WO1991001992A1 - Nucleation of ice - Google Patents
Nucleation of ice Download PDFInfo
- Publication number
- WO1991001992A1 WO1991001992A1 PCT/GB1990/001269 GB9001269W WO9101992A1 WO 1991001992 A1 WO1991001992 A1 WO 1991001992A1 GB 9001269 W GB9001269 W GB 9001269W WO 9101992 A1 WO9101992 A1 WO 9101992A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cholesterol
- methanol
- ice
- medium
- acetic acid
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
Definitions
- 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.
- 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.
- 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.
- temperatures such as about -5°C snow making machines are not effective, whilst at lower temperatures the process is inefficient.
- Ice nucleators have been known for several years, either as physico-chemical curiosities or in meteoro ⁇ logical applications as initiators of rainfall.
- 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).
- 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.
- 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.
- cryopreservation 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.
- Cryoprotectants such as DMSO or glycerol
- other additives such as salts
- 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.
- 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.
- 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.
- 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.
- a second aspect of the present invention relates to an ice nucleating composition
- 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.
- 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.
- a cryoprotectant for example dimethyl sulphoxide (DMSO) and/or glycerol.
- 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.
- the medium preferably contains from 0.25 to 1.5 mg/ml of the cholesterol, optimally from 0.75 to 1.25 mg/ml.
- 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.
- Figs. 1 and 2 both prior art
- cholesterol crystals are typically both small, compact and spherulite in appearance.
- 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.
- 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.
- 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.
- 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.
- 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) .
- 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.
- 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) .
- 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) .
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- a method of cryopreserving a biological sample 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.
- 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.
- cholesterol e.g. analytical or reagent grade
- 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. ⁇ 5 g).
- 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.
- 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.
- 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.
- suitable cryopreservation vessel such as an ampoule, tube, straw or bag, or a particular carrier, for example glass, polymer or other beads.
- 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.
- 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/mm 2 , up to a limit determined by practical convenience. Coating densities are suitably at least 0.001 mg/mm 2 , and preferably below 0.1 mg/mm 2 , with an optimum of about 0.0035 mg/mm 2 .
- 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.
- 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.
- the aqueous medium will be, for the sake of convenience, water.
- the cholesterol may be dispersed in the medium either before, at the same time as or after the medium is sprayed into the atmosphere.
- the cholesterol may be dispersed into a reservoir of the medium to be sprayed.
- a double nozzle arrangement may be used to cause the cholesterol to be dispersed into the nascent medium droplets.
- 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.
- solute present in the medium such as water
- the medium may be sprayed as a very fine spray, that is to say, an aerosol.
- a spray may be generated by means of a compressor, which for convenience will compress air.
- a gas .vapourising from a liquid for example a liquified atmospheric gas such as nitrogen.
- 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.
- 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.
- Figure 1 is a view of cholesterol under a microscope (xl05) , obtained commercially from Sigma Chemicals;
- Figure 2 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;
- 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.
- the horizontal broken line labelled "A" represents the melting point of 0.5M NaCl (-1.7°C) .
- Bovine sperm diluent (Milk Marketing Board) (0.25ml in straws) 43.3 24 -4.0 ⁇ 0.40 -8.1 ⁇ 2.64 TABLE 2 (contd.)
- 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.
- 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.
- 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:
- 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.
- Example 1 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.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU61792/90A AU654112B2 (en) | 1989-08-11 | 1990-08-13 | Nucleation of ice |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB898918370A GB8918370D0 (en) | 1989-08-11 | 1989-08-11 | Material for use in biological cryoprotection |
