EP0486585A1

EP0486585A1 - Nucleation of ice

Info

Publication number: EP0486585A1
Application number: EP90912547A
Authority: EP
Inventors: Brian William Wilson 1 Bethel Cottage Grout
Original assignee: CELL SYSTEMS Ltd
Current assignee: CELL SYSTEMS Ltd
Priority date: 1989-08-11
Filing date: 1990-08-13
Publication date: 1992-05-27
Also published as: JPH05502664A; WO1991001992A1; AU654112B2; AU6179290A; CA2064708A1; GB8918370D0

Abstract

Le surrefroidissement d'un milieu aqueux (et donc les dommages à un échantillon biologique qui se trouve dans le milieu) peut être minimisé en provoquant une nucléation de glace dans le milieu au moyen de cholestérol possédant une structure cristalline pouvant être obtenue par la cristallisation à partir du méthanol et/ou de l'acide acétique. Ce cholestérol cristallin peut être ajouté au milieu de cryoconservation (qui peut également contenir un cryoprotecteur) ou enduit sur une surface intérieure d'une ampoule, d'un tube de cryoconservation ou d'une paille. Le cholestérol peut également être utilisé pour provoquer une nucléation de glace dans l'atmosphère afin de créer de la grêle ou de la neige, ou bien de la neige artificielle dans des bombes aérosols contenant le milieu aqueux et destinées à l'utilisation sur les pistes de ski.Supercooling of an aqueous medium (and therefore damage to a biological sample in the medium) can be minimized by inducing ice nucleation in the medium using cholesterol having a crystal structure obtainable by crystallization at from methanol and/or acetic acid. This crystalline cholesterol can be added to the cryopreservation medium (which may also contain a cryoprotectant) or coated on an interior surface of an ampule, cryopreservation tube or straw. Cholesterol can also be used to cause ice nucleation in the atmosphere to create hail or snow, or artificial snow in aerosol cans containing the aqueous medium and intended for use on runways ski.

Description

NUCLEATION OF ICE

The present invention relates to the nucleation of ice using a novel form of a known substance which has been unexpectedly found to possess improved ice nucleating properties. The invention covers methods of ice nucleation in aqueous media, such as in the manufacture of 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 important 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 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 0°C small droplets may undercool to temperatures as low as -40°C before freezing. Thus at high ambient temperatures such as about -5°C snow making machines are not effective, whilst at lower temperatures the process is inefficient.

Currently the addition of bacterium Pseudomonas syrinσae (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. 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. Che . 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. Phγs. 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 compounds 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 sample 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 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 DMSO 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 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 level 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 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 be 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 NaCl) to maintain a physiological balance; a sugar such as sucrose; or a cryoprotectant, for example dimethyl sulphoxide (DMSO) and/or glycerol. If salts or electrolytes are employed, then these may be present at a concentration of from 0.1M to 10.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) for the cholesterol to have a significant effect on the prevention of undercooling. 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 the present invention (see for example Figs. 3 and 4) the crystal structure 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. 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 later. In any case the amount of cholesterol of the present invention will be negligible in comparison with the 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 described in the International 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 undercooling, as do other factors 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 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.

Thus a fourth aspect of 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 aqueous 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 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 2.0°c/minute, such as from 0.5° to 1.5°C/minute. Preferably the sample is cooled to between -15° to -40°C 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.

Preferably 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 appropriate 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) . 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%, of cholesterol. Other preferred characteristics of the seventh aspect are as for the sixth aspect mutatis 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) . 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 of 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 cholesterol 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 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. However, 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 invention 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 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.

The 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 (xl05) , obtained commercially from Sigma Chemicals;

Figure 2 (prior art) is a view of cholesterol under a microscope (xl05) obtained from Sigma Chemicals and crystallised from ethanol;

Figure 3 is a view of cholesterol under a microscope (xl05) in accordance with the present invention crystallised from acetic acid;

Figure 4 is a view of cholesterol under a microscope (xl05) in accordance with the present invention crystallised from methanol; and

Figures 5 and 6 are plots of temperature against time showing nucleation profiles during cooling of sucrose and saline solutions 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 0.6?§g in 100ml. The solutions were then warmed in a dust-free atmosphere at 70°C 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 vessels and to each was added 20ml of 10%w/v sucrose. Three of the tubes then had 0.2g of one of the three cholesterol preparations added to it. A type T thermocouple 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. 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 from 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 0.625g in 100ml. Five cryotubes (Nunc Ltd) were placed in a water bath at 70°C in a dust-free atmosphere and 0.1ml of an appropriate cholesterol solution added to each of them. The tubes were held in the bath until the contained solvent had completely evaporated. A sample of 0.5ml of 0.5M NaCl was added to each tube and a type T thermocouple inserted, connected to a datalogger. Cooling and nucleation profiles were recorded as in comparative Example 1 (see Fig.6)

In Figure 6 the nucleation profiles represent cholesterol crystallised from the following solvents:

1 methanol ;

2 acetic acid;

3 ethanol ;

4 none (uncoated cryotubes, control, with no cholesterol) ; and

5 ether.

