EP0486602A1

EP0486602A1 - Biological cryopreservation

Info

Publication number: EP0486602A1
Application number: EP19900913048
Authority: EP
Inventors: Brian William Wilson Grout; Ian Robert Bruce Mcfadzen
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: AU653708B2; JPH05502663A; WO1991001637A1; AU6277690A; CA2064745A1

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.Bioassays for pollutants or poisons can be carried out all year round, even out of season, using unfertilized and cryopreserved eggs of the phylum Mollusca, for example oysters and molluscs. The eggs are cryopreserved in a medium which contains a cryoprotective agent such as dimethyl sulfoxide in contact with cholesterol, and which can be coated on an ampoule or on a straw or a cryopreservation tube.

Description

BIOLOGICAL CRYOPRESERVATION

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.

Various assays and techniques are known for the detection and quantification of pollutants in water, especially sea water. 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:

C.E. Woelke, "Development of a receiving water quality bioassay criterion based on the 48hr Pacific oyster (Crassostrea iαas) embryo", Washington Dept. of Fisheries Technical Report , 1-93(1982);

"Conducting static acute toxicity tests with larvae of four species of bivalve molluscs", Designation E724-80, Annual Book of ASTM Standards, American Society for Testing and Materials, 1-17 (1980) ;

A.R.D. Stebbing et a L , "The Effects of Stress and Pollution on Marine Animals", Bioassay, New York, 133- 140 (1985) ; and

J.E. Thain and J. Watts, "The use of a bioassay to measure changes in water quality associated with a bloom of Crvodinium aureolum_f Hulburt Rapp. P-v Reun, Cons.Int.Explor.mer 82, 103-107 (1987). The cryopreservation of sperm is disclosed in the following documents:

J.E. Lannan, Genetics 6_8:599-601 (1971), Experimental self-fertilisation of the Pacific Oyster Crassostrea Virσinica; and

J.B. Hughes, Cryobiology i0.:342-344 (1973), An examination of eggs challenged with cryopreserved spermatazoa of the American Oyster, Crassostrea Virσinica; and

S.R. Zell, M.H. Barnford and H. Hidu, Cryobiology 1.6:448-460 (1979), Cryopreservation of the spermatazoa of the American Oyster, Crassostrea Vir inica.

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.

One known bioassay 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. However, such 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. Thus the basic problem of inadequate availability of molluscan embryos outside the breeding season remains. Furthermore, 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.

Therefore, according to a first aspect of the present invention there is provided a process comprising cryopreserving unfertilised eggs of the phylum Mollusca. By cryopreserving (freezing) the eggs they can be stored for long periods of time, sufficient for survival of the eggs through the unseasonal periods of the year. The eggs can then be thawed at a convenient time prior to use in a hatchery or a bioassay.

By this process the availability of eggs can be extended to any time of the year, and in particular to these periods when the eggs are not naturally abundant. In addition, 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. Thus 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. In addition, 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.

Further benefits of cryopreservation and storage of eggs may be realised in the form of conservation of genetic material and the maintenance of disease-free stocks. In addition 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. Arσopecten. Venus, Mercenaria, Tapes or Crassostre . such as mussels, molluscs and oysters can be used, especially eggs of the genus Crassostrea.

Particularly preferred species include:

Crassostrea qiqas Pacific oyster Tapes phillipinarum Manila clam Crassostrea virqinica American oyster Mercenaria mercenaria Hard clam Tapes decussata Native palourde Arqopecten irradians Bay scallop

Generally the eggs are cryopreserved in any suitable aqueous medium which advantageously contains a cryoprotectant. However, 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. Thus, for example, 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.

There is no precedent for selecting eggs at this stage, prior to release, since the eggs are arguably ^•"unready*^* for fertilisation at this stage. .Furthermore, there is no precedent for keeping eggs away from the medium in which they would normally continue their development (that is to say sea water) and still be receptive to fertilisation.

Prefered methods of cryopreservation are disclosed in EP-A-0246824 and International Patent Application entitled "Nucleation of Ice" filed on 13th August 1990 in the name of Cell Systems Ltd. , although any suitable cryoprotection protocol may be employed that ensures survival of a sufficient proportion of the eggs for the protocol to be practical. Thus 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. Preferably the medium contains from 0.25 to 1.5 mg/ml of cholesterol, optimally from 0.75 to 1.25 mg/ml. However, conveniently 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/mm², for example within a range 0.001 to 0.1 mg/mm², with about 0.0035 mg/mm² 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. Preferably the aqueous medium is provided with a cryoprotectant, for example glycerol and/or dimethyl sulphoxide. Suitably 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.

