Boutron(~),

P.

Coquilhat(~),

J-L-

Descotes(~),

G.

Faure(~),

M.

Ferrari(~),

L.

Kay(~),

J.

Mazuer(~),

P.

Monod(~),

J.

Odin(~)

and A.

Ray(~)

(~)

Laboratoire de

physiologie

cellulaire

cardiaque,

Universitd J. Fourier, BP

53X,

38041 Greno- ble

Cedex,

France

(~)

Laboratoire Louis

Ndel/CNRS

BP 166, 38042 Grenoble Cedex 9, France

(~)

Service

urologie,

Centre

Hospitalier Rdgional

Universitaire, BP 217X, 38043 Grenoble Cedex, France

(~)

Centre de Recherches _sun (es Trks Basses

Tempdratures/CNRS,

BP 166, 38042 Grenoble Cedex 9, France

(Received

8

August

1992,

accepted

in final form 6

September 1992)

R4sumd. Avant de

prdsenter

les rdsultats

prdliminaires

obtenus _par notre groupe, nous

passons d'abord _{en revue} les principaux problbmes h rdsoudre _{pour conserver} h trbs basse tem-

pdrature des organes en vue de leur transplantation. Cette

cryoprdservation

est _une voie de recherche actuellement

explorde

_pour augmenter la durde de conservation des

greffons

et per-

mettre ainsi de mieux contr61er la

compatibilitd

donneur-receveur. Nous

rappelons

_que la _con-

servation des cellules iso16es I la tempdrature de l'azote liquide, actuellement rdalisde _avec succks h l'dchelle

industrielle,

_{ne peut se} faire qu'en prdsence de substances plus _ou moins toxiques dites

cryoprotectrices,

et h condition de respecter ^{des vitesses} de refroidissement et de rdchauflement

adaptdes

h

chaque

_type de cellule. Nous montrons ensuite que l'extension de la

cryoprdservation

au cas des _organes entiers _{ne pourra se} faire

qu'au

_moyen de la

vitrification,

seule solution _pour dviter toute formation de

glace.

Cette vitrification _sera l'aboutissement de 2 _axes de

recherche,

l'un _sur l'dlaboration de solutions

cryoprotectrices

les moins toxiques possibles, ^l'autre _sur l'ob- tention de vitesses de refroidissement et de rdchauflement suffisamment dlevdes et

homogbnes.

Aprbs

avoir bribvement rdsumd l'dtat des recherches

sur le _coeur et le rein de

petits mammi&res,

nous

prdsentons

les

premiers

rdsultats _{que nous avons} obtenus _sur la

perfusion

I 4 °C _{et l'auto-}

transplantation

de reins de lapin, _sur la toxicitd _sur le _coeur de rat d'un _nouveau cryoprotecteur le

2,3-butanediol,

et _sur le refroidissement de

systkmes

modkles expdrimentaux

d'organes.

Abstract, Before

reporting

the

preliminary

results obtained by _{our group, we} first review the main

problems

to be solved in the preservation of organs ^at very lo,v temperature~ ^before

being transplanted.

This

cryopreservation

is

being presently explored

in order to increase the preservation time of

transplants

and to contribute to _a better control of the donor

recipient

_com-

patibility.

We recall that~ for the isolated cells to be

preserved

at

nitrogen liquid

temperatures,

as now

successfully performed

at industrial

scale,

it is necessary ^to immerse the cells in

a solu- tion

containing

more or less toxical additives

(so-called cryoprotectants).

Furthermore

cooling

and

warming

rates trust be

specific

of each type ^{of cells. We then show that}

cryopreservation

could be

extrapolated

to whole organs

by

_means of vitrification, the only _way to avoid _any ice

crystallization.

This vitrification will be the result of two directions of research, the _{one on} the elaboration of

cryoprotective solutions,

the least toxic

possible,

the other _on the obtention of

high enough

^and

homogeneous cooling

^and

warming

rates. After

having briefly

summarized

the state of research _on the heart and kidneys of small mammals~ _we present ^{the first results}

that we have obtained

on perfusion at 4°C and the auto-transplantation of rabbit

kidneys,

_on

the toxicity ^of _{a new} cryoprotectant, 2,3-butanediol, on the heart rate, ^and _on ^the

cooling

of experimental models of organs.

This

cross-disciplinary

research group was initiated in 1989

by

R. Rammal. He succeeded in

con

vincing

all of _us of the

importance

in

merging

our various

specialities

in order to

attempt

to progress in this field where _many

problems

_are linked.

1 Introduction.

The number of _organ

transplantations

has

steadily

increased since 1950.

They

have enabled the survival and relative comfort of _a number of

patients.

When

taking

into account the

development

of

surgical techniques

and their

iiuprovement during

the

past

_years

one1i1ight

think that the most

important activity

in the next decades will be _organ

transplantation.

At the _same

time, increasing knowledge

in

immunology improves

the control of

rejection [I].

To-

day

the short and

long

term

prognostic

for organs

grafts

is

already good 90i~

of

kidney grafts

are still functional after I _year and in the best _{cases one}

hopes

the

graft

will last _more than 16 _years [2].

Similarly,

_more recent liver and _pancreas

grafts

have _an

improving prognostic _[3].

Nevertheless,

these

satisfactory aspects

must not hide the

daily problems

encountered

by

surgeons in

preserving

_organs.

