AU6274390A - Biological cryopreservation - Google Patents

Description

BIOLOGICAL CRYOPRESERVATION

This invention relates to the cryopreservation of larvae of the phylum Mollusca for storage so that they can be used outside the breeding season, for example in hatcheries , or in bioassays.

In hatchery operations and other places where molluscs are reared, the seasonal nature of the reproductive cycle restricts the availability of embryos and larvae at certain times during the year. This can be inconvenient when it is desired to breed such animals throughout the year.

On e attempt at s o lv ing th is prob l em i s by "conditioning" the animals which involves keeping mature animals in a controlled water system with a high nutritional input and a raised water temperature appropriate for induction of the breeding condition in the species being used. Over a period of time , usually in the order of weeks , a proportion of the animals may be brought into a reproductive condition. However , this is a time consuming exercise , is not inexpensive and is therefore commercially inefficient. In addition , the results are often variable and inconsistent.

It is known to cryopreserve embryos of the mussel Choromyt i lus chorus at a very early stage of fertilisation (Gallardo , C . S . , del Campo , M. R. and Filun, L. , "Preliminary trials of the cryopreserving of marine mollusc embryos as illustrated with the marine mussel Choromytilus chorus" , Cryobiology .25, : 565 (Abstract) , 1988 ) . This work originates from a university department in Chile where the embryos consist of 1 to 8 cells, which would occur a few hours after fertilisation at 15°C. Success is reported but mysteriously no data is given, suggesting that in fact the success rate is very low.

J.D. Toledo et al, Nippon Suisan Gakkaishi 55(A), 1661 (September 1989) discusses the cryopreservation of blue mussel (Mytilus edulis) embryos at the cleavage or trocophore stage with different cooling protocols and reports on the survival rates found.

An example of conducting bioassays is given in "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) .

When considering more developed embryos the complexity of multicellular structures compounds the problems of cryopreservation significantly (see Low temperature preservation of cells, tissues and organs, in Low Temperature Preservation in Medicine and Biology, Eds. M.J. Ashwood-Smith and J. Farrant, Pitman Medical Press, Tunbridge Wells, pp. 19-44 (1980) and Low temperature preservation in medicine and veterinary science, in The Effects of Low Temperatures on Biological Systems, Eds. B.W.W. Grout and G.J. Morris, Edward Arnold Ltd, London, pp. 432-450 (1987)). Logic thus indicates that it would be disadvantageous to select a larval stage for cryopreservation which contains many hundreds of cells, when a single cell or a small number of cell stages are suitable. For similar reasons of complexity it has been found that when cryopreserving mammalian embryos the limit for success is about 100 cells (see Principles of embryo preservation, in Low Temperature preservation in Medicine and Biology, supra. D.G. Whittingham (1980)). Therefore there is still no acceptable way of preserving viable embryos, such as molluscan embryos, in suspended development, so that stocks can be gathered during the breeding season and held until some other convenient time of year (for example outside the breeding season) when it is desired to produce larvae.

According to a first aspect of the present invention there is provided a process comprising cryopreserving larvae of the phylum Mollusca which are at, or beyond, the trocophore stage.

By this process the availability of larvae can be extended to any time of the year, and in particular to those periods when the larvae are not naturally abundant. In addition, the present invention may provide larval 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 also has the advantage of providing a level of comparability and cross-referencing (such as between bioassays) that is not possible with prior art technigues which use fresh larvae each time. Thus the present invention allows one to work with the same batch of larvae in assays over a long period of time, even years, since the cryopreservation method allows a batch of larvae to be stored for such a time. However, 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 resulting advantages regarding storage may allow larvae (for example from the same batch) to be transported, such as for export, worldwide.

Further benefits of cryopreservation and storage of larvae 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 larvae stocks against disease or pollution and may allow for the maintenance of genetically valuable larvae such as the collection and storage of exotic species, foreign species and certified disease-free populations.

