Note: Descriptions are shown in the official language in which they were submitted.
WO 91/01992 PCI/GB90/01269
NUCLEATION OF ICE
The present invention relates to the nucleation of ice
using a novel form of a known substance which has been
unexpectedly ~ound to possess improved ice nucleating
properties. The invention covers methods of ice
nucleation in aqueous media, such as in the manufacture
o~ artificial snow, freeze drying processes, seeding
water droplets in the atmosphere to induce rain, snow
or hail or in the freezing of water-containig
foodstuffs. The invention also contemplates methods of
cryopreserving a biological sample, in particular to
minimise the effects of undercooling during
cryopreservation in order to alleviate or avoid damage
to the biological sample. In the latter respect the
invention may find particular use in the
cryopreservation of human embryos and embryos of other
animals.
The production of ice in the form of snow is
commercially importan~ in the skiing industry. Natural
snowfall is often erratic and the artificial production
of snow is employed to ensure suitable skiing
conditions. Thus heavily used ski runs may be
recovered and rendered usable by application of fresh
snow. Artificial ski slopes may be produced from snow
rather than fibre matting. The production of snow is
also potentially important for the insulation of crop
plants in extreme winter conditions. In addition, it
has been suggested that ice structures, such as
platforms, for exploratory drilling in the polar
regions could be constructed by spray freezing of sea
water.
Snow making machines usually produce an aerosol of
WO 91/01992 PCI/GB90/01269
~ 8
water into the air which then freezes naturally.
However, a major problem with this is that ice
nucleation in small water droplets is a very
inefficient process; whilst the melting point of ice is
0C small droplets may undercool to temperatures as low
as -40C before freezing. Thus at high ambient
temperatures such as about -5C snow making machines
are not effective, whilst at lower temperatures the
process is inefficient.
Currently the addition of bacterium Pseudomonas
syrinaae (produced by Eastman Kodak under the trade
name Snowmax) is used in some circumstances to initiate
ice formation. This organism has a surface structure
similar to the ice crystal lattice and can be an
efficient ice nucleator. When added to a snow making
machine it can increase output by up to 150~. However,
there are disadvantages to its use:
a) it is an expensive product ~at the time of
filing this could be as much as 411 per kilo);
and
b) secondly, the release of bacteria, albeit a
naturally occuring micro-organism which has been
processed to be non-viable, into the environment
has serious legislative and environmental
problems.
Thus there exists a need for improved water nucleation
techniques, particularly in snow or ice particle
production processes.
WO91/01992 PCT/GB90/01269
2r~
Ice nucleators have been known for several years,
either as physico-chemical curiosities or in meteoro-
logical applications as initiators of rainfall. For
cloud seeding meteorological purposes, inorganic
substances such as silver iodide have probably been
studied the most, although Head discussed the use of
steroids as ice nucleators in cloud seeding operations
some 25 years ago (Head, Nature 1058 (9th September
1961) and J. Phvs. Chem. Solids 23 (1962) 1371). In
these studies it was proposed that nucleation of ice
would act as a catalyst for cloud formation, which
would result in rainfall: the production of ice or
snow, other than as incidental to cloud seeding
operations was not proposed. Fukuta and Mason studied
the epitaxial growth of ice on organic crystals (J.
Phvs. Chem. Solids 24 (1963) 715), but did not suggest
any useful application for their observations. It
should be pointed out that nucleation of ice by organic
com~unds represents a very small proportion of the
work carried out on ice nucleation for meteorological
purposes. For example, the textbook "Ice Physics" by
Hobbs (Clarendon Press, Oxford, 1974), which is almost
1000 pages long has only a page or two on organic ice
nucleators; in all of these studies, ice nucleation was
examined on a microscope cold stage, not in bulk or
dispersed fluids.
The present invention also has application in the
sphere of biological sa~ple cryopreservation. The
storage of biological samples, such as cells, by
cryopreservation (freezing) is a common method of
maintaining the viability of cells for long periods of
time. However, a major problem with cryopreservation
WO91/Ot992 PCT/GB90/01269
Z~ 4
is that during cooling of the biological sample the
surrounding medium tends to supercool before ice
nucleation occurs below the freezing point of the
surrounding medium. (This is also known as
undercooling.~ This undercooling causes damage to the
biological sample and excessive undercooling can
prevent survival of embryos, as shall be explained.