After

being

extracted froiu _a

cerebrally

dead

individual,

the organ ^must be

preserved

in _an

optimal

_way. At the moiuent,

preservation

is

performed by flushing

_organs with

adapted

solu- tions

(Euro-Collins, U.~f.)

and

cooling

them down to 4 °C [4].

Hypothermia

slows the cellular

iuetabolisms and

delays

cellular

death,

which is unavoidable due to ischemia

[5]. Besides,

the increase in cellular oedeiua in connexion with

hypotheriuia implies

the _use of

preservation

_so-

lutions which enhances the

organ's ability

to withstand the deleterious elsects of cold.

To-day,

these methods allo,v the

preservation

of the heart for 6

hours,

the liver and _pancreas for 12

hours,

and the

kidney

for 48 hours

or even 72 hours. These

preservation

times _{are so} short that

occasionally they

_may be

responsible

of the immediate failure of the

graft. They

limit organ transfer and do _not allow the

optimization

of the choice of the

recipient

with respect to the

immunological

characteristics of the

transplant.

In order to

satisfy

the

compatibility problems, preservation

for _a

longer

time is _necessary.

One _way which _{concerns us} here is

cryopreservation,

which is

already successfully applied

to isolated-cellular

systeius

_[6] and

paucicellular

systems. Another _way

presently

less

developed

could be the

complete dehydration

of organs and their conservation in _a

dry living

state

(so

called

anhydrobiosis) _[7].

It is also _to be noted that _a

completely

dilserent _way is

eXplored,

the

xenografts

which _may also

palliate

the lack of organs and contribute to the settlement of organs banks. All these methods would eliminate the

biggest

constraint for surgeons the too short

delay

between

harvesting

and

transplantation.

The

principle

of

cryopreservation

is to cancel _{or at} lea-St _to

strongly

reduce the metabolism

by

lowering

the

temperature

well below 0 °C. Metabolism is the ensemble of

enzymatic

reactions

:

these reactions increase _{more or} less

quickly

as a function of

temperature.

In the

living

cells there _{are many} such

enzymatic

reactions and the

analysis

of the whole mechanism is _very

complicated.

To illustrate in _a

simple

_way the influence of the

temperature

let _us take _a

simple

firts order reaction. In this _case the decrease of the concentration of _a

species

A is

proportional

to the concentration

[Al itself,

I-e-

d[A]/dt

₌

-k[A].

The

parameter

^{k is the} rate constant of reaction.

Enzyiuatic

reactions _are

thermally

activated and therefore k

obeys

the Arrhenius law (8j

k =

ko

_exp

(-E/RT);

ko

is

a

constant,

^{E the activation} _energy

(characteristic

of the

reaction),

R the ideal _gas constant and T the

temperature

^{in Kelvin. The}

point

of

cooling

is then obvious _as it results in

slowing

the _enzyme

activity.

We

give

in table I

examples

of the reduction of the _{rate constant} k when

temperature

^{is divided}

by

I-b and

3,

for several activation

energies typical

of human

metabolism. To succeed in

cryopreserving

cells it is _necessary to slow down the

enzymatic

reactions of lowest acti,,ation energy, I-e- to cool down to _a

sufficiently

low temperature

(Note

however that the Arrhenius law is well verified

only

_{over a} limited range of

temperature).

Table I. Reduction ratios of the rate constant k for

typical

activation energy of human metabolism. For

example,

when E

= 40

kJ/mole,

_a reaction which needs I _s at T

= 300 K

will take _m I hour at 200 K and 2 _x 10~ _years at 100 K!..

Activation energy 20

(kJ/mole)

40

(kJ/mole)

60

(kJ/mole)

80

(kJ/mole)

When

temperature

is divided

by

1.5 55 3 _x 10~ 1.7 _x 10~ 9 _x 10~

(from

300 K to 200

K)

When

temperature

^{is divided}

by

3 9 _x 10~ 8 _x 10~~ 7.6 _x

10~°

_6.9

x

10~~

(from

300 K to 100

II)

Most of the mammal cells in their natural environment _are killed when

they

_are frozen in

liquid nitrogen,

whatever _may be the

cooling

and

heating

rates.

They

_can survive

only

when the saline solutions

(mainly

with

Nacl)

in which

they

_are immersed contain also _an additive

called

cryoprotectant (CP)

_or

cryoprotective agent (CPA).

The survival is

yet

^not

systematic

it

depends essentially

on the

cooling

and

heating

rates. Cells _can

stay

_years at -196

°C,

the

liquid nitrogen temperature,

which is not

dangerous

and where the metabolism is

stopped _[9].

The main

problem

with CP is their

toxicity

all

presently

known CP _{are more or} less toxic and _we will discuss further how isolated cells _can be

successfully cryopreserved

and what _are the difficulties in

extending cryopreservation

to organs. We also summarize the researches of

Boutron _on CP for organs and the state of world research _on mammalian heart and

kidney.

We will end

by reporting

the

preliminary

results of _our group in Grenoble.

2,

Cryopreservation

of

living

cells.

2. I THE CRYOPROTECTIVE AGENTS. Most of

commonly,

the used CA'S

belong

to two

main families natural sugars and others.

Nlannitol,

trehalose and sorbitol

belong

to the

first;

they mainly

act

by allowing

the cells to accommodate to the ice.

They

don't

penetrate

^the cells. The second

faiuily

includes the two most classical

CP'S, glycerol

and

dimethylsulfoxide (DMSO)

and _more recent CP'S such _as

1,2-propanediol, levo-2,3-butanediol, 1,3-butanediol.