To ensure cryopreservation 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:

(a) optionally cooling the eggs at a rate of from 2 to 6°C/minute to a temperature of from +3 to -5°C;

(b) optionally maintaining the eggs at about that temperature for from 4 to 6 minutes;

(c) cooling the eggs at a rate of from 2 to 6°C/minute to a temperature of from -15 to -25°C;

(d) maintaining the eggs at about that temperature for from 3 to 7 minutes;

(e) further cooling the eggs at a rate of from 2 to 6°C/minute to a temperature of from -30 to -40°C;

(f) optionally maintaining the eggs at that temperature for up to 2 minutes; and

(g) optionally plunging the eggs into liquid nitrogen. The most preferred cooling protocol comprises:

(a) cooling eggs of the phylum Mollusca. at a rate of about 3°C/minute to a temperature of about 0°C;

(b) maintaining the eggs at about 0°C for about 5 minutes;

(c) further cooling the eggs at a rate of about 3°C/minute to a temperature of about -20°C;

(d) maintaining the eggs at a about that temperature for about 5 minutes;

(e) cooling the eggs at a rate of about 3°C/minute to a temperature of about -35°C;

(f) maintaining the eggs at about -35°C for no longer than one minute; and

(g) plunging the eggs into liquid nitrogen.

According to a second aspect of the present invention there is provided 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.

Thus it will be appreciated that the bioassay may be conducted on the cryopreserved eggs themselves, or the eggs fertilised and the bioassay conducted on the resultant larvae.

Thus a preferred method for conducting a bioassay comprises:

(a) cryopreserving unfertilised eggs of the phylum Mollusca:

(b) thawing the eggs;

(c) optionally fertilising the eggs to produce larvae of the phylum Mollusca;

(d) contacting a sample to be assayed with the eggs, or if (c) is conducted , with the larvae.

Although once the eggs are fertilised they develop through several stages, inducing embryo, veliger and prodissoconch stages, 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) . Usually both the median lethal concentration (LC50) and median effective concentration (EC50) , based on abnormal shell development, are measured.

During the bioassay 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. Advantageously 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. However, it will be appreciated that the term "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.

The unfertilised eggs will have suitably been first cryopreserved in accordance with the first aspect of the present invention. In a preferred embodiment a method of conducting a bioassay comprises:

(a) cryopreserving unfertilised eggs of the phylum Mollusca:

(b) thawing the unfertilised eggs;

(c) fertilising the eggs to produce larvae; and

(d) contacting the larvae with a sample to be assayed, for example one suspected of comprising a pollutant or toxicant.

Any suitable 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.

Thus for preference the bioassay is conducted on larvae of the phylum Mollusca. Thus in order to allow such bioassays to be conducted outside of the breeding season 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 . However, 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.

Thus in a particularly preferred embodiment a method of conducting a bioassay comprises:

(a) separately cryopreserving unfertilised eggs and sperm of the phylum Mollusca:

(b) thawing the eggs and sperm;

(c) fertilising the eggs with the sperms to give larvae of the phylum mollusca;

(d) contacting the larvae with a sample to be assayed.

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. Thus 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.

Other preferred features and characteristics of the fifth aspect are as for previous aspects mutatis mutandis.

The invention will now be described by way of example, which is profided by way of illustration and not to be contsrued as being limiting to the scope of the present invention.

Example

Mature animals of the species Crassostrea qiσas 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: 0.85M Trehalose and 15%v/v DMSO in distilled water.

Eggs were loaded at between 0.5 to 1.0 million per ml into 0.5ml plastic straws coated with cholesterol. At exactly 10 minutes after the introduction of the eggs into the CPA the cooling protocol was initiated. The protocol was:

Cool at 3°C per minute from 20°C to 0°C;

Hold at 0°C for 5 minutes;

Cool at 3°C per min from 0°C to -20°C;

Hold -20°C for 5 minutes;

Cool at 3°C per minute to -35°C;

Hold at -35°C for no more than 1 minute; and

Plunge into liquid nitrogen.

Thawing was by totally immersing the straw in water at

80°C.