The larvae at the trocophore stage possess cilia which differentiates it from younger larvae. The larvae are preferably at, or beyond, a post-trocophore stage such as at the prodissoconch I stage when they are termed straight hinge ^,D' larvae, 'D'-shell larvae or hinge larvae (since they possess a shell) . Thus suitable larvae are at least about 24 hours after fertilisation which with development (eg in sea water) at an appropriate temperature such as from 22 to 28°C, are likely to be prodissoconch I. At this stage the larvae already have a number of well differentiated organs including heart, stomach, mouth, anus and shell gland, together with a rudimentary shell and associated musculature. Also the larvae, unlike embryos, are capable of independent movement. The total cell number is in the order of thousands and so cryopreservation at this and later stages coupled with survival is contrary to the teaching of the prior art, for example Whittingham supra. Although cryopreservation is preferred at the prodissoconch I stage, older D^/ larvae can be used, such as those at the prodissoconch II stage. • Thus the invention contemplates the cryopreservation of larvae at from 24 to 96 hours post- fertilisation (for example after incubation at a temperature of from 22 to 28°C) . Nevertheless, preferably the larvae that are cryopreserved are those at or older than 24 hours, such as 48 hours, after fertilisation although larvae as old as 2 or 3 weeks can be employed. Optimally larvae are at the prodissoconch II stage. This usually occurs at about 48 hours post-fertilisation, such as after incubation at about 25°C.

Figure l illustrates the early larval development in a typical bivalve mollusc (not drawn to scale) . It is provided by way of example and is not to be construed as being limiting on the present invention. Embryos A to D show a number of cleavage stages from the fertilised egg A (approximately 25-40 microns in diameter) to D which is a multicellular stage. At E the larva possesses cilia and is termed a mobile trocophore (about 50-70 microns in width) . F is the veliger that secretes the larval shell (prodissoconch I) by the shell gland which, once complete, indicates that the larva is at the straight-hinge or D-shell stage (G, about 100-130 microns wide) . Secretion of a second larval shell (prodissoconch II) begins immediately after the prodissoconch I stage. After fertilisation the growing stage to produce larvae can be any suitable protocol known to those in the art, although it is preferred that the larvae are grown in the presence of an amino acid, such as glutamic acid. The amino acid will usually be provided in an aqueous medium in which the larvae are placed. Preferably the amino acid is provided at a concentration of from 5 to 15 cM (micromolar) , such as from 8 to 12 mcM, optimally about 10 mcM. The larvae are advantageously grown in the presence of the amino acid from at least 2 hours post-fertilisation. This can greatly improve the viability of the larvae after cryopreservation and subsequent thawing.

It is preferred that the larvae are fed on an algal diet during growth; this is suitably from 18 hours post-fertilisation to the time of cryopreservation.

Also preferred is that the larvae are grown in salt water, such as at a temperature of from 15 to 25°C. The water is suitably aerated and the larvae are preferably agitated frequently. This must be performed with care since stirring may damage the larvae; thus agitation with a perforated plunger is preferred.

The larvae are preferably of the class Bivalvia (Pelecypoda) , for example the sub-class Lamellibranchia such as the super-order Pteriomorphia. Heterodonta or Pa1aeohe erodonta such as of the orders Mvtilidae. Pectimidae. Ostreidae or Veneridae. Especially preferred larvae belong to the genera Mvtilus. Ostrea. Tapes. Patinopecten. Arαopecten. Venus, Mercenaria or (especially) Crassostrea. Particularly preferred species include:

Crassostrea σiσas Pacific oyster

Tapes phillipjnarum Manila clam

Crassostrea vircrinica American oyster

Mercenaria mercenaria Hard clam

Tapes decussata Native palourde

Arσopecten irradians Bay scallop

Cryopreservation of the larvae suitably takes place in an aqueous medium. Particularly preferred cryopreservation techniques are those 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. Thus the larvae are advantageously cryopreserved in an aqueous medium to be nucleated at, or close to the freezing point of the medium to minimise damage to the larvae using an organic solid ice nucleator. Suitable organic solids include steroids, amino acids, oligomeric or polymeric amino acids and polyhydroxylated compounds. Cholesterol is particularly preferred, and especially cholesterol crystallised from methanol or acetic acid. Cholesterol crystallised form methanol is the ice nucleator of choice.

The organic solid may be added to the aqueous medium for example to give 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 onto 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 substrate may also be polymer beads such as acrylic beads available from Bio-Rad, such as Bio-Beads SM7.

Coating densities of the organic solid on the substrate are suitably above 0.0007 mg/mm , for example within the range 0.001 to 0.1 mg/mm", with about 0.0035 mg/mm" being optimal.