GB8918370.1 | 1989-08-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1991001992A1 true WO1991001992A1 (en) | 1991-02-21 |
Family
ID=10661496
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1990/001269 WO1991001992A1 (en) | 1989-08-11 | 1990-08-13 | Nucleation of ice |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0486585A1 (en) |
JP (1) | JPH05502664A (en) |
AU (1) | AU654112B2 (en) |
CA (1) | CA2064708A1 (en) |
GB (1) | GB8918370D0 (en) |
WO (1) | WO1991001992A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8222027B2 (en) | 2006-06-20 | 2012-07-17 | Cook General Biotechnolgy, LLC | Systems and methods for cryopreservation of cells |
US8709797B2 (en) | 2006-06-20 | 2014-04-29 | Cook General Biotechnology Llc | Systems and methods for cryopreservation of cells |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5186676B2 (en) * | 2006-06-20 | 2013-04-17 | クック・ジェネラル・バイオテクノロジー・エルエルシー | Apparatus and method for cryopreservation of cells |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH274845A (en) * | 1949-05-03 | 1951-04-30 | Organon Labor Ltd | Process for the isolation of cholesterol from the mixture of the unsaponifiable components of the wool fat. |
FR1040894A (en) * | 1950-07-21 | 1953-10-19 | Organon Labor Ltd | New cholesterol extraction process |
US2688623A (en) * | 1950-07-21 | 1954-09-07 | Organon Labor Ltd | Process for the isolation of cholesterol from wool-wax alcohols |
DE2751392A1 (en) * | 1976-11-19 | 1978-05-24 | Fallis | METHOD OF ISOLATING CHOLESTEROL FROM EGGS |
EP0206705A1 (en) * | 1985-06-19 | 1986-12-30 | Louis Handfield | Machine for making snow |
EP0267026A2 (en) * | 1986-11-04 | 1988-05-11 | Board Of Regents, The University Of Texas System | Apparatus and method for cryopreparing biological tissue for ultrastructural analysis |
DE3818591A1 (en) * | 1988-06-01 | 1989-12-07 | Martin Prof Dr Ing Herrmann | Process for the production of reduced cholesterol fat products with simultaneous removal of contaminating substances with the possibility of obtaining cholesterol |
-
1989
- 1989-08-11 GB GB898918370A patent/GB8918370D0/en active Pending
-
1990
- 1990-08-13 JP JP2511810A patent/JPH05502664A/en active Pending
- 1990-08-13 CA CA002064708A patent/CA2064708A1/en not_active Abandoned
- 1990-08-13 AU AU61792/90A patent/AU654112B2/en not_active Ceased
- 1990-08-13 EP EP90912547A patent/EP0486585A1/en not_active Ceased
- 1990-08-13 WO PCT/GB1990/001269 patent/WO1991001992A1/en not_active Application Discontinuation
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH274845A (en) * | 1949-05-03 | 1951-04-30 | Organon Labor Ltd | Process for the isolation of cholesterol from the mixture of the unsaponifiable components of the wool fat. |
FR1040894A (en) * | 1950-07-21 | 1953-10-19 | Organon Labor Ltd | New cholesterol extraction process |
US2688623A (en) * | 1950-07-21 | 1954-09-07 | Organon Labor Ltd | Process for the isolation of cholesterol from wool-wax alcohols |
DE2751392A1 (en) * | 1976-11-19 | 1978-05-24 | Fallis | METHOD OF ISOLATING CHOLESTEROL FROM EGGS |
EP0206705A1 (en) * | 1985-06-19 | 1986-12-30 | Louis Handfield | Machine for making snow |
EP0267026A2 (en) * | 1986-11-04 | 1988-05-11 | Board Of Regents, The University Of Texas System | Apparatus and method for cryopreparing biological tissue for ultrastructural analysis |
DE3818591A1 (en) * | 1988-06-01 | 1989-12-07 | Martin Prof Dr Ing Herrmann | Process for the production of reduced cholesterol fat products with simultaneous removal of contaminating substances with the possibility of obtaining cholesterol |
Non-Patent Citations (3)
Title |
---|
J. Phys. Chem. Solids, Volume 23, 1962, Pergamon Press, (GB), R.B. HEAD: "Ice Nucleation by some Cyclic Compounds", pages 1371-1378 see the whole article (cited in the application) * |
J. Phys. Chem. Solids, Volume 24, 1963, Pergamon Press, (GB), N. FUKUTA et al.: "Epitaxial Growth of Ice on Organic Crystals", pages 715-718 see the whole article (cited in the application) * |
Nature, Volume 191, 9 September 1961, (London, GB), R.B. HEAD: "Steroids as Ice Nucleators", pages 1058-1059 see the whole article (cited in the application) * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8222027B2 (en) | 2006-06-20 | 2012-07-17 | Cook General Biotechnolgy, LLC | Systems and methods for cryopreservation of cells |
US8709797B2 (en) | 2006-06-20 | 2014-04-29 | Cook General Biotechnology Llc | Systems and methods for cryopreservation of cells |
US8936905B2 (en) | 2006-06-20 | 2015-01-20 | Cook General Biotechnology Llc | Systems and methods for cryopreservation of cells |
US9565854B2 (en) | 2006-06-20 | 2017-02-14 | Cook General Biotechnology Llc | Systems and methods for cryopreservation of cells |
US9877475B2 (en) | 2006-06-20 | 2018-01-30 | Cook General Biotechnology Llc | Systems and methods for cryopreservation of cells |
Also Published As
Publication number | Publication date |
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EP0486585A1 (en) | 1992-05-27 |
GB8918370D0 (en) | 1989-09-20 |
AU654112B2 (en) | 1994-10-27 |
JPH05502664A (en) | 1993-05-13 |
AU6179290A (en) | 1991-03-11 |
CA2064708A1 (en) | 1991-02-12 |
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