All

these

polyalcohols

and DMSO

penetrate

the

cells; they chiefly

act

by decreasing

the

quantity

of ice formed.

To be _a

cryoprotectant,

_a

conipound

must be

amongst

other

things

of very low

toxicity

and soluble in water.

Figure

I

represents

a schematic

phase diagram

of _a

binary system containing

water and another solute

forming

no

hydrate _[10].

The

water-glycerol phase diagram [Ill

».ould be almost identical. Like _any

solute,

_a

cryoprotectant

first lowers the

temperature

of ice

crystallization

at

thermodynamic equilibrium.

Instead of

crystallizing

at 0 °C at

equilibrium,

ice would

crystallize

from _a

teiuperature Tm

to _a

temperature Te

where _an eutectic mixture of ice and solute

crystals

_can foriu.

(°ci

o °c Tm

' Tn

Te ...-..I..

~g

I

H2^{0 50 E} 100

SOLUTE CONCENTRATION (%)

Fig.

i. Schematic phase

diagram

of _a biiiarj, system water with another solute

forming

_no

hydrate (Ex: water-glycerol).

Furthermore,

_even at low

cooling

rate,

thermodynamic equilibrium

is not reached. The eutectic does not

crystallize.

The ice

begins

to

crystallize only

at the still lower

temperature Tn

<

Tm.

Due to the heat

liberated,

the

temperature

of the residual

liquid

first increases. Then it decreases

again

while the solution continues to be cooled but

equilibrium

is not reached. Ice itself

crystallizes incompletely

_{or even} not at all when

sufficiently

fast

cooling

rates _are

applied, leaving

the solution in _a

partially

_or

wholly aiuorphous

_state. The

liquid

becomes _iuore and

more viscous and

finally

as

rigid

_{as a} solid. The iuolecules remain in the _same disorder _as in _a

liquid

_a

glass

is obtained.

2. 2 THE _{COOLING AND WARMING RATES.} The

general shape

of variation of survival with

cooling

rate, for _a

given warming

i-ate, is

represented figure

2

[12].

^A survival

peak

is

observed,

which is called the classical

peak,

followed

by

_a second increase of survival at the

highest cooling

rates for solutions with

high

concentrations of CP. As the cells _are

cooled,

_pure ice

first

crystallizes

outside the cells. Mammalian cells

can live

only

in solutions

containing salts, mainly

Nacl. As extracellular ice

forms,

extracellular salts concentration increases. Due to the

resulting

osmotic _pressure, the cells have _a

tendency

to loose ,vater and shrink. At the lowest

cooling

rates the cells have

plenty

of tiiue to shrink. Too much

shrinkage

kills the cells. This

too

«

~

~i

_soiuiion

_opum>i

intracellular

Complete

vitrification

~

» effects

cooling

rate

freezing

4

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Cooling

rate

20°C

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£~

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£~

^~

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~ ^{* ~}

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Slight

wternal

contraction contraction crystallization

~ ~ ~

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£j~

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-196°C

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Lethal _Viable Irreversible

Wholly amorphous

contraction cells

damages

_state

Fig.

2. Variation of survival of cells with

cooling

rate

(from

Ref.

[12]).

is called _a "solution elsect". It had first been

suggested

that

damage

_was caused

by

the _too

large

increase in salts

[13].

^{The mechanism of}

damage

is _more

complicated

to

explain _[14].

At the

cooling

rates

corresponding

to the

right

side of the classical

peak,

cells

shrinkage

is small due to lack of time.

Consequently,

ice

crystallizes

also inside the cells which _are killed. At the intermediate

cooling

_rates, cells

survive,

because

shrinkage

is sufficient to avoid intracellular

crystallization

but insufficient to be

damaging

in itself.

At the

highest cooling

rates, ^{cells have} not

enough

time to

shrink,

but

they

survive when ice has nevertheless not

enough

time to form inside _as well _as outside the cells. This

corresponds

to the second increase of survival. The solution becomes

wholly amorphous

inside and outside the cells.

When the

cryoprotectant

concentration is

increased,

the survival

peak

becomes

higher

and

moves to slower

cooling

rates

(Fig. _{3) [15].}

At the slow

cooling

rates

corresponding

to the left

side of the

peak,

the survival rate increases because with _more

cryoprotectant,

^{there is} less extracellular ice at each

temperature,

and the cell shrinks less. The

right

side of the

peak

almost does not _move due to two

opposite

elsects. Since there is less extracellular

ice,

the cells shrink

less,

which favors intracellular ice

formation,

but

they

contain also _more

cryoprotectant,

which

impedes

intracellular ice formation.

When the

warming

rate is

increased,

the survival

peak increases,

and _moves to

larger cooling

rates

(Fig. 4) [9].

^{When survival is}

high

after fast

warming

rates but low after slow

warming rates,

^{this shows that} it is _on

warming

that intracellular ice

crystallizes

and

damages

the cells.

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_{', "} 0.4M ~

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e 0

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0 0

o-I I 0 100 1000 10 100 1000

cooling Rate (°c/mnj C°°'i~g Rate (°c/mnj

Fig.

3.

Fig.

4.

Fig. 3. Effect of

cooling

rate _on the survival of

mouse marrow stem cells

containing

the indicated

concentrations of

glycerol,

cooled to 77 K and thawed _at io00

K/min (from

Ref.

[15]).

Fig.