The aqueous medium may contain additional soluble components (such as a cryoprotectant, a sugar, a 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 contains a cryoprotectant, for example glycerol or dimethyl sulphoxide, in an appropriate amount, for example 1. to 60% v/v, such as from 5 to 15% v/v, especially about 10% v/v. Alternatively or in addition the medium can contain a sugar, such as glucose and/or trehalose, preferably at a concentration of from 0.1M to 10.0M, more preferably from 0.8 to 1.2M, optimally about 1.0M.

The success rate of larvae cryopreservation can be affected by the freeze/thaw protocol employed. In the present invention it is advisable to cool the larvae to a temperature of at least -30°C, and preferably at least -35°C. 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 with mechanical refrigerators. This is preferably achieved using liquid nitrogen, which has a boiling point of -196°C. By cryopreserving the larvae at a temperature of -196°C or less one may be able to achieve complete cessation in larvae development. In addition, this may allow extremely long cryopreservation (and therefore storage) periods, for example several decades.

The cooling protocol optimally includes an isothermal hold, usually from 1-10 minutes, such as for 4-8 minutes. Good results can be obtained using the following cooling protocol:

proceeding by either (al) or (a2) ;

(al) cooling larvae of the phylum Mollusca at a rate of from 12-17°C/minute to a temperature of from -15 to -25°C;

(a2) cooling a larva of the phylum Mollusca to a temperature of from -40 to -50°C at a rate of from 40 to 50°C/minute and then warming the larva to a temperature of from -15 to -25°C at a rate of from 5 to 15°C/minute;

(b) maintaining the larvae at about that temperature for from 5 to 7 minutes;

(c) further cooling the larvae at a rate of from 12 to 17°C/minute to a temperature of from -30 to -40°C;

(d) optionally maintaining the larvae at that temperature for up to 2 minutes; and

(e) optionally plunging the larvae into liquid nitrogen.

A preferred cooling protocol comprises:

proceeding by either (al) or (a2) ;

(al) cooling larvae of the phylum Mollusca at a rate of about 15°C/minute to a temperature of about -20°C;

(a2) cooling larvae of the phylum Mollusca to a temperature of from -44 to -46°C, eg about -45°C, at a rate of about 45°C/minute and then warming the larva to a temperature of about -20°C at a rate of about 10°C/minute;

(b) maintaining the larvae at about -20°C for ^•about 6 minutes;

(c) further cooling the larvae at a rate of about 15°C/minute to a temperature of about -35°C;

(d) maintaining the larvae at about -35°C for no more than one minute; and

(e) plunging the larvae into liquid nitrogen.

The preferred cooling protocol will naturally depend on the initial temperature of the larvae and the nature of any aqueous medium in which they are suspended. In particular, it should be noted that when cryopreserving larvae in straws, it is preferable to start the cooling protocol with option (al) . Here it is particularly preferable to use an aqueous medium having 15% v/v DMSO and 1.0M trehalose, using 0.5 mg/ml of cholesterol crystallised from methanol as an ice nucleator. The volume of aqueous medium is suitably about 0.5ml.

In the cooling protocol option (a2) is more suitable when cooling in a bag. Such a bag is preferably made of aluminium foil and also suitably heat-sealable.

According to a second aspect of the present invention there are provided cryopreserved larvae of the phylum Mollusca that are at, or beyond, the trocophore stage. Preferred features and characteristics of the second aspect are as for the first aspect of the present invention mutatis mutandis.

The invention in its broadest terms contemplates providing the cryopreserved larvae at periods outside the natural breeding season, for example for hatcheries or biassays. Thus a third aspect of the invention relates to a method of breeding larvae of the phylum Mollusca comprising:

(a) cryopreserving larvae of the phylum Mollusca that are at, or beyond, the trocophore stage; and

(b) thawing the larvae.

Any suitable cryopreservation or thawing protocol may be used provided that sufficient larvae survive for the protocol to be practical. For example, thawing may take place at a temperature of from 22 to 28°C, such as about 25°C. This may be in air or a suitable liquid medium such as water, which is suitably saline, in particular 'sea water. Other preferred features and characteristics are as for the first aspect of the present invention mutatis mutandis.

The present invention in its broadest terms contemplates using cryopreserved larvae of the phylum Mollusca in bioassays. Thus according to a fourth aspect of the present invention there is provided a method of conducting a bioassay comprising contacting cryopreserved larvae of the phylum Mollusca that are at, or beyond, the trocophore stage with a sample to be assayed.

