RU2295566C2 - Method for cryoconservation of dog's tail beroe ovata - Google Patents

Abstract

FIELD: biotechnology, method for cryoconservation of biological cells for prolonged storage.

SUBSTANCE: claimed method includes selection of fertilized dog's tail Beroe ovata eggs in step of 2-4 blastomeres. Then eggs are placed in barrier solution, frozen by dipping in liquid nitrogen followed by storage therein. Further eggs are defrosted followed by washing thereof in washing solution. Sea water used as physiological medium for barrier and washing solutions is preliminary sterilized by filtration through membrane filter.

EFFECT: increased yield of non-damaged dog's tail eggs being capable of further development.

2 tbl, 8 ex

Description

The present invention relates to the field of biotechnology, in particular to methods of cryopreservation for long-term storage of biological cells, and can be used in fish farming for bio-cultivation and production of Beroe ovata ctenophores at the right time. The crest Beroe ovata is known as a stenophage capable of suppressing and regulating the size of the population of its prey, the ctenophore Mnemiopsis leidyi A. Agassiz. The latter causes great damage to fisheries, eating away the food base of valuable species of planktophagous fish [1].

The mass development of the predator Mnemiopsis leidyi A. Agassiz is observed in spring and early summer, while the mass development of the ctenophore Beroe ovata begins in the 2nd half of summer, when damage to the biota has already been done.

This implies the task of timely artificial population of ctenophore Beroe ovata into the developing population of ctenophore Mnemiopsis leidyi A. Agassiz to suppress it, without relying on the natural and belated course of events.

The maintenance of a live culture of Beroe ovata ctenophore in artificial conditions is a high-cost technology, therefore a less costly method of obtaining culture in the required time was developed using cryopreservation of fertilized Beroe ovata eggs in the early stages of ontogenesis with their subsequent revitalization.

A known method of storing cells by freezing [2], in which the primary cells or cultured cells of animals or humans in the presence of vitrified (vitrified liquid) is introduced dropwise into liquid nitrogen.

A known method of manufacturing a solution for preservation of cells by freezing [3], in which glycerol and polyethylene glycol (PEG) are added to the main solution. The amount of glycerol is about 10-20 v / v%. The amount of PEG is about 5-10% w / v.

Known methods of cryopreservation for the long-term storage of objects are unsuitable for cryopreservation of ctenophores. This is due to the fact that cryopreservation involves deep cooling of objects to the temperature of liquid nitrogen (-196 ° C). The main problem when freezing objects is free water. The resulting ice crystals damage the intracellular and tissue structures of objects, therefore, during thawing, decompartition of individual structures occurs, which leads to the development of necrotic changes and death of objects. Being the most watered of aquatic animals, ctenophores are one of the most difficult objects for cryopreservation. The task is also complicated by the fact that in ctenophores, both in natural and in artificial conditions, from a large number of eggs being swept out, only a small fraction (usually less than 1%) is capable of further division and development.

Closest to the proposed invention is the selected as a prototype method of cryopreservation of the larvae of the ctenophore Beroe ovata [4], in which the fertilized eggs (embryos) of the ctenophore are placed in the enclosing solution in a volume equal to 50% of the sample volume and mixed. The enclosing solution contains 40 g of mannitol and 300 ml of glycerol in 1000 ml of filtered seawater as cryoprotectants. Sea water is used as a physiological medium that maintains the desired pH value.

Then carry out freezing by immersion in liquid nitrogen, followed by storage in it.

When defrosting, the desired container is removed from the storage and thawing is carried out in a bath with water at a temperature of + 30 ° С. After thawing, thawed eggs of the Beroe ovata ctenophore are washed in two stages in a washing solution, which is marine filtered water with sodium added (22 g of sodium chloride in 1000 ml of marine filtered water).

The yield of viable ctenophore eggs obtained in a known manner is less than 1%.

The disadvantage of this method is the relatively low proportion of the output of undamaged ctenophore eggs capable of further development.

The objective of the invention is to increase the yield of intact ctenophore eggs capable of further development.

This object is achieved by the fact that in the known method, comprising placing fertilized ctenophore eggs in the enclosing solution, freezing by immersion in liquid nitrogen, followed by storage in it, thawing, followed by washing thawed ctenophore eggs in two stages in a washing solution and using it as physiological environment for enclosing and washing solutions of sea water, according to the invention for cryopreservation, fertilized ctenophore eggs are selected at the stage of 2-4 blastomeres, and seawater is pre-sterilized by filtration at a Komovsky pump through a membrane filter.