4. Survival _curves of chinese hamster cells

corresponding

to several

warming

_rates. The cells

were cooled to 77 K before

warming

at the rates indicated

(from Ref.[9]).

The

cooling

rate

corresponding

to maximum survival

depends

_on the kind of cells

(Fig. 5) [16]. For,

the

higher

the

permeability

of the cells

membranes,

the

higher

the

cooling

rate

corresponding

to _an average

shrinkage.

Maximum survival

corresponds

to the

highest cooling

rate for red blood cells the membranes of which _are the most

permeable.

3.

Cryopreservation

of _organs,

3.I FROM CELLS TO ORGANS.

Long

term conservation at

liquid nitrogen temperature

has been

performed

with _success at

laboratory

scales and

applied

for _years to many kinds of isolated cells

(red

blood

cells, lymphocytes,

_marrow stem

cells, fibroblasts, spermatozoa,

etc...)

and also isolated cells from organs

(heart cells,

liver

cells,

_pancreas

cells,

and cells of the

paratyroid).

Small _groups of cells such _as

embryos

_or islets of

Langerhans

_can be frozen

70

fi /

~ ^>

)

~~

l' )., _~

_~

*

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~~

3 50

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.~

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Cooling Rate (°C/mn)

Fig.

5. Comparative effects of

cooling velocity

_on various cells cooled _to 77 K and thawed rapidly

(from

Ref.

[16]). Copyright

1971

by

the AAAS.

without

damage.

One _can freeze also without

damage skin, veins, arteries,

cardiac

valves~

nerves~ and small organs such _as the hearts of chicken

embryos,

and

frog

hearts.

The

commonly

used method is to immerse the

biological

material in _a

cryoprotective

solution in

plastic bags

tubes _or straws which _are cooled

by pulverization

of

liquid nitrogen drops

in

a

gas flow mixture of air and

nitrogen

which _sweeps the

plastic bags.

The

liquid nitrogen

flux is monitored to _ensure the desired

cooling rate~ corresponding

to the

top

of the classical

peak.

Despite

about 30 _years of

research,

_one is yet ^{not able} to freeze without

damage

the main organs~ such _as the

heart~ kidney~

_or the liver of _man and mammals.

Many attempts

have been made to freeze organs

using

^the classical

peak. Only

_a few

successes have been obtained _on

dog kidneys~ (see

Sect.

3.3)

which have not been

reproduced

[17].

^{The three} reasons for failure _are the

following:

I)

Due to heat transfer

probleius

the

cooling

rate is not the _same at the center and _on the

edges

of the organ. It _can therefore not be

optimal everywhere.

2)

The organ may be constituted of cells of dilserent

kinds, requiring

dilserent

cooling

rates

(Fig. 5).

3)

The main _reason is that extracellular ice~ ^{which is innocuous for isolated}

cells,

is

injurious

for organs. It breaks the vessels

similarly

to water

pipes

in winter.

The

only

_way to cryopreserve organs without

damage

is to

vitrify

them: to cool them to very low

temperatures

^without any

crystals

of ice _or

hydrate formation~

_or ^else to _use the classical

peak

in such conditions that the

quantity

of extracellular ice formed will be small

enough.

In material

science,

to obtain _an

amorphous

state, it is well known that _{one must}

perform

a very fast

cooling

from the

liquid

state. So _{one can}

produce amorphous

ice from _pure water

only

in the form of _a thin

film,

and

provided

_one is able _to cool at _a rate _as

high

_as 10~

°C/mn [18j.

In the _case of organs, vitrification

(or

reduction of the

quantity

of

crystallized ice)

necessi- tates

raising

the concentration of CP in order to decrease the

cooling

rates to realistic values.

But the

toxicity

of these CP increases with their concentration in

perfusions.

It is then of interest to work at the fastest

cooling

rates

possible,

which is _a heat transfer

problem,

^with _a CP

having

the lowest

toxicity

_as

possible

and

having

in _aqueous

solution,

for the

same solute contents, ^the

highest possible glass-forming tendency.

3.2 CRYOPROTECTIVE AGENTS FOR ORGANS.

3.2. I Research

on

cryoprotective

solutions. To obtain

vitrification,

_one must reach

high

concentrations of

cryoprotectants

of

particularly

low

toxicity

and

particularly

efficient at im-

peding

ice

crystallization.

Two kinds of research have been done.

Firstly,

the

toxicity

is minimized

by mixing

^several

cryoprotectants.

The two best known such CP'S _are:

.

VSI,

which contains

1,2-propanediol, DMSO,

acetamide and

polyethylene glycol [19],

.

VS4,

which contains

1,2-propanediol,

DMSO and formamide

[20]

in _a carrier solution.

In these solutions the DMSO neutralizes the

toxicity

of the amide and the amide neutralizes the

toxicity

of DMSO.

Secondly,

_{one can} also examine the CP of _very low

toxicity

which

gives

for _same solute contents the

highest glass-forming tendency

_on

cooling

and the

highest stability

of the

amorphous

state _on

warming.

3.2.2

Glass-forming tendency

_on

cooling.

In

figure

6 _we show the variations of the ratio

(init)

of

crystallized

ice in the solution with

cooling

rate, on

cooling

to well below the _tem-

peratures

of ice

crystallization

for several concentrations of

levc-2,3-butanediol

in water

[21].

One _sees that the

glass forming tendency

increases

rapidly

with

concentration,

but

toxicity

also. The continuous lines _are theoretical _curves. There

is,

_as

usual,

a very

good agreement

between

theory

and

experiment.