As ice forms in a surrounding (aqueous) medium the
concentration of any solutes in the remaining liquid
medium increases. By osmotic pressure the cells will
thus dehydrate by water moving to the more concentrated
medium. If the cells have insufficient time to
dehydrate then intracellular ice may form, which is
generally lethal to the cell.
Cryoprotectants ~such as DM50 or glycerol) and other
additives (such as salts) which depress the freezing
point are often added to cryopreservation media.
However, in the case of embryos survival depends on
the cells of the embryo being shrunk by dehydration
before freezing occurs.
There is a general need to prevent or minimise the
effects of undercooling; these aims may be achieved by
introducing ice nucleators into (or in contact with)
the medium. For example, EP-A-0246824 teaches that a
wide range of solid materials, when in contact with the
medium, can cause water in the aqueous medium to be
nucleated at, or close to, the freezing point of the
medium.
Special care has to be taken with certain biological
WO 91/01992 PCltGB90/01269
Z ~
S
samples, such as embryos, where the degree of
undercooling in the surrounding medium can be critical
to the survival of the sample. There is thus still a
desire for reducing the amount of undercooling in the
medium. The present applicants have discovered, quite
unexpectedly, that one particular organic solid, when
specially treated, can reduce undercooling even further
to a l~vel where undercooling temperatures are within
the important range of only a few degrees less than the
freezing point of the medium, allowing a reduction in
the degree of undercooling and concomitantly reduced
damage to the sample.
Furthermore, the present invention improves over the
prior art by several degrees the amount of undercooling
during cryopreservation, which is significant in cases
where the difference of a few degrees in undercooling
can determine whether or not an embryo survives.
Thus according to a first aspect of the present
invention there is provided cholesterol having a
crystal structure obtainable by crystallising
cholesterol from methanol or acetic acid. Cholesterol
crystallised from methanol is the cholesterol of
choice.
The Applicants have found that by crystallising (which
term is used to include recrystallising) cholesterol
from acetic acid or methanol the undercooling effect
can be reduced in comparison with crystallisation from
other solvents, such as ethanol. Thus the cholesterol
of the present invention has been found to be a
particularly effective ice nucleator, that is to say
W091/01~92 PCT/GB90/Ot269
Z~
the cholesterol is effective at inducing ice nucleation
(ice formation). The invention, in its broadest terms,
thus contemplates the use of cholesterol obtainable by
crystallisation from methanol or acetic acid as an ice
nucleating agent.
The cholesterol of the present invention is suitably
prepared by forming a solution of cholesterol in
methanol or acetic acid, and then evaporating the
methanol or acetic acid, thereby yielding the
cholesterol crystals. Such evaporation is preferably
rapid. The methanol or acetic acid is preferably
reagent or even analytical grade, so that it is
substantially (if not completely) free of water;
optimum results are obtained if the cholesterol is not
contacted with water during the preparation process.
Thus a second aspect of the present invention relates
to an ice nucleating composition comprising cholesterol
having a crystal structure obtainable by crystallising
cholesterol from methanol or acetic acid. The
composition may be the cholesterol alone, or may be
provided on an inert carrier (as shall be decribed
later) although in a preferred embodiment the
composition is an aqueous medium. The aqueous medium
may he water (either tap or distilled), but may contain
additional soluble components at a concentration range
of from almost zero concentration (infinite dilution)
up to a concentration where there is still free water
available to be frozen. Thus the composition may
contain one or more salts or electrolytes (such as
WO91/01992 PCT/GB90/01269
NaCl) to maintain a physiological balance; a sugar such
as sucrose; or a cryoprotectant, for example dimethyl
sulphoxide (DMS0) and/or glycerol. If salts or
electrolytes are employed, then these may be present at
a concentration of from O.lM to lO.OM, such as 0.4M to
0.5M, preferably about 0.5M. In the case of sugars the
concentration may be from 1 to 50%w/v, such as from 5
to 15%W/V, preferably about 10%w/v. Where a
cryoprotectant is present, the concentration is
suitably from 1 to 60% v/v, such as from 5 to 15% v/v
and optimally about 10% v/v.
The concentration of the cholesterol in the medium will
vary depending upon the presence of any other
substances in the medium, but can be as low as from
0.0001 to 0.001 g/ml ~or g/cc if the medium is a solid)
fcr the cholesterol to have a significant effect on the
prevention of undercoolin~. However, in practice the
medium preferably contains from 0.25 to 1.5 mg/ml of
the cholesterol, optimally from 0.75 to 1.25 mg/ml.