The proposed method improves the safety of ctenophore eggs due to their selection at the optimal development stage for cryopreservation, i.e. when the ratio of the volume of the nucleus to the volume of the cytoplasm in the cell is minimal, which facilitates the "binding" of intracellular fluid by cryoprotectors and reduces the risk of damage to subcellular structures.

Sterilization of seawater used in enclosing and laundering solutions by filtration at a Komovsky pump through a membrane filter increases the survival of ctenophore eggs by protecting them from contact with bacteria, fungi and protozoa that can destroy them in a matter of hours after thawing.

The set of distinctive features of the described method ensures the achievement of the task.

Comparison of the prototype with the claimed solution showed that the above signs are distinctive, and therefore, the claimed method meets the criterion of "novelty."

The method is as follows.

A suspension of individually selected fertilized Beroe ovata ctenophore eggs at the stage of 2-4 blastomeres is placed in the enclosing solution, and then immersed in liquid nitrogen, followed by storage in it. At the right time, defrosting is carried out, and then the thawed ctenophore eggs are washed in two stages in a washing solution. At the same time, seawater is used as physiological medium for the enclosing and washing solutions, which are pre-sterilized by filtration on a Komovsky pump through a membrane filter.

Example 1

The individually selected fertilized eggs (embryos) of Beroe ovata, which are at different stages of crushing and do not have visible damage and deformities, were divided into groups corresponding to the stages of crushing in 1, 2, 4, 8 and more blastomeres.

The sea water used to prepare the media during freezing and thawing of each group of Beroe ovata ctenophore eggs is filtered through a regular narrow-pore blue ribbon filter.

The selected Beroe ovata ctenophore eggs, which are at the stage of crushing, are placed in 1 blastomere in a plastic container with 2-3 ml of sea water. Then add the enclosing solution in a volume equal to 50% of the sample volume, and mix. The enclosing solution contains 40 g of mannitol and 300 ml of glycerol in 1000 ml of sea filtered water. The container is closed with a lid, placed in a cuvette and quickly frozen, immersed in a Dewar vessel with liquid nitrogen.

Thawing is carried out by transferring the container to the bath for defrosting with a water temperature of + 30 ° C. Thawed ctenophore eggs are washed in two stages. At the first stage, the solution for washing the sample contains 22 g of sodium chloride in 1000 ml of sea filtered water. This solution is added to thawed ctenophore eggs. The ratio of the added solution to the sample volume is 1: 1. Stand for 15 minutes, then add a solution for the second stage of washing, containing 12 g of sodium chloride in 1000 ml of sea filtered water. The ratio of the added solution volume to the total sample volume is 1: 1. After 15 minutes, sterilized sea water is added in a ratio of 1: 1 with a total volume of water. After 10-15 minutes, the test with thawed ctenophore eggs is transferred to a large volume of marine aerated water and the further development of embryos is monitored.

The results of monitoring the development of thawed eggs of the ctenophore Beroe ovata are given in table 1.

Examples 2-4

According to example 1, freezing and thawing of ctenophore eggs was carried out, which are at the stages of crushing of 2, 4, 8 blastomeres and have no visible damage and deformities.

The results of monitoring the development of thawed ctenophore eggs 1, 2, 4, 8 blastomeres are given in table 1.

Table 1 No. Stage crushing eggs of the ctenophore Beroe ovata before
frozen in blastomeres Intact Beroe ovata eggs 1 hour after defrosting,% The surviving eggs of Beroe ovata 24 hours after defrosting,% Eggs of Beroe ovata, which continued their further development,% one one four 0 0 2 2 fifteen four 0.6 3 four twenty 6 one four 8 12 2 0

From table 1 it is seen that during cryopreservation of Beroe ovata eggs at the stage of crushing in 1 blastomere after defrosting, the number of intact eggs with no visible signs of violation of the outer shell was 4% of the initial one. However, all surviving Beroe ovata eggs turned out to be non-viable and collapsed within 24 hours after defrosting.

During cryopreservation of ctenophore eggs at the crushing stage of 2 blastomeres after thawing, the number of undamaged Beroe ovata eggs that do not have visible signs of damage to the outer shell and blastomeres amounted to 15% of the initial one. After 24 hours, the number of preserved embryos was 4% of the original. The development of 0.6% of the initial number of eggs continued.