It has been demonstrated that the

glass-forming tendency

is very

dependent

_on the solute for the _same concentration

[21].

q we>

50

~ .

40

~

. 20%

30 _"

. 25%

. 30%

20 ^. 35%

+ + 40°la

i o

o

i io ioo iooo

Cooling Rate (°C/mn)

Fig.

6. Ratio _q

(in $l)

of

crystalized

ice _{as a} function of coitcentration and

cooling

rate in levo-

2,3-butanediol

solutions. The percentages of

polyalcohol

_are in

weight by weight.

Isolated

points:

experimental points;

continuous lines: theoretical _curves

(from

Ref.

[21]). (q

^is

actually

the number of grams ofice whose solidification at 273 K would liberate the _{same amount} of heat

as that from loo g

of solution. In these units, _q is _a

rough

estimate of the real quantity of ice crystalized in percentage

(w/w)

of

solution).

In table II _are

given

critical

cooling

rates for which the

quantity

of ice formed _on

cooling

is considered _as

negligible _[22].

Table II. Theoretical critical

cooling

rates

(°C/mn)

for which the

quantity

of ice formed

on

cooling

is

negligible.

it (w /w) levo-2,

3- racemic

2,

3-

2,

3-butanediol

2,

3-butanediol

2,

3-butanediol

alcohol butanediol butanediol 97~ dl in water

97it

dl in bulser

97it

dl in Euro-

in water in water Collins

20 2000

25 1400 1800

30 410 350 270 150 150

35 26 22 38 23

40 5

Most of these

cooling

rates _are deduced from the theoretical _curves.

They

_{are very}

dependent

both _on the solute and

on the concentration. The most efficient solute

by

far is

levo-2,3- butanediol,

followed

by 1,2-propanediol. Glycerol, though classically

used in

cryobiology

is far behind. We will discuss in _iuore details in section 5 the _case of

2,3-butanediol.

3.2.3

Stability

of the

wholly amorphous

state. When _a

droplet

of _a

wholly amorphous

solution is rewarmed in _a differential

scanning caloriineter~

when it forms _no

hydrate,

and the

warming

rate is not fast

enough

to avoid ice

crystallization

_on

warming,

the

thermogram

has the

shape given

in

figure

7. The solution becomes

a

supercooled liquid

above the

glass

transition at

Tg.

^{Then ice}

crystallizes

at the

peak,

the

top

^{of which is} at

temperature Td. X-ray

diffraction shows that it is first nietastable cubic ice which forms. At

higher temperatures

this transforms into

ordinary hexagonal

ice. It then melts until _a

temperature Tm.

The faster is the

wariiiing rate,

^the

higher

is

Td

In the range of

warming

rates that has been used in

calorimetry, Td

varies

linearly

with

Log

V within _{a very}

good approximation ([21]

and Ref.

herein).

At

sufficiently high ,varming rates,

the

Td Peak

meets the

Tm peak, overlaps,

and then the two

peaks disappear:

ice has _not

enough

time to

crystallize.

One _can define _a critical

warming

rate

l~r

above which the aiuount of

crystallized

ice becomes

negligible [23].

In table III _are

given

critical

warming

rates

[22, 23].

One notes that the critical

warming

rates _are much

higher

than the critical

cooling

rates: ice

crystallizes

much _more

easily

_on

warming

than _on

cooling.

The most efficient solutes _{are, as on}

cooling, levo-2,3-butanediol

and then

1,2-propanediol _[23].

Table III. Critical

ivarming

rates

(°C/mn)

for which the

quantity

of ice formed _on

warming

is

negligible.

~ (w /w) levo-2,

3- racemic 2, 3-

2,

3-butanediol

2,

3-butanediol

2,

3-butanediol alcohol butanediol butanediol 97it dl in water

97it

dl in buffer

97it

dl in Euro-

in water in water Collins

30 3 _x 10~ 1.8 x

10~

_x

10~

_{4.9 x}

10~

_1-1x 10~

35 3700 6800 8800 960

~~

_melting ^T~fi

dt

ice solidification Peak

Glass

~~ ~~~.