Although the invention is not limited to what is
believed to be the reason for the cholesterol's
unexpected improved ice nucleating properties, the
Applicants believe that it is due to the crystalline
structure of cholesterol when obtainable on
crystallisation from methanol or acetic acid.
It will be seen from Figs. 1 and 2 (both prior art)
that commercially supplied cholesterol crystals are
typically both small, compact and spherulite in
appearance. However, when cholesterol is crystallised
from either acetic acid or methanol, in accordance with
W091/Ot992 PCT/GB90/Ot269
the present invention (see for example Figs. 3 and 4)
the ~rystal str~cture changes significantly, and the
crystals are needle-like in appearance. This marked
difference in crystal structure is believed to be
responsible for reducing the undercooling during
cryopreservation by inducing ice nucleation at a higher
temperature.
A third aspect of the present invention relates to a
method of inducing ice nucleation in an aqueous medium,
the method comprising cooling the aqueous medium while
in contact with cholesterol having a crystal structure
obtainable by crystallising cholesterol from methanol
or acetic acid.
Here the term "aqueous medium" means any substance or
material that contains water in which it is desired to
induce ice nucleation. This not only includes liquids
but also gases that contain water and even water-
containing solids. In addition, the term "aqueous
medium" is to be interpreted as covering not ony
continuous (one phase) media but also two phase media
such as liquid-solid, liquid-liquid (for example
emulsions) or gas-liquid, for example a gas (eg air)
with the aqueous medium in the form of a dispersion or
suspension (eg an aerosol). The term also covers three
phase gas-liquid-solid systems. Indeed, the method of
the present invention may additionally comprise forming
an aerosol of the aqueous medium, for example by
spraying the aqueous medium, such as into the
atmosphere.
W091/Ot992 PCT/GB90/01269
7~3
.. . . ~
In one embodiment the aqueous medium is preferably a
food or a foodstuff that is desired to be frozen.
The foodstuff may be solid or semi-solid and may have
a considerable number of components other than water.
Contemplated foodstuffs include ice lollies, sorbet,
yoghurt, diary products such as cream and ice cream,
fruit, foodstuffs that are often sold frozen such as
meat and seafood (eg fish) products, as well as
suitable combinations of these (and other edible
foodstuffs as appropriate) subject to taste, for
example cake and gateau (which often inlude cream and
fruit). Although cholesterol is sometimes not a
desirable component of food, very little of the
cholesterol of the present invention will be required
in order to assist ice nucleation, as will be described
latèr. In any case the amount of cholesterol of the
present invention will be negligible in comparison with
th~ cholesterol content of some foods (eg gateau).
The cholesterol of the present invention can be added
to the foodstuff at any suitable time but will
generally be during the preparation process. Thus the
invention may allow one to influence customer
acceptability, storage properties and/or process time
of the foodstuff by being able to exert a degree of
control over the freezing process. One particular
benefit envisaged is to reduce the freezing process
time.
The cooling of the aqueous medium may be achieved by
use of specific cooling apparatus such as a passive
cooler (such as that describéd in the International
WO91/01992 PCT/GB9n/Ot269
~r~
Patent Application filed on 7th August, 1990 in the
name of Cell Systems Ltd) , a freezer (preferably
programmable, for example available from Planer
Products, Sunbury on Thames, Middx) or through exposure
of the aqueous medium to cooling environmental
conditions (for example exposing the aqueous medium to
air or an atmosphere which is at a temperature less
than the melting point of the aqueous medium).
It should be realised that almost any aqueous medium
will undercool before freezing albeit to a greater or
lesser extent, and that the degree of undercooling is
dependent upon the nature of the aqueous medium. In
particular substances that are soluble (such as
solutes) have a considerable influence on the degree of
u~dercooling, as do other ~actors such as the volume of
the aqueous medium being cooled and the rate of cooling
of the aqueous medium.
The nature of the aqueous media with which the
cholesterol of the present invention can be used is
both wide and varied, provided that the use of
cholesterol as a chemical additive is not, for some
reason, precluded.
For example, the aqueous medium may simply be water,
where one wishes to induce ice nucleation to make snow
or ice. This aspect of the invention is discussed
later.
