EP0486602A1 - Cryoconservation biologique - Google Patents

Cryoconservation biologique

Info

Publication number
EP0486602A1
EP0486602A1 EP19900913048 EP90913048A EP0486602A1 EP 0486602 A1 EP0486602 A1 EP 0486602A1 EP 19900913048 EP19900913048 EP 19900913048 EP 90913048 A EP90913048 A EP 90913048A EP 0486602 A1 EP0486602 A1 EP 0486602A1
Authority
EP
European Patent Office
Prior art keywords
eggs
temperature
larvae
unfertilised
minute
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP19900913048
Other languages
German (de)
English (en)
Inventor
Brian William Wilson Grout
Ian Robert Bruce Mcfadzen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CELL SYSTEMS Ltd
Original Assignee
CELL SYSTEMS Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB898918370A external-priority patent/GB8918370D0/en
Priority claimed from GB898919250A external-priority patent/GB8919250D0/en
Application filed by CELL SYSTEMS Ltd filed Critical CELL SYSTEMS Ltd
Publication of EP0486602A1 publication Critical patent/EP0486602A1/fr
Ceased legal-status Critical Current

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
    • 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
    • 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
    • A01N1/0278—Physical preservation processes
    • A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
    • 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

Definitions

  • This invention relates to the cryopreservation of eggs belonging to the phylum Mollusca. and in particular to the cryopreservation of unfertilised eggs, so that they may be used outside the breeding season, for example in hatcheries or for use in bioassays.
  • Bioassays employ living matter and usually expose a living organism to the pollutant at a particular stage in its development, which is then monitored. Such bioassays are already known from the following art:
  • sperm In a modified assay sperm alone is exposed to the pollutant and thereafter used to fertilise eggs, see P.A. Dinnel et ai, "Improved methodology for a sea urchin sperm cell bioassay for marine waters". Arch.Environ.Contam.Toxicol. .16, 23-32 (1987). Sperm is used to fertilise eggs and then two parameters are recorded, namely the percentage fertilisation and the pattern of development of the surviving embryos.
  • bioassays for water pollutants monitors the development of molluscan embryos from fertilisation to the prodissoconch stage (straight-hinge 'D' larvae) , see Annual Book of ASTM standards, supra.
  • assays are limited in that they can only be performed at those times of the year when the molluscs concerned are in a reproductive condition.
  • the use of some laboratory conditioning techniques to induce unseasonal reproduction can extend availability but this is at the expense of cost and inconvenience.
  • the basic problem of inadequate availability of molluscan embryos outside the breeding season remains.
  • the use of bioassays is restricted to locations where reproductive molluscs are available, which precludes its use in organisations and places that do not have the appropriate wet facilities.
  • the present invention seeks to overcome or at least mitigate these problems, and seeks to allow bioassays to be conducted conveniently all year round and without a significant increase in costs.
  • a process comprising cryopreserving unfertilised eggs of the phylum Mollusca.
  • cryopreserving freezing
  • the eggs can then be thawed at a convenient time prior to use in a hatchery or a bioassay.
  • the present invention may provide egg stocks for hatcheries out of season, the capability to extend the stocking season and allow increased control of production cycles.
  • the process of the present invention may have the additional advantage of providing a level of comparability and cross-referencing (such as between bioassays) that is not possible with prior art techniques which use fresh eggs each time.
  • the present invention allows one to work with the same batch of eggs in assays over a long period of time, even years, since the cryopreservation method allows a batch of eggs to be stored for such a time, whereas prior art techniques allow only a few hours in which to obtain consistent experimental results.
  • the process of cryopreservation of the present application and the advantages that may result regarding storage allow eggs (for example from the same batch) be transported, such as for export, worldwide.
  • cryopreservation and storage of eggs may be realised in the form of conservation of genetic material and the maintenance of disease-free stocks.
  • the invention may assist in the protection of valuable stocks of eggs against disease or pollution and may allow for the maintenance of genetically valuable eggs such as the collection and storage of exotic species, foreign species and certified disease-free populations.
  • Particularly suitable eggs are those belonging to the class Bivalvia (Pelecypoda) , for example the subclass Lamellibranchia. such as the super order Pteriomorphia. Heterodonta or Palaeoheterodonta such as the orders Ostridae, Mytilidae. Pectimidae or Veneridae. Preferred eggs are from the genera Mvtilus . Ostrea . Patinopectin. Ar ⁇ opecten. Venus, Mercenaria, Tapes or Crassostre . such as mussels, molluscs and oysters can be used, especially eggs of the genus Crassostrea.
  • Particularly preferred species include:
  • the eggs are cryopreserved in any suitable aqueous medium which advantageously contains a cryoprotectant.
  • the aqueous medium is preferably not saline.
  • the eggs may be suitably removed from the animal, for example by surgical excision,before they are exposed to sea water.
  • the eggs are removed before natural spawning and placed directly into a cryopreservation medium. If the eggs are liberated into sea water before addition to the medium then this may lead to low viability following freezing and thawing. It is speculated that this is due to changes in permeability of the egg membrane which occurs on exposure to sea water which may result in a lowered permeability to the cryoprotectant.
  • the excised female gonadal material which may contain other debris as well as the eggs, may need to be purified before cryopreservation, for example by filtration.
  • the eggs are preferably cryopreserved in an aqueous medium in contact with an organic solid.
  • the solid may act as a nucleating agent which causes water in the aqueous medium to be nucleated at or near the freezing point of the medium, to minimise damage to the eggs.
  • Suitable organic solids include steroids, amino acids, oligomeric or polymeric amino acids, and polyhydroxylated compounds. Cholesterol is especially preferred, especially if it has been crystallised from either methanol or acetic acid. Cholesterol crystallised from methanol is the ice nucleator of choice.
  • the organic solid may be added to the aqueous medium for example at a concentration of from 0.0001 to 0.001 g/ml, such as 0.2mg/ml or above.
  • the medium contains from 0.25 to 1.5 mg/ml of cholesterol, optimally from 0.75 to 1.25 mg/ml.
  • the organic solid can be coated on to a substrate that is in contact with the aqueous medium.
  • the substrate will usually be a vessel in which cryopreservation takes place, such as an ampoule, straw, bag or tube.
  • the organic solid may also be provided on polymer beads for example acrylic beads, such as are available from Bio-Rad, eg. Bio-Beads Sm7.
  • Coating densities of the organic solid on the substrate are suitably at least 0.0007 mg/mm 2 , for example within a range 0.001 to 0.1 mg/mm 2 , with about 0.0035 mg/mm 2 being optimal.
  • the aqueous medium may contain additional soluble components (such as a cryoprotectant, sugar and/or salt at a concentration range of from almost zero concentration (infinite dilution) up to high concentrations provided that there is still free water available to be frozen.
  • additional soluble components such as a cryoprotectant, sugar and/or salt at a concentration range of from almost zero concentration (infinite dilution) up to high concentrations provided that there is still free water available to be frozen.
  • a cryoprotectant for example glycerol and/or dimethyl sulphoxide.
  • the cryoprotectant is present in an amount of from 1 to 60%v/v, for example from 5 to 15% v/v, such as about 10%v/v.
  • the aqueous medium may also contain a sugar, such as mannitol and/or trehalose, for example at a concentration of from 0.1M to 10.0M, more preferably from 0.8M to 1.2M, with about 1.0M being optimal.
  • a sugar such as mannitol and/or trehalose
  • the eggs should be cooled to at least -30°C. However, once cooled the eggs can then be transferred to liquid nitrogen for long term storage if desired. Short term storage can be achieved in deep freezes at about -80°C.
  • the preferred storage temperature is below -135°C, below the glass transition temperature of water, which can be attained in mechanical refrigerators. This is preferably achieved using liquid nitrogen, which has a boiling point of -196°C. It should be noted that the eggs should be cooled with care; rapid cooling can be fatal.
  • the cooling protocol optimally includes at least one isothermal hold, and preferably two or three, each of which usually lasts from 1 to 10 minutes, such as from 4 to 8 minutes.
  • the eggs will usually be cooled starting from room temperature, for example about 20°C.
  • the preferred cooling protocol is as follows:
  • the most preferred cooling protocol comprises:
  • cryopreserved unfertilised eggs of the phylum Mollusca Prefered features and characteristics of the second aspect of the invention are as for the first mutatis mutandis.
  • the invention also extends to providing cryopreserved unfertilised eggs of the phylum Mollusca. the process comprising a process of the first aspect and subsequent thawing the eggs.
  • the invention in its broadest terms contemplates various bioassays for the detection of, eg. pollutants.
  • a third aspect of the present invention relates to a method of conucting a bioassay, the method comprising contacting cryopreserved unfertilised eggs of the phylum Mollusca or larvae resulting from the fertilisation of a cryopreserved unfertilised egg of the phylum Mollusca. with a sample to be assayed.
  • bioassay may be conducted on the cryopreserved eggs themselves, or the eggs fertilised and the bioassay conducted on the resultant larvae.
  • a preferred method for conducting a bioassay comprises:
  • the term "larvae” here is intended to cover all these stages; that is, the organism resulting from the fertilisation of egg with sperm, from the moment of fertilisation onwards.
  • the bioassay (contact with the sample) preferable lasts for no longer than 48 hours, since there is a danger that the larvae will starve without feeding.