Transition

~'~~

Tg ^Td

~~~~~~

liquid Super

cooled

wholly

liquid amorphous

Tempernture

Fig.

7. Schematic

thermogram

obtained with

a differential

scanning

calorimeter for _aqueous solution observed

on

warming

after _{a very}

rapid cooling

to 77 Il. The derivative of the heat received

by

the sample is represented, _versus temperature.

The

high

critical

warming

rate

might

be not so easy ^to reach in organs, even

by

microwave

thawing. Experiments

_on

erythrocytes ([24]

and Ref.

herein)

have shown that in

warming

at 5000

°C/mn

where intracellular ice forms but remains cubic _a

high

survival is observed while

on

warming

at 200

°C/mn

where ice has

enough

tiiue to become

hexagonal

_a low survival is observed.

This, together

with other

experiments, suggests

that _{a way} to cryopreserve organs without

damage

could be to avoid _any ice formation _on

cooling

and to avoid the transition from cubic to

hexagonal

ice _on

,varming.

3.3 HEAT TRANSFER IN ORGANS. The

cooling

of human organs faces _a

thermodynamic

problem:

how _{can we}

quickly

_remove heat from inside _a

large

volume of _a

biological tissue,

the thermal

conductivity

of which is low? Indeed _we know that water is the main component, between 60 and

90~

of the _{organ mass}

[25].

^So one may think that the thermal

conductivity

of

water,

about 5.5

mW/cm.K just

above 0

°C,

will

give

_a rather

good

order of

magnitude

for heat transfer in organ. As _we have _{seen, a} CP solution must be

perfused

in organ ^to obtain

vitrification. The thermal

conductivity

I of the CP solutions has almost the _same value _as water before

freezing.

At the

phase change,

I increases

by

_a factor of about 3 and continues to

increase,

_{up to,} for

example,

40

iuw/cm.I(

at 120 K in the _case of _a saline solution of 2M

glycerol _[26].

Measurements _on various bovine tissues show that I lies in this range, with

a

slight tendency

to decrease with

decreasing temperature

in the frozen state

[27].

^{The heat}

capacity

of water is also

a

good approximation

to that of CP solutions. For

example

the heat

capacity

of the above

glycerol

^{solution is about} 4

kJ/kg.K

at 300 K and 2.5 at about 120 K.

With these values,ve _can

get

_a

rough

estiiuate of the

refrigeration

_{power necessary} for

cooling

a human

kidney

(Se 150

g).

We obtain 6 kW at _a

cooling

rate of10 K

Is

which would be

necessary for vitrification ,vith _a low concentration of

a

typical

CP.

Realization of such _a i-ate at the external surface of the

kidney

could

likely

be obtained. The transmission of

cooling

to the inner of the organ has been

widely

studied _on

experimental

and theoretical models

[18, 25, 26, 28, 29].

It has been shown that the

cooling

rate decreases when

entering

the volume in

a

homogeneous phase,

_a well known result for solids

[30].

But when there is

propagation

of _an ice front in the

volume,

the

cooling

rates increase with

depth

to the middle of the

sample _[29, 31].

The

goal

^of vitrification

being precisely

to avoid ice formation this _means that the _conve- nient solution for

perfusion

will be to make the _organ

macroscopically homogeneous.

As _a

consequence we are

waiting

for lower

cooling

rates in the organ. We have _seen that the value necessary ^to obtain

vitrification,

the sc-called critical

cooling

rate is

a CP characteristic _pa- rameter. It is

currently stimulating

_a number of

experimental

and theoretical

analyses [32-35].

Attempts

to

vitrify

volumes of about _one litre of CP have shown that fractures _are difficult _to avoid

[36].

Another _way to cool has been

explored

in the

past

on

dog kidneys

with

a relative _success

[17].

In each

experiiuent

the

kidney

_was frozen

by nitrogen

_gas at its surface and

simultaneously by perfusion

^of cooled helium _gas

through

the vascular

system.

^A

temperature

of -80 °C

was reached. Then the

kidney

_was thawed

by

_a microwave

system

^and

autografted.

Several weeks

later,

the normal controlateral

kidney

_was removed.

Fifty percents

^{of the}

operated dogs

survived 2-14 months _on their

single kidney. Unfortunately

this _{success was}

irreproducible although experiments

had been

previously attempted

under _a

quite

similar

protocol [37] except

that the

high

flow rate of He could not be reached in this 2nd series. Theoretical

analysis

of the

potentialities

of

cooling by perfusion

deiuonstrated that the classical known coolants could not

operate

due to their

rapid

thermalization when

reaching

the small

capillaries _[38].

4.

Summary

^of researches _on heai~t and

kidney throughout

the world.

4.I HEART. The first

report

on the

resumption

of cardiac

activity

after

freezing

_was

published by

Gonzales and

Luyet

in 1950

(see

^review _paper

[39]). They observed,

in chick

embryos

incubated in _a

30it ethylene-glycol solution,

frozen in

liquid nitrogen

and

rapidly thawed,

that the heart of _some chicks

regained

_a contractile

activity.

A

systematic study

_on the effects of

freezing

_on the mammalian heart _was

performed by Audrey

Smith in 195i

[40].

^She

pointed

out that isolated hamster

hearts,

frozen below -5

°C,

never recovered mechanical

activity

^but that _a

previous perfusion

of the hearts with 15~

glycerol

solution allowed _some hearts to _{recover a} reduced contractile

activity

after

cooling

to -20 °C. Thus the need of the _use of _a

cryoprotective agent during freezing

_was established.

Rapatz [41] applied

this

principle

to the

freezing

of the

frog

heart: after

perfusion

with

a I-I M

ethylene glycol solution,

the isolated hearts _were cooled to -78 °C for 5 minutes then

gradually

rewariued before

reperfusion

with _a

physiological

^solution. 9 out of10 hearts

spontaneously

recovered _a contractile

activity.

The _success of

Rapatz's experiment

stimulated research _on

freezing

and

cryoprotection

of

the mammalian heart but this research

calve up

against

_a

great difficulty:

the

toxicity

of the

cryoprotective agents.

Experiiuents

showed that

perfusions

of isolated rat hearts at 37

°C,

with

15~ [42]

or

20it [43]

^{DMSO solutions did not}

produce

irreversible alterations of cardiac

contractility

but that

higher

DMSO concentrations

strongly

decreased the

contractility

in _an irreversible _way and induced alterations of

iuyocardial

intracellular components

[42].

Attempts

to freeze rat hearts

perfused

with IS to

20it cryoprotectant

^solution showed that

cryoprotected