In contrast, the aqueous medium may contain liquid
and/or solid waste, toxic, hazardous and/or noxious
WO91/01992 PCT/GB90/01269
11
substances and materials, eg. radioactive materials, so
that solidification of the aqueous medium using ice
nucleation may stabilise the aqueous medium (or
substantially solid medium as it may then be) so that
it is more suitable for further treatment or for
storage purposes.
Thus the invention finds particular use in freeze
stabilisation processes where it is desired to produce
a solid (albeit a frozen one) from an aqueous medium.
The resulting substance which will usually be a solid
(although it may be an, e.g. two-phase, combination of
both solid ice and aqueous medium, for example having a
slushy consistency) may thus be rendered easier to
handle or transport.
~hus a fourth aspect o~ the present invention relates
to a substance, comprising ice, where the ice has been
formed by process according to the third aspect. Thus
the substance will generally comprise the aqueous
medium although it will be appreciated that in some
situations all, and not just some, of the aqueous
medium will be frozen into a solid.
The term "aqueous medium" is preferably as discussed in
the third aspect mutatis mutandis. - It is not to be
interpreted as being limited to a single phase,
although often it will be and various soluble
substances may be present; indeed, the present
invention specifically contemplates the presence of
solids (that will usually be water-insoluble) in the
WO91/01992 PCT/GB90/01269
7~3
agueous medium, and in particular a biological sample,
such as in a method of cryopreserving a biological
sample. The substance may also be a foodstuff as
previously described.
Thus in a particularly preferred (fifth) aspect of the
present invention there is provided a method of
cryopreserving a biological sample, the method
comprising cooling the sample in an aqueous medium in
contact with cholesterol having a crystal structure
obtainable by crystallising cholesterol from methanol
or acetic acid. The aqueous medium is preferably as
described for the second aspect mutatis mutandis.
The term "biological sample" includes cells (both
eukaryotic and prokaryotic), organs and tissue composed
of cells, embryos, viruses, all of which can be natural
or modified genetically or otherwise, and biologically
active molecules such as nucleic acids, proteins,
glycoproteins, lipids and lipoproteins.
Commercially available cholesterol (e.g. analytical or
reagent grade) may be obtained from various sources,
for example from Sigma Chemicals. This will generally
be suitable for recrystallisation from methanol or
acetic acid for use in the present invention.
- The amount of cholesterol employed to assist ice
nucleation need only be small, for example about 0.02mg
(2 x 10. 5g).
The cholesterol may be brought into contact with the
medium (or the sample itself) in the form of discrete
WO91/01992 PCT/GB90/01269
7~3
13
crystals or on an inert carrier.
A preferred cooling protocol for the biological sample
includes an isothermal hold and/or plunging into liquid
nitrogen. Suitably cooling is at a rate of from 0.3 to
2.0C/minute, such as from 0.5 to 1.5C/minute.
Preferably the sample is cooled to between -15 to
-40C before plunging into liquid nitrogen.
Therefore, according to a sixth aspect of the present
invention there is provided a substrate at least
partially coated with cholesterol having a crystal
structure obtainable by crystallising cholesterol from
methanol or acetic acid. Such a coated substrate may
be used in a method of cryopreserving a biological
sample in an aqueous medium in accordance with the
fifth aspect.
Prefera~ly the substrate is an inner surface of
suitable cryopreservation vessel such as an ampoule,
tube, straw or bag, or a particular carrier, for
example glass, polymer or other beads. Suitably the
substrate is made of a biologically inert material such
as glass or an appropria~e polymer, which may be a
plastics material (such as polypropylene or polyvinyl
chloride) or an acrylic polymer. Modified dextran
beads are a particularly appropriate substrate, which
are available from Pharmacia under the trade mark
CYTODEX. Also preferred are acrylic polymer beads
which are available from Bio-Rad as Bio-Beads SM7.
These beads, suitably coated, can simply be added to
the cryopreservation medium (such as according to the
second aspect of the present invention).
WO91/01~2 PCT/GB90/01269
~ J`~ ~ a 14
The coating density of cholesterol on the substrate
will usually be at least 0.0007 mg/mm2, up to a limit
determined by practical convenience. Coating densities
are suitably at least 0.001 mg/mm2, and preferably
below 0.1 mg/mm2, with an optimum of about 0.0035
mg/mm2. Other preferred features of the sixth aspect
of the invention are as for the previous aspects,
mutatis mutandis.