  • Preferred larvae for bioassays are 48 hours old, or in the prodissoconch 1 stage (ie. post-trocophone stage) .
  • LC50 median lethal concentration
  • EC50 median effective concentration
  • the larvae are preferably kept in an aqueous medium, such as saline, suitably at from 15 to 25°C.
  • the aqueous medium is preferably aerated.
  • the medium is agitated frequently, preferably with a perforated plunger to avoid damaging the larvae.
  • the density of the larvae should preferably be below 100 per ml, and suitably above 1 per ml. Preferred ranges are from 10 to 50 per ml, optionally from 15 to 30 per ml.
  • Bioassays assay by biological function, productivity, development or performance to determine the effect of a substance or subtances in the sample or conditions presented to the biological material involved in the bioasay.
  • the sample may comprise a toxicant or pollutant, or toxicant although bioassays are not limited to substances which may have a negative effect on biological activity.
  • Bioassays can be used to determine the effects (if any) of various additives, for example paint additives, on various life forms which might be relevant for paint that is to be painted on oil rigs.
  • the sample may be taken from the environment suspected of being polluted, for example sea water, optionally diluted, or it may be a suspension of a sample, usually in water.
  • the terms "pollutant” and “toxicant” include any substance that may be considered harmful, noxious, dangerous or even toxic to any living matter especially wildlife and humans, and not necessarily harmful, noxions, dangerous or toxic only to unfertilised eggs of the phylum Mollusca.
  • sample not only encompasses pollutants, but any substance whose effect on biological activity is to be investigated, including positive effects. Indeed, some bioassays are not conducted with environmental samples and so “sample” here includes analytical or reagent grade chemicals that mey be obtained commercially. The exact practical procedure of such bioassays will be well known to those skilled in the art.
  • a method of conducting a bioassay comprises:
  • cryopreservation and thawing protocol may be employed provided that sufficient unfertilised eggs survive for the protocol to be practical.
  • Thawing may take place at a temperature of from 15 to 25°C, for example about room temperature, either in air or suitably in a liquid medium such as water.
  • the eggs may be filtered gently, for example on a 15 micron filter, before use in the bioassay.
  • the bioassay is conducted on larvae of the phylum Mollusca.
  • the fertilising sperm is for preference also cryopreserved.
  • the cryopreservation techniques described for the first aspect in relation to eggs are applicable to sperm mutatis mutandis .
  • a particularly preferred cooling protocol for the sperm is to add the sperm to the aqueous medium (preferably 1.0M mannitol, 1.0M trehalose, 15% v/v DMSO) in a volume ratio of about 1:1.
  • the mixture may then be placed in a 0.5ml straw and cholesterol crystallised from methanol (eg. 0.2mg/ml) added.
  • This may then be cooled from room temperature to -100°C at a rate of from 40 to 60°C/minute, such as about 50°C/minute. It may then be plunged into liquid nitrogen.
  • the sperm is preferably thawed by immersion in, eg. water, at about 40°C. This protocol may give 88% and above fertilisation rates when compared with unfrozen controls.
  • a fourth aspect of the present invention relates to a bioassay kit comprising cropreserved unfertilised eggs of the phylum Mollusca and means for contacting a sample with the unfertilised eggs or larvae resuting from fertilisation of the eggs.
  • the kit may comprise one or more containers or wells in which the eggs or lavae and sample can be brought into contact with each other.
  • the kit may also possess a surface at least partially coated with an organic solid ice nucleator as described in the first aspect (such as on the inside of a container or well) .
  • the kit preferably also comprises cryopreserved sperm. This allows bioassays to be conducted whenever and wherever it is convenient to do so, without having to rely on the availability of either eggs or sperm.
  • Mature animals of the species Crassostrea qi ⁇ as were selected from a laboratory maintained and conditioned population that had been established as being in reproductive condition. The animal had its external surfaces blotted dry and was then opened. All internal fluids were blotted dry and the gonadal tissue exposed.
  • the outer membrane of the gonad was pierced with a clean, fine-bore glass pasteur pipette and the enclosed material removed gently by the application of negative pressure. Material was extracted until it was no longer easy to fill the pipette without marked mechanical agitation.
  • the gonadal material was transferred to a dry petri dish during collection and the final gonadal suspension layered onto a 15 micron seive and, as far as practicable, the ovarian fluid removed.
  • the eggs were then placed directly into the appropriate cryoprotective agent (CPA) .
  • CPA cryoprotective agent
  • CPA 0.85M Trehalose and 15%v/v DMSO in distilled water.
  • the eggs were left to rehydrate for a minimum of 30 minutes prior to insemination of 25°C in filtered seawater.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Environmental Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