hearts cooled to -20 °C for _a short time

regained

_a weak contractile

activity

but that hearts cooled below -20 °C _never recovered _any mechanical

activity [44, 45].

These low concentrations of

cryoprotective _agent

did _not

prevent

^the formation of ice which

produced

mechanical

damage

to the

myocardial

tissue.

The ice formation and the

toxicity

of

cryoprotectants injured

cardiac muscular cells and reduced the heart

contractility

but

they

also

impaired strongly

the function of coronary vessels.

The _coronary flow of isolated rat hearts

[46]

^showed a

great

^reduction

following cryopreservation

and

cooling

to -20 ^°C. A _coronary flow _so reduced did not allow the heart to maintain _a normal contractile

activity.

The decrease of the flow would have been

produced by

the formation of

an intracellular cedema which arises

during thawing

and

cryoprotectant

removal.

Reports

_on

freezing

and

cryopreservation

of the mammalian heart ceased after 1977 and from this time almost all

reports

on

freezing

and

cryoprotection

of mammalian _organs have been devoted to the

kidney.

4.2 KIDNEY.

Presently,

the two main teams

working

_on rabbit

kidneys

_are those of Dr.

G.

Fahy

in U.S. and of Dr. D.E.

Pegg

in

England. Fahy

has been able to

perfuse

rabbit

kidneys

_up to the

huge cryoprotectant

concentration of vitrification solution VS4 of

49it (w Iv)

without

damage [20, 47].

^After

autc-transplantation,

the

kidneys

had excellent

long-term

life

support

function and

histology.

However

(Faliy, private communication)

the

kidneys

do not survive after

cooling

to -30 °C in this

solution, though

_no ice forms: there is _a cold shock.

Stoiancheva et al.

perfused dog kidneys

without

damage

_up to 30it

(w/v) 2,3-butanediol [48]. Jacobsen, Pegg

et al.

perfused

rabbit

kidney

with up ^to 3M

1,2-propanediol (m 23it

w/w)

without

damage: they

survived _a

long-time

^after

transplantation _[49]. However,

_even

on

cooling

to

only

-6

°C,

^the

kidneys

sulsered cold shock. In earlier studies in _a solution

containing

^also

albumin,

there _{was no} cold shock

[50]. Histological analysis recently performed

on an

autotransplantated

rabbit

kidney

has shown

a

satisfactory

maintenance of tubules for

kidneys

cooled down to -20 °C whereas blood flow _was not restored in

kidneys

cooled down tO -70 °C

[51].

5.

Expei~iments

iii _{Grenoble: first} results of the group.

5. I SURGICAL TECHNIQUES AND PRE-COOLING DOWN TO 4 °C. The

goal

of the

surgical

experimentation

_on rabbit

kidneys

is to

perform

_a contralateral

homograph.

After

harvesting

the

kidney

is cooled

by perfusion

down to 4 to 2 °C. A schematic

drawing

of the cooled

perfusion experimentation

on rabbit

kidney

is shown in

figure

8. The increase and decrease of the CP concentration in the

perfusion

solution _can be monitored

linearly

with time in _{a very}

simple

way,

only

with _a

peristaltic

_pump and

by applying

the

communicating

vessels

principle.

The coil of the heat

exchanger

is bonded to the _cap and is

easily

removed for sterilization. After about

twenty

atteiupts ^of contralateral

kidney hoiuografts,

the

surgical technique

is at

present satisfactory.

It _{uses a} small tubular

prostheses,

the _{salve as} those tried

by

the Rockeville _group

[52]

^{and also}

experimented

with _on rat livers

[53]. During

this

preliiiiinary phase

8

kidneys

_were

perfused

with _a solution

containing

dilserent concentrations of

2,3-butanediol (between 20it

and

30~)

and

prepared

for

histological analyses.

5

kidneys

exhibited

a well

preserved histological

structure which _{reassures us} in

trying 2,3-butanediol

_as CP in the future

experiments.

5.2 STUDY OF TOXICITY OF SOME CRYOPROTECTANTS ON RAT HEART We have _com-

pared

the toxicities of _{a new} CP

[21],

^of a CP

presently

used _on rabbit

kidney [44, 54]

and of two CP'S

previously

used _on the heart

[13].

^The

perfusion protocol

that _we have settled is described _on the

drawing figure

9. The

toxicity

is characterized

by

the values of the func-

~p ~

Cryoprotectant

~°~~~~°"

Pressure gauge

Kidney

2°C-4°C Peristaltic

Pump

Heat

exchanger

Freezing

_Thermost.

group bath

Fig.

8. Schematic

drawing

of the cooled

perfusion

experimentation _on ^rabbit kidney.

tional parameters

(coronary flow,

left ventricular

developed pressure),

metabolic

parameters (intramyocardial

concentrations of ATP and

glycogen)

and

histological

observations. As _an

example

_we show in

figure

10 the influence

on the left ventricular

developed

_pressure of the concentration ofvarious CP'S. All other observations show _a similar

high

chemical and osmotic

toxicity

for the concentrated solutions

(>

3

M).

We think that the chemical

toxicity

could be reduced

by adding

_a

protectant

for the cellular membrane

(sugars,

amino

acids)

and the _os- motic stress could be attenuated

by

_more

slowly varying

the CP concentration

(see Fig. 9)

and

by

the _use of _an

impermeant agent (mannitol)

to avoid intacellular cedema.

5.3 VITRIFICATION CONDITIONS. We have tested the

glass forming tendency

of solutions in which CP is not _a pure levo

2,3-butanediol

isomer. This _{was a} batch of Aldrich

[22]

which

contains _a

96.7it

racemic mixture of the levo and dextro

isomers,

and

only

3.

lit

of the _meso form. It is

cheap, _contrary

to the _pure isomers which _are about 100 times _more

expensive,

and its aqueous solutions have

almost,

^for the same water

content,

the _same

glass-forming tendency

and

stability

of the

amorphous

state _{as aqueous} solutions of

levo-2,3-butanediol _[21].

Our results _are shown in

figure

II: when Euro-Collins takes the

place

of _{water as}

solvent,

the critical

cooling

rates are divided

by

about

2,

_an

interesting

result for further

application

to the rabbit

kidney.

5.4 COOLING TECHNIQUE, it is obvious that the heat transfer cannot

easily

be

experi-

mentally

studied

directly

in real organs, due to

preservation problems,

and

stability

and _re-

producibility

of the

experimental

situation. We defined and realized solid

cylindrical

models

having

a low thermal

dilsusivity.

~fe have studied the influence _on the local

cooling

rate of _a network of

good

thermal conductors.

Figure

12 shows _our

preliirinary

results. It _may be _seen

intra0eritoneai Pentobarbltai

W'50

_{mg Kg-160dy Wt}

out

^.

^*'

~

(Heart

excised cooled (+4"C)

)

introduction and removal of the cr oprotective aoent

37 37

~~ ~