A seventh aspect of the present invention relates to a
process for the preparation of a substrate of the sixth
aspect, the process comprising contacting a substrate
with a solution of cholesterol in methanol or acetic
acid, and evaporating the methanol or acetic acid. The
solution preferable contains at least 0.2%, preferably
0.4%, ~f cholesterol. ~ther preferred characteristics
of the seventh aspect are as for the sixth aspect
mu~atis mutandis.
An eighth aspect of the present invention relates to a
method of producing rain or snow or ice particles in
the atmosphere, (such as hail, snow or ice), the method
comprising contacting particles of an aqueous medium in
the atmosphere with cholesterol having a crystal
structure obtainable by crystallising cholesterol from
methanol or acetic acid to cause water in the aqueous
particles to be nucleated to form snow or ice, the
aqueous particles and/or the atmosphere being at a
temperature of less than the freezing point of aqueous
particles. Rain will generally result from melting of
the snow or ice particles (if this occurs).
WO91/01992 PCT/GB90/01269
The aqueous particles will usually be water droplets,
for example those found in the stratosphere, such as in
clouds. The cholesterol thus may act as an ice
nucleator in order to "seed" clouds to induce snow or
hail. The cholesterol will generally be contacted with
the aqueous particles by using an aircraft, such as an
aeroplane or a (meteorological) balloon. This may be
achieved by release of the cholesterol (for example
under gravity) or by spraying.
The method may additionally comprise generating the
particles of the aqueous medium in the atmosphere.
Thus in a preferred method the present invention in a
ninth aspect relates to a method of producing snow or
ice particles, the method comprising spraying an
aqueous medium into the atmosphere, either the medium
or atmosphere or both being at a temperature of less
than the melting point o~ the medium, and dispersing in
the medium cholesterol having a crystal structure
obtainable by crystallising cholesterol from methanol
or acetic acid, to cause water in the aqueous medium to
be nucleated to form snow or ice particles.
Usually the aqueous medium will be, for the sake of
; convenience, water. It will be appreciated that the
cholesterol may be dispersed in the medium either
before, at the same time as or after the medium is
sprayed into the atmosphere. In the case of dispersing
the cho}esterol into the medium before spraying, the
cholesterol may be dispersed into a reservoir of the
medium to be sprayed. In the case of dispersing the
cholesterol into the medium at the time of spraying, a
double nozzle arrangement may be used to cause the
W091/01992 PCT/GB90/Ot269
2r~ 3 16
cholesterol to be dispersed into the nascent medium
droplets. In the case of the cholesterol being
dispersed in the medium after spraying, a spray of the
cholesterol may be caused to pass through an area of
small particles of the medium (such as droplets), which
can then become dispersed in the particles.
It will be understood that if water is used, it need
not be completely pure. Indeed, an element of impurity
may be preferred. For example, a certain amount of
solute present in the medium (such as water) may aid
nucleation. However, it is generally preferred that
the amount of solute present, if any, be kept below an
excessive amount, so as not to cause too severe a
depression of the freezing point of the medium.
The medium may be sprayed as a very fine spray, that is
to say, an aerosol. Such a spray may be generated by
means of a compressor, which for convenience will
compress air. ~owever, it may be possible to produce a
suitable aerosol using a gas.vapourising from a liquid,
for example a liquified atmospheric gas such as
nitrogen. The use of freons will for preference be
avoided because of the environmental impact.
Mechanical methods of generating aerosols may also be
employed.
A tenth aspect of the inYention relates to an apparatus
suitable for producing snow or ice particles, the
apparatus comprising means for spraying an aqueous
medium into the atmosphere and means for dispersing in
the medium cholesterol having a crystal structure
obtainable by crystallising cholesterol from methanol
WO91/01992 PCT/GB90/01269
1~
or acetic acid which causes water in the medium to be
nucleated to form snow or ice particles.
According to an eleventh aspect of the invention, there
is provided (artificial) snow or ice particles whenever
produced by a method of the ninth aspect and/or by an
apparatus of the tenth aspect.
It will be appreciated that preferred features and
characteristics of one aspect of the present invention
are as for another, mutatis mutandis.