During cryopreservation of Beroe ovata ctenophore eggs at the crushing stage of 4 blastomeres after defrosting, the number of intact Beroe ovata embryos that did not have visible signs of damage to the outer shell was 20% of the original. Of these, 6% of Beroe ovata ctenophore eggs survived 24 hours after defrosting, and 1% of the initial number of eggs continued to develop.

During cryopreservation at the stage of crushing of 8 blastomeres after defrosting, the number of intact Beroe ovata ctenophore eggs that do not have visible signs of a violation of the outer shell and blastomeres amounted to 12% of the initial one. After 24 hours, 2% of Beroe ovata ctenophores were preserved. However, not one of the surviving ctenophore eggs continued its further development.

Thus, it can be seen from Table 1 that during cryopreservation at the stage of 2-4 blastomeres, the number of apparently intact and viable ctenophore eggs is the largest and only at these stages of crushing does a part of the embryos continue to develop after defrosting.

This is explained by the lowest total water cut and the minimum ratio of the nucleus volume to the cytoplasm volume in the blastomeres at this stage of fragmentation, which facilitates the “binding” of intracellular fluid by cryoprotectants and reduces the risk of damage to subcellular structures.

Examples 5-6.

To prepare the physiological environment for the enclosing and laundering solutions, sea water was used, filtered through an ordinary narrow-porous blue-ribbon type paper filter. Beroe ovata ctenophore eggs were used as samples at the crushing stages of 2 and 4 blastomeres, in which ctenophore eggs survived 24 hours after defrosting.

Freezing and thawing of the samples was carried out as described in examples 1-4.

Example 7-8.

As physiological medium for the enclosing and washing solutions, sterilized sea water was used, which was obtained by filtration at a Komovsky pump through a membrane filter.

Beroe ovata ctenophore eggs were used as a sample at the crushing stage in 2 and 4 blastomeres, which had the best survival rates after defrosting and continued their further development. Freezing and thawing of the samples was carried out as described in examples 1-4.

Table 2 shows the results for Beroe ovata ctenophore eggs at a 2.4 blastomere crushing stage, where sea water filtered through a conventional narrow-band blue-ribbon paper filter and sterilized sea water, which was obtained, were used as enclosing and washing solutions. filtration on a Komovsky pump through a membrane filter.

table 2 Sea water The stage of crushing eggs of the ctenophore Beroe ovata before freezing in blastomeres Intact Beroe ovata eggs 1 hour after defrosting,% The surviving eggs of Beroe ovata 24 hours after defrosting,% Eggs of Beroe ovata, which continued their further development,% Filtered 2 fifteen four 0.6 Sterilized 23 7 1,0 Filtered four twenty 6 1,0 Sterilized thirty 10 1,5

From table 2 it is seen that during the sterilization of seawater, the proportion of intact viable embryos in unreserved material increases.

Using the proposed method will increase the yield of intact embryos of ctenophore Beroe ovata, capable of further development.

Information sources

1. The book "Grebnevnik Mnemiopsis leidyi A. Agassiz in the Sea of Azov and the Black Sea: biology and the consequences of the introduction" under the scientific. ed. S.P. Volovik. - Rostov-on-Don, BKI, 2000 - pp. 423-449-ISBN 5-85796-057-6.

2. Japanese Patent No. 3044323, MKI C 12 N 5/06.

3. Japan patent No. 3025931, MKI C 12 N 5/06.

4. Collection of scientific papers (2000-2001) "The main problems of fisheries and the protection of fish-breeding ponds of the Azov-Black Sea basin." - M., 2002. The State Committee of the Russian Federation for Fisheries, FSUE AzNIIRKh, pp. 238-243.

Claims (1)

Method for cryopreservation of BEROE OVATA ctenophore, including the placement of fertilized ctenophore eggs in the enclosing solution, freezing by immersion in liquid nitrogen and storing in it, thawing, followed by washing the thawed eggs in two stages in the washing solution and using as a physiological medium for the enclosing and washing solution water, characterized in that for cryopreservation, fertilized ctenophore eggs are selected at the crushing stage of 2-4 blastomeres, and sea water is pre-sterilized filtration on a Komovsky pump through a membrane filter.