The horizontal broken line labelled "A" represents the melting point of 0.5M NaCl (-1.7°C) .

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.

COMPARATIVE EXAMPLE 3

Five different aqueous 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 of lmg/ml. The second set (controls) were conducted without any cholesterol present. The results are shown in Table 2.

TABLE 2

No. of tubes

Solution frozen Cooling or Mean & solution rate straws nucleation volume (°Cmin^""1) cooled point^ ±sd)

Cholesterol crystallised from MeOH Control

10% v/v Dimethyl sulphoxide in distilled water (0.5ml in straws) 1.0 25 -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 (0.5ml in straws) 0.3 23 -4.9io.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 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 + 1.0M sucrose

(lml in 2ml vials) 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 is considerably reduced by the use of the cholesterol, giving a much greater reproducibility between samples.

EXAMPLE 1

Cryopreservation of bacterial cells.

Five different species of bacteria were harvested from culture slopes in 10ml 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 Thames, Middx) or to a passive freezing device (Cell Systems, Cambridge) and cooled at -1°C per minute to -70°C, 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 25°C 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

Staphylococcus aureus 80.70 81.45

Neisseria meninqitidis 63.85 59.45

Haemophilus influenzae 59.50 70.65

Vibrio 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 incorporated into 5 straws to a concentration of about 0.5mg/ml which were cooled in the passive freezer as used in Example 1, configured to provide a cooling rate of -0.3°C per minute to -35°C, before plunging the straws into liquid nitrogen. The remaining 5 straws were cooled in a Planer R206 controlled rate freezer and seeded manually at -6°C.

The cooling profile for the machine was:

1. cool @ 5.0 °C per minute from 20 to -5 °C;

2. cool § 0.2 °C per 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 30°C, 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 transp1anting.

EXAMPLE 3

Cryopreservation of mammalian cell lines.

Three types of cultured mammalian cells were suspended in 91%FBS 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 -18°C 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

NRK-49F 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 methanol.

Claims

1. Cholesterol having a crystal structure obtainable by crystallising cholesterol from methanol or acetic acid.

2. An ice nucleating composition comprising cholesterol having a crystal structure obtainable by crystalling cholesterol from methanol or acetic acid.

3. A composition as claimed in claim 2 which is an aqueous medium.

4. A composition as claimed in claim 3 containing a cryoprotectant.

5. A composition as claimed in claim 4 containing glycerol or dimethyl sulphoxide.

6. 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 crystalling cholesterol from methanol or acetic acid.

7. A substance comprising ice wherein the ice has been formed by a method as claimed claim 6.

8. A substrate at least partially coated with cholesterol having a crystal structure obtainable by crystallising cholesterol from methanol or acetic acid.

9. A substrate as claimed in claim 8 which is an ampoule, tube, bag, straw or glass or polymer beads.

10. A substrate as claimed in claim 8 wherin the coating density of cholesterol is at least 0.0007 mg/mm².

11. A process for the preparation of a substrate at least partially coated with cholesterol having a crystal structure obtainable by crystallising cholesterol from methanol or acetic acid, the process comprising contacting a substrate with a solution of cholesterol in methanol or acetic acid and evaporating the methanol or acetic acid.

12. A method of cryopreserving a biological sample, the method comprising cooling the sample in an aqeous medium in contact with cholesterol crystallised from methanol or acetic acid.

13. A method as claimed in claim 12 wherein the biological sample is a cell, or organ or tissue composed of cells, an embryo, virus, all of which may be natural or modified genetically or otherwise, a biologically active molecule, nucleic acid, protein, glycoprotein, lipid or lipoprotein.

14. A method of producing rain or snow or ice particles in the atmosphere, the method comprising contacting particles of an aqueous medium in the atmosphere with cholesterol having a crystal structure obtainable from 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 the aqueous particles.

15. 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 freezing point of 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 from snow or ice particles.

16. 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 or acetic acid which causes water in the medium to be nucleated to form snow or ice particles.

17. Snow or ice particles when produced by a method according to claim 15 and/or by an apparatus as claimed in claim 16.