T~e invention will now be described by way of example,
with reference to the accompanying drawings, in which:
Figure 1 ~prior art) is a view of cholesterol under a
microscope (x105), obtained commercially from Sigma
Chemicals;
Figure 2 (prior art) is a view of cholesterol under a
microscope (x105) obtained from Sigma Chemicals and
crystallised from ethanol;
Figure 3 is a view of cholesterol under a microscope
(x105) in accordance with the present invention
crystallised from acetic acid:
Figure 4 is a view of cholesterol under a microscope
(x105) in accordance with the present invention
crystallised from methanol: and
Figures 5 and 6 are plots of temperature against time
WO 91/01992 PCI'/GB90/01269
Z~s ~ 18
showing nucleation profiles during cooling of sucrose
and saline solut ions respectively using
cryopreservation techniques both known in the art and
according to the present invention.
COMPARATIVE EXAMPLE 1
Samples of cholesterol (Sigma Chemicals) were
separately dissolved in methanol, ethanol, and ethanoic
acid at 0.62Sg in lOOml. The solutions were then
warmed in a dust-free atmosphere at 70C to dryness.
The crystals from each of these treatments were
mechanically removed from their containers and stored
under anhydrous conditions at room temperature. Four
plastic universal tubes were used as experimental
ves~els and to each was added 20ml of lO~w/v sucrose.
Three of the tubes then had 0.2g of one o~ the three
cholesterol preparations added to it. A type T
~hermocouple was attached to a computerised datalogger
and placed at the internal midpoint of each tube which
was sealed. All the tubes were cooled in a domestic
freezer and cooling rate, temperature of exotherm
formation and latent heat of fusion were recorded
(Fig.5). In Figure 5 the key to the nucleation profiles
are:
1 represents a control with no cholesterol;
2 represents cholesterol crystallised from ethanol;
3 represents cholesterol crystallised from acetic
acid; and
4 represents cholesterol crystallised from methanol.
WO91/01992 PCT/G890/01269
;~a~. 5 ~
The data clearly show that the extent of the
undercooling is reduced significantly in the presence
of cholesterol crystallised from methanol and that
improvement over the control and cholesterol
crystallised ~rom ethanol was also achieved with
cholesterol crystallised from acetic acid.
COMPARATIVE EXAMPLE 2
Samples of cholesterol (Sigma Chemicals) were
separately dissolved in ethanol, methanol, acetic acid
and ether at O.625g in l00ml. Five cryotubes (Nunc
Ltd) were placed in a water bath at 70C in a dust-free
atmosphere and O.lml of an appropriate cholesterol
solution added to each of them. The tubes were held in
the bath until the contained solvent had completely
e~aporàted. A sample of 0.5ml of 0.5M NaCl was added
to sach tube and a type T thermocouple lnserted,
connected to a datalogger. Cooling and nucleation
pro~iles were recorded as in comparative Example l (see
Fig.6)
In Figure 6 the nucleation profiles represent
cholesterol crystallised from the following solvents:
l methanol;
2 acetic acid;
3 ethanol;
4 none (uncoated cryotubes, control, with no
cholesterol); and
s ether.
The horizontal broken line labelled "A" represents the
W091/01992 PCT/GB90/Ot269
7~3
melting point of 0.5M NaCl (-1.7C).
The data clearly show that cholesterol crystallised
from methanol or acetic acid is more efficient in
nucleating the cooling sample than cholesterol derived
from ethanol. Cholesterol crystallised from ether
appears inactive.
COMPARATI~E EXAMPLE 3
Five different agueous solutions were cooled at
diierent cooling rates in cryopreservation tubes or
straws until they froze. The cooling experiments were
divided into two sets; in the first set each solution
had added to it cholesterol crystallised from methanol
~as prepared in Comparative Example 2) to a
concentration o~ lmg/ml. The second set (controls) were
conducted without any cholesterol present. The results
are shown in Table 2.
WO91/01~2 PCT/GB~/01269
21
TABLE 2
No. of
tubes
Solution frozen Cooling or Mean
& solution rate straws nucleation
volume (Cmin~1) cooled pointC(+sd)
Cholesterol
crystallised
from MeOH Control
10% v/v Dimethyl
sulphoxide in
distilled water
(0.Sml in straws) 1.025 -4.6+0.5 -8.7+2.8
10% v/v Dimethyl
sulphoxide in
distilled water
(0.5ml in straws) 10.0 25 -5.3+0.7 -11.3+1.4
10% v/v glycerol
in bovine embryo
culture medium
~0.Sml in straws) 0.323 -4.9+0.64 -7.8+1.77
Bovine sperm
diluent (Milk
Marketing Board)
(0.25ml in straws) 43.3 24-4.0+0.40-8.1+2.64
W091/Ot992 PCT/GB90/01269
Z 22
TABLE 2 (contd.)