By bioassay it is meant an assay by biological function, productivity or development to determine the effect of a substance in the sample or condition presented to the biological material (larvae) in the bioassay. The sample may comprise a toxicant or pollutant whose effect on the larvae is to be investigated. For example, 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 the sample. Although the sample may be an environmental sample it may also be a substance or chemical from the laboratory whose effect on the larvae is to be investigated; thus "sample" includes analytical or reagent grade chemicals. The terms "pollutant" or "toxicant" include any substance that may be considered harmful, noxious, dangerous or even toxic to any living matter, especially wild life and humans, although it should be appreciated that the pollutant may not have the same effect on larvae of the phylum Mollusca. Indeed, it should be noted that the sample may not comprise substances that may be harmful to the larvae, but may contain substances that may have positive effects on the larvae.

The bioassay (the contact time between the sample and the larvae) preferably 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 I stage (ie, they are in a post-trocophore stage) . Usually both the median lethal concentration (LC50) and median effective concentration (EC50) , based on abnormal shell development, are measured.

The larvae may be prepared by either contacting sperm and eggs that have been obtained (such as by excision) from appropriate adults or by spawning. Induction of spawning may be achieved by any of the following methods:

(a) rapid raise of water temperature (in which the adults are placed) by from 5 to 10°C;

(b) injection of potassium chloride into an adult; or

(c) contact of the female with sperm or hydrogen peroxide. 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 one per ml. Preferred ranges are from 10 to 50 per ml, optimally from 15 to 30 per ml.

A fifth aspect of the present invention relates to a bioassay kit comprising cryopreserved larvae of the phylum Mollusca at, or beyond, the trocophore stage, and means for contacting a sample with the larvae. Thus the kit may comprise one or more containers or wells in which the larvae 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) .

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

The invention will now be described with reference to the accompanying drawings, in which:

FIGURE 2 is a graph illustrating the growth of manila clam larvae (Tapes philippinaru ) , both as in frozen controls and cryopreserved larvae in accordance with the present invention; and

FIGURE 3 is a graph illustrating the survival of pacific oyster larvae (Crassostrea αiqas) , both as unfrozen controls and cryopreserved larvae in accordance with the present invention.

The invention will now be described by way of example with reference to the following Examples, which are provided by way of illustration only and are not to be construed as being limiting on scope of the present invention.

Comparison Example 1

Mature animals of Tapes philippinarum (manila clam) were spawned by thermal shock and the sperm from several males was mixed. This was used to fertilise eggs of individual females which were then taken into culture. The fertilised eggs were maintained in sea water at a density of 50 per ml at a temperature of 25°C, without aeration. The sea water was freshly filtered to 0.45 microns immediately prior to the incubation, and had been UV sterilised. Chloramphenicol was added at lppm (as an antibiotic to control bacterial contamination of larvae) . At 2hrs post-fertilisation 10 microM glutamic acid was incorporated into the sea water. At 24hrs post- fertilisation the larvae were fed with an appropriate algal diet and collected at 48 hours post-fertilisation on a 35 micron sieve. Larvae were rinsed in clean sea water (without chloramphenicol) prior to incubation in cryoprotectant. The concentrated larvae were placed onto a 15 micron sieve and then as far as practicable all the sea water removed. The larvae were placed into a cryoprotective agent (CPA) 'at 20°C.

CPA: 0.425M Glucose, 0.425M trehalose, 15% v/v DMSO in distilled water.

Larvae were loaded into 0.5ml plastic straws coated with cholesterol and the cooling protocol started exactly 15 minutes after the start of the incubation in CPA.

The cooling protocol employed was:

20°C to -20°C at 15°C/minute hold at -20°C for 6 minute

-20°C to -35°C at 15°C/minute after no more than 1 minute at -35°C plunge into liquid nitrogen.

Thawing was by immersion of the straw in water at 75- 80°C until the last region of ice disappeared. The straw was removed immediately and wiped dry. The end of the straw was cut off and the contents expelled into 0.5ml of sea water at 20°C.

A further 1ml of sea water was added after 1 minute and then another 2ml at 1-2 minutes after that. After a further 2 minutes the supernatant was removed to reduce the suspension to a minimal volume without loss of material, and then 5ml of sea water added. After settling, the supernatant was again removed and replaced with sea water ready for final use.

Control larvae were obtained as outlined above except they were not subjected to cryopreservation. The size of the larvae were measured four times during growth (0,7, 26 and 35 days) either from thaw (in the case of cryopreserved larvae) or from the time that they would have been cryopreserved, had they not been controls (in the case of the controls) . The results are given in Figure 2. The survival rate of the cryopreserved larvae on day 35 after thawing was 84% of the uncryopreserved controls.