When the last region of ice had just disappeared the end of the straw was snipped off and the contents blown into 0.5ml of sea water at room temperature. After 30 sec 1ml of sea water was added and left for 5 min. Then a further 1ml of sea water was added every 5 min up to a volume of lOml. The eggs were pipetted onto a 15 micron filter and then resuspended, above the filter, in dec. sea water for 1 min with very gentle agitation. The eggs were then taken off the filter and resuspended in 200ml of clean sea water.

The eggs were left to rehydrate for a minimum of 30 minutes prior to insemination of 25°C in filtered seawater.

Using eggs of Crassostrea σiσas. 55% ofthe intact, mature oocytes that had recovered from cryopreservation were fertilised using fresh spermatazoa stripped from an approprately conditioned male. The derived embryos were cultured in filtered, UV treated seawater at 25°C for 24 hours. Development to D-larvae was comparable for control material, where eggs were unfrozen, and experimental material where eggs were cryopreserved.

Claims

1. A process comprising cryopreserving eggs of the phylum Mollusca.

2. A process as claimed in claim 1 wherein the eggs belong to the genus Crassostrea.

3. A process as claimed in claim 1 wherein the eggs are cryopreserved in an aqueous medium.

4. A process as claimed in claim 3 wherein the aqueous medium is in contact with cholesterol.

5. A process as claimed in claim 4 wherein the cholesterol is coated on a substrate.

6. A process as claimed in claim 1 wherein the eggs are cooled to a temperature of at least -30°C.

7. A process as claimed in claims 1 to 6 comprising:

(a) optionally cooling the eggs at a rate of from 2 2 ttoo 66°°(C/minute to a temperature of from +3 to -5°C;

(b) optionally maintaining the eggs at about that temperature for from 4 to 6 minutes; (c) cooling the eggs at a rate of from 2 to 6°C/minute to a temperature of from -15 to -25°C;

(d) ma inta ining the eggs at about that temperature for from 3 to 7 minutes;

( f) optionally maintaining the eggs at that temperature for up to 2 minutes ; and

(g) optionally plunging the eggs into liquid nitrogen.

8. A process as claimed in claim 7 comprising:

(a) cooling the eggs at a rate of about 3°C/minute to a temperature of about 0°C;

(b) maintaining the eggs at about 0°C for about 5 minutes ;

(c) further cooling the eggs at a rate of about 7°C/minute to a temperature of about -20°C;

(d) ma intaining the eggs at about that temperature for about 5 minutes; (e) cooling the eggs at a rate of about 3°C/minute to a temperature of about -35°C;

(f) maintaining the eggs at about -35°C for no longer than one minute; and

(g) plunging the eggs into liquid nitrogen.

9. A process as claimed in claim 7 further comprising thawing the eggs.

10. Cryopreserved eggs of the phylum Mollusca.

11. Eggs as claimed in claim 10 of the class Bivalvia.

12. Eggs as claimed in claim 10 or 11 which are cryopreserved according to a process as claimed in any of claims 1 to 9.

13. A process for conducting a bioassay comprising contacting unfertilised eggs of the phylum Mollusca , or lavae resulting from the fertilisation of a cryopreserved unfertilised egg of the phylum Mollusca. with a sample to be assayed.

14. A process as claimed in claim 13 comprising:

(a) cryopreserving unfertilised eggs of the phylum Mollusca;

(b) thawing the unfertilised eggs; (c) fertilising the eggs to produce larvae of the phylum Mollusca: and

(d) contacting the larvae with a sample to be assayed.

15. A method as claimed in claim 13 wherein the eggs or larvae and sample are contacted for no longer than 48 hours.

16. A method as claimed in claim 13 wherein the larvae are at, or beyond, the prodissoconch 1 stage.

17. A method as claimed in claim 13 wherein the larvae are kept in saline at a temperature of from 15 to 25°C.

18. A method as claimed in claim 17 wherein the saline is frequently agitated using a perforated plunger.

19. A method as claimed in claim 18 where the density of the larvae is from 15 to 30 per ml of saline.

20. A process as claimed in claim 14 wherein the cryopreservation is by a process as claimed in any of claims 1 to 9 and/or where the unfertilised eggs used are according to claim 10.

21. A bioassay kit comprising cryopreserved unfertilised eggs of the phylum Mollusca and means for contacting a sample with the unfertilised eggs or larvae resulting from fertilisation of the eggs.

22. A kit as claimed in claim 21 additionally comprising cryopreserved sperm.