Des bioessais pour les polluants ou les poisons peuvent être réalisés pendant toute l'année, même hors de saison, au moyen d'oeufs non fertilisés et cryoconservés du phylum Mollusca, par exemple les huîtres et les mollusques. Les oeufs sont cryoconservés dans un milieu qui contient un cryoprotecteur tel que le sulfoxyde diméthylique en contact avec du cholestérol, et qui peut être enduit sur une ampoule ou sur une paille ou un tube de cryoconservation.
EP19900913048 1989-08-11 1990-08-13 Cryoconservation biologique Ceased EP0486602A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB898918370A GB8918370D0 (en) 1989-08-11 1989-08-11 Material for use in biological cryoprotection
GB8918370 1989-08-11
GB8919250 1989-08-24
GB898919250A GB8919250D0 (en) 1989-08-24 1989-08-24 Biological cryopreservation

Publications (1)

Publication Number Publication Date
EP0486602A1 true EP0486602A1 (fr) 1992-05-27

Family

ID=26295736

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19900913048 Ceased EP0486602A1 (fr) 1989-08-11 1990-08-13 Cryoconservation biologique

Country Status (5)

Country Link
EP (1) EP0486602A1 (fr)
JP (1) JPH05502663A (fr)
AU (1) AU653708B2 (fr)
CA (1) CA2064745A1 (fr)
WO (1) WO1991001637A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05502662A (ja) * 1989-08-11 1993-05-13 セル・システムズ・リミテッド 生物学的凍結保存
WO1992012722A1 (fr) * 1990-01-17 1992-08-06 The Regents Of The University Of California Compositions de glycopeptides resistantes a la congelation, destinees a proteger des cellules et des tissus durant leur congelation
JP5186676B2 (ja) * 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 細胞の凍結保存のための装置及び方法
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
CA2647664C (fr) * 2008-12-17 2015-06-30 Erik John Woods Systemes et methodes de cryoconservation cellulaire

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8611894D0 (en) * 1986-05-15 1986-06-25 Cell Systems Ltd Biological cryo-protection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9101637A1 *

Also Published As

Publication number Publication date
WO1991001637A1 (fr) 1991-02-21
CA2064745A1 (fr) 1991-02-12
AU6277690A (en) 1991-03-11
JPH05502663A (ja) 1993-05-13
AU653708B2 (en) 1994-10-13

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