Comparison Example 2

The procedure of Comparative Example 1 was repeated except using Crassostrea qiqas (Pacific oyster) larvae. For the non-control larvae, the cooling protocol was commenced 24 hours post-fertilisation, and four replicates were used. The survival rates of each of the four (cryopreservation) replicates and control were measured 10 days after thawing (from 24 hours post- fertilisation of the control) , the results of which are given in Figure 3.

Example 3

A bioassay was conducted on two species of larvae using elutriates from sediment collected at nine sampling sites in the North Sea in the Spring of 1990. A reference was also conducted using clean water supplied by MAFF, Burnham on Crouch.

The larvae employed had been cryopreserved in 0.5ml straws and thawed as described in Comparative Example 1.

In each bioassay a 40ml sample was used, with a concentration of 10 larvae per ml, and incubated at 20°C. After exposure all the samples were formalin fixed for assessment.

Percentage Survival Sampling Station

Larvae Used 1 2 3 4 5 6 7 8 9 Ref

24 hr Crassostrea qjqas 78 80 86 92 78 86 92 94 92 98 48 hr Tapes philippinarum 54 74 92 - 80 - 94 - 98 98

Claims (21)

1. A process comprising cryopreserving larvae of the phylum Mollusca which are at, or beyond the trocophore stage.

2. A process as claimed in claim 1 wherein the larvae are of the class Bivalvia.

3. A process as claimed in claim 1 wherein the larvae are of the genus Mytilus. Ostrea. Tapes or Crassostrea.

4. A process as claimed in claim 1 wherein the larvae are at least 24 hours post-fertilisation.

5. A process as claimed in claim 1 wherein the larvae are cryopreserved in an aqueous medium.

6. A process as claimed in claim 5 wherein the aqueous medium is in contact with an cholesterol.

7. A process as claimed in claim 6 wherein the cholesterol is coated on a substrate.

8. A process as claimed in claim 7 wherein the cholesterol is coated on an ampoule, straw, bag or tube.

9. A process as claimed in claim 1 comprising:

proceeding either by (al) or (a2) ; (al) cooling the larvae at a rate of from 12 to 17°C/minute to a temperature of from -15 to -25°C;

(a2) cσoling the larvae to a temperature of from -40°C to -50°C at a rate of from 40 to 50°C/minute and then warming the larvae to a temperature of from -15 to -25°C at a rate of from 5 to 15°C/minute;

(b) maintaining the larvae at about that temperature for from 5 to 7 minutes;

(c) further cooling the larvae at a rate of from 12 to 17°C/minute to a temperature of from -30 to -40°C;

(d) optionally maintaining the larvae at that temperature for up to 2 minutes; and

(e) optionally plunging the larvae into liquid nitrogen.

10. A process as claimed in claim 9 comprising:

proceeding either by (al) or (a2) ;

(a) cooling the larvae at a rate of about 15°C/minute to a temperature of about -20°C;

(a2) cooling the larvae to a temperature of from -44 to -46°C at a rate of about 45°C/minute and then warming the larvae to a temperature of about -20°C at a rate of about 10°C/minute;

(b) maintaining the eggs at about -20°C for about 6 'minutes;

(c) further cooling the larvae to a temperature of about -35°C;

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

(e) plunging the larvae into liquid nitrogen.

11. Cryopreserved prodissoconch larvae of the phylum Mollusca that are at, or beyond, the trocophore stage.

12. Larvae as claimed in claim 11 which have been cryopreserved by a process as claimed in any of claims 1 to 10.

13. A method of breeding larvae of the phylum Mollusca comprising:

(a) cryopreserving larvae of the phylum Mollusca that are at, or beyond, the trocophore stage; and

(b) thawing the larvae.

14. A method as claimed in claim 13 using a process as claimed in any of claims 1 to 10.

15. A method of conducting a bioassay, the method comprising contacting cryopreserved larvae of the phylum Mollusca that are at, or beyond, the trocophore stage, with a sample to be assayed.

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

17. A method as claimed in claim 15 wherein the larvae are at, or beyond, the prodissoconch I stage.

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

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

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

21. A bioassay kit comprising cryopreserved larvae of the phylum Mollusca at, or beyond, the trocophore stage, and means for contacting a sample with the larvae.