~~~~~°P~~9'~

2 _mn Coronary ^flow

arrest _and

0,llm/mn contractile activity

0,llm/mn Washoutj

30 mn lo

nls0

^mn

Measurement of coronary samples for

~~°~ ~~~ ~°~~~~~~"~ ~~~~~~~Y

Biochemistry

Elec~~$~$~~~~coPY

^{~~~~' ~~~~°~~~~}

Fig.

9.

Experimental protocol

for perfusion and removal of cryoprotective agents ^with the isolated rat heart.

that the network reduces

by

_a factor 2.7 the time _necessary to reach the final

temperature.

The maximum

cooling

rate is increased from 31

°C/mn

to 72

°C/mn.

These results have been obtained

by plunging

the models in

liquid nitrogen.

The

corresponding

thermal

gradients

cannot be

applied

without

damage

to real organs. Further

experiments

_are in progress in _an

industrial

apparatus

to freeze the cells

(NICOOL

PLUS from Air

Liquide)

whose

cooling

rate

can be monitored from 0

°C/mn

to 60

°C/mn.

6 Conclusion.

From the

analysis

of the results of the

experimental

and theoretical researches _on

cooling

of organs, it _appears that

cryopreservation might

be _a solution for

long

term

preservation only

when _one will be able to

vitrify

and

devitrify

_organs without

damage. Up

to _now vitrification

runs up

against

two main

problems:

the

toxicity

of CP above _a

given

concentration and the low

~ 70

X

E 60

E

]

50

b

a 40

30

~o

Glycdrol

. D&Ec i o ^. Propanediol

> Butanediol o

0 0.5 1.5 2 2.5 3 3.5

Concent. (more/Jitre)

Fig.

10. Infuence

on the left ventricular

developed

_pressure

(LVDP)

of the concentration of various cryoprotective agents.

q (%)

50

.

30 .

. 30%

~~

>

u EC30%

1o

o

io

300

+ 250

I

«

(

.

E 200 °

« .

~ .

iso

ioo

50

0

5

Time

[26] ^{RUBINSKY B. and CRAVALFIO}

E-G-, Cryobiology

21

(1984)

303.

[27] ^VALVANO

J-W-,

Low

Temperature Biotechnology,

J-J- Mc Grath and II-R- Diller Eds-

(The

American

Society

of Mechanical

Engineers,

N-Y.,

1988).

[28] ^RUBINSKY

B.,

The

Biophj,sics

of

Organ Preservation,

D-E-

Pegg

and A-M-Jr, Karow Eds.

(Plenum Press, N-Y-,

London

1987).

[29j

HARTMANN U-, NUNNER

B-,

I(bRBER Cit. and RAU

G., Cryobiology

28

(1991)

lls.

[30] ^{ECKERT E-R-J- and DRAI<E} R-M-Jr-,

Analysis

of heat and _mass transfer

(Mc

Graw Hill, N-Y-,

1972).

[31] ^RUBINSKY

B.,

Low Temperature

Biotechnology,

J-J- Mc Grath and K-R- Diller Eds.

(The

American Society of Mechanical

Engineers,

N-Y.,

1988).

[32] ^BOUTRON

P., Cryobiology

23

(1986)

88.

[33] ^REN H-S-, HUA T-C-, YU

G-X-,

CHEN X-H-,

Cryogenics

30

(1990)

536.

[34] ^SUTTON

R-L-,

J. Diem. Soc. Faraday Trans. 87.1

(1991)

101.

[35] SUTTON

R-L-, Cryo-Letters

11

(1990)

49.

[36j

FAHY G-M-, SAUR J- and WILLIAMS

R-J-, Cr_yobiology

27

(1990)

492.

[37j

^PEGG D-E-, GREEN C-J- and WALTER C-A-,

Cryobiolog_Y

15

(1978)

618.

[38] RUBINSI<Y

B., Cryobiology

24

(1987)

537.

[39] ^LUYET B.,

Cryobiology

8

(1971)

190.

[40] ^SMITH A-U-, Proc. Roy. Soc. Ser. 8147

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