Distilled water
(0.5ml in straws) 1.0 12 -2.2+0.8 -9.2+2.1
0.5M DMSO + 0.5M
glycerol + l.OM
sucrose
(lml in 2ml uials) 1.0 12 -9.8+0.28 -17.5+3.63
This example demonstrates two advantages of using
cholesterol crystallised from methanol. The first is
that the nucleation temperature is significantly closer
to the theoretical value, resulting in a reduction of
undercooling. The second is that the standard deviation
i5 considerably reduced by the use of the cholesterol,
giving a much greater reproducibility between samples.
AMPLE 1
Cryopreservation of bacterial cells.
Five different species of bacteria were harvested from
culture slopes in lOml of nutrient broth with 10~ v/v
glycerol and the resulting suspended bacterial
population was measured into lml aliquots in
polypropylene cryotubes (2ml). Cholesterol crystallised
from methanol (about 0.02mg) was added to each aliquot
to induce ice nucleation.
The tubes were transferred either to a conventional
programmable freezer (Planer Products, Sunbury on
WO9t/01992 PCT/GB90/01269
23
Thames, Middx) or to a passive freezing device (Cell
Systems, Cambridge) and cooled at -1C per minute to
-70C, when the tubes were removed and plunged into
liquid nitrogen. Sample temperatures were monitored
using a Type T thermocouple/electronic thermometer
combination with the probe immersed in the sample.
The tubes were thawed by immersion in water at 25C and
the samples spirally-plated onto nutrient broth to
provide a viable cell count.
Bacterium % viable cells (mean of
duplicate cultures)
Planer freezer Passive freezer
Escherichia_coli 82.45 82.70
Sta~hvlococcus aureus 80.70 81.45
Neis~eria menlnitidis 63.85 59.45
Haemo~hilus influenzae 59.50 70.65
~ibrio cholerae 75.70 72.45
This example demonstrated the successful
cryopreservation of certain bacteria using cholesterol
crystallised from methanol.
EXAMPLE 2
Cryopreservation of bovine embryos.
Bovine embryos at the 4-cell stage of development were
incubated in ovum culture medium with 10% v/v glycerol
and then loaded individually into ten 0.25ml plastic
straws. Cholesterol crystallised from methanol was
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24
incorporated into 5 straws to a concentration of about
O.Smg/ml which were cooled in the passive freezer as
used in Example 1, configured to provide a cooling rate
of -0.3C per minute to -35C, before plunging the
straws into liquid nitrogen. ~he remaining 5 straws
wère cooled in a Planer R206 controlled rate freezer
and seeded manually at -6C.
The cooling profile for the machine was:
1. cool Q S.0 C per minute from 20 to -5 C;
2. cool Q 0.2 C pPr minute from -5 to -6 ;
3. seed during the second stage;
4. cool ~ 0.5 C per minute from -6 to -32 C;
5. then plunge into liquid nitrogen.
The frozen embryos were thawed by immersion of the
straws in water at 30C, rinsed in several washes of
culture medium with decreasing concentrations of
cryoprotectant and incubated in culture medium
overnight.
Of the 5 embryos frozen in the passive freezer 4 were
in excellent condition after culture and the fifth was
still of an acceptable quality for transplanting. The
embryos cooled in the Planer freezer were scored as 3
excellent and 2 still viable but not acceptable for
transplanting.
EXAMP~E 3
Cryopreservation of mammalian cell lines.
Three types of cultured mammalian cells were suspended
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2s
in 91%F8S culture medium with 10% v/v DMSO and
cholesterol crystallised from methanol placed in 2.5ml
plastic ampoules and then frozen in the passive freezer
as used in Example 1 configured to cool at -1.5 C per
minute. The cells were removed from the freezer when
the samples had reached -18C and were plunged directly
into liquid nitrogen for a minimum storage period of 24
hours. Recovered cells were cultured in vitro and
viable cell counts taken, based on the mean of 2
ampoules.
cell line % viability
NRx-4sF 97
Rat fibroblast
COS-7 98
Monkey kidney cells
3T3-Li 95
Mouse fibroblast
This example demonstrates that certain mammalian cell
lines may be successfully cryopreserved using
cholesterol crystallised from ~ethanol.
