RU2314687C1 - Method for cryoconservation of cells in sea invertebrates - Google Patents

Abstract

FIELD: medicine, biotechnology.

SUBSTANCE: the present innovation deals with treating embryonic cells of sea invertebrates with cryoprotector mixture that includes lipids, sugars, antioxidants and saline solution. As lipids one should apply lipid extracts of sea hydrobionts at low temperatures of phase transitions which should be pre-treated with ultrasound. Cryoprotector mixture should used at a certain ratio of the components taken, weight%. Moreover, cryoprotector mixture should be additionally supplemented with 5-10%-dimethyl sulfoxide (DMSO). The innovation enables to increase the degree of conservation of cells in sea invertebrates without affecting their function activity after freezing-defrosting due to keeping the integrity and the stability of their cell membranes and provides the chance for evaluating cell conservation quantitatively.

EFFECT: higher efficiency.

1 cl, 2 dwg, 7 ex, 2 tbl

Description

The invention relates to biotechnology, in particular to methods of cryopreservation of biological material, namely cells of marine invertebrates, and can be used in cryobiology, cell biology and marine biotechnology to preserve marine invertebrate cells.

The problem of cryopreservation of cells of marine invertebrates is very relevant. In conditions of increasing anthropogenic impact on the oceans and the animals inhabiting it, it is necessary to preserve the gene pool of marine organisms. Cryopreservation of germ cells and embryos is one of the promising ways to preserve the gene pool of commercial, as well as rare and endangered species of animals and plants. The development of cryopreservation methods for marine invertebrates will remove geographical restrictions and dependence on the breeding season, which may open the way for the creation of new biotechnologies.

While sperm freezing is a routine for many species, freezing of oocytes and embryos remains problematic today. Conventional cryoprotectants used in freezing mammalian cells are ineffective for these objects. Perhaps this is due to the fact that marine invertebrates are poikilothermic animals, and their thermal acclimation occurs in a fairly wide temperature range. Marine invertebrates are not able to quickly adapt the viscosity of the membrane lipid matrix to sudden changes in temperature (Sanina NM, Kostetsky E. Ya. Thermotropic behavior of major phospholipids from marine invertebrates: changes with warm acclimation and seasonal acclimatization. Comp. Biochem. Physiol. 2002. 133B: 143-153). The special fatty acid composition of the cell membranes of marine animals and plants, in which unsaturated fatty acids predominate, requires fundamentally new cryoprotectants.

Known methods for cryopreservation of cells, embryos and larvae of marine invertebrates by introducing into the cryoprotective mixture of various biologically active substances: mono - and disaccharides (Crowe JH, Crowe LM. Preservation of Membranes in Anhydrobiotic Organisms: The Role of Trehalose. Science, 1984, V.223 : 701-703), antifreeze proteins (Theede H., Schneppenheim, Beress L. .. Antifreeze Glycoproteins in Mytilus edulis. Marine Biology, 1976, V. 36: 183-189; Rubinsky B., Arav A., Devries AL Cryopreservation of Oocytes Using Directional Cooling and Antifreeze Glycoproteins. Cryo-Letters, 1991, V.12: 93-106; Arav A., Rubinsky B., Fletcher G., Seren E. Cryogenic Protection of Oocytes with Antifreeze Proteins, Molecular Reproduction and Development, 1993, V.36: 488-493), lipids (Simpson AM, Swan MA, White I .G. Action of phosphatidylcholine in protecting ram sperm from cold shock. Gamete Res., 1986, V.15: 43-56; Graham JK, Foote RH Effects of several lipids, fatty acyd chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing. Cryobiology, 1987, V.24: 42-52; Odintsova N.A., Kiselev K.V., Sanina N.M., Kostetsky E.Y. Cryopreservation of primary cell cultures of marine invertebrates. Cryo-Letters, 2001, V.22: 299-310; Odintsova NA, Ageenko NV, Kiselev KV, Sanina NM, Kostetsky EY. Analysis of marine hydrobiont lipid extracts as possible cryoprotective agents. Int. J. Refrigeration, 2006.29: 387-395). The introduction of antioxidants into cryoprotective mixtures is also known, for example, alpha-tocopherol (vitamin E) by freezing immature hematopoietic cells (Matthes G, Hubner U, Granz M, Schutt M, Hackensellner HA. Antioxidants increase the cryoresistance of GM-CFC. 1987. Folia Haematol Int Mag Klin Morphol Blutforsch. 114 (4): 482-483), ascorbate for freezing mouse embryos (Lane M, Maybach JM, Gardner DK. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. 2002. Human Reproduction 17 ( 10); 2686-2693), echinochrome - naphthainoid pigment from sea urchins when freezing oyster larvae and sea urchins (Naidenko TKh. Cryopreservation of Crassostrea gigas oocytes, emb ryos and larvae using antioxidant echinochrome A and antifreeze protein AFP1. 1997. Cryo-Letters 18: 375-382).

However, in the above methods, only a negligible effect of the cryoprotectants used on the biological activity of frozen cells and larvae was noted.

There is a method of cryopreservation of marine invertebrate larvae by using a mixture of dimethyl sulfoxide (DMSO), trehalose and antioxidants, in particular echinochrome (area of tested concentrations: 1-100 μg / ml) for oyster cells and sea urchins (Naydenko T.Kh., Koltsova E.A. Use of the antioxidant echinochrome A during cryopreservation of embryos and larvae of sea urchins. Sea Biology 1998, T.24, N 3, S.198-201).

The disadvantage of this method is only a qualitative assessment of the viability, mobility and development of embryos and larvae of marine invertebrates, which largely reflects the subjective approach of the study. As a result, it is impossible to assess the degree of preservation of embryos and larvae of marine invertebrates after thawing. In addition, our quantitative experiments on the freezing of marine invertebrate embryonic cells using the same cryoprotective mixture showed that the positive effect of echinochrome is absent or extremely insignificant compared to cells frozen simply in the presence of DMSO, as can be seen on the cell viability graphs presented in FIG. .1 and 2. Figure 1 presents data on the viability of mollusk cells, and figure 2 - echinoderms, where 1) unfrozen cells; 2) frozen in the presence of 5% DMSO; 3) in the presence of DMSO + 1% echinochrome (natural); 4) frozen in the presence of DMSO + 1% echinochrome (synthetic). The number of viable cells after freezing and thawing in the presence of cryoprotectants used in this experiment is almost the same: about 5% of live mussel cells frozen in the presence of DMSO only (Fig. 1, column 2) and the same number when using a mixture of DMSO and echinochrome, both synthetic and natural (Fig. 1, columns 3 and 4). The number of living cells of the sea urchin after freezing in the presence of DMSO only (Fig. 2, column 2) was about 10%, while the number of living cells frozen in the presence of DMSO and echinochrome ranged from 9 to 11% (Fig. 2, columns 3 and 4).

The closest in number of essential features and technical result achieved to the invention is a method for cryopreservation of mollusk larvae, including treating them with a cryoprotective mixture containing 0.1-10 M (0.3-30%) aqueous solution of polysaccharides, in particular sugar, glucose or trehalose, and / or cryoprotectants such as DMSO or glycerin (5-15%), as well as cholesterol (0.25-1.5 mg / ml). (WO 91/01636, IPC A01N 1/02, G01N 33/50, 1991).

An aqueous solution of polysaccharides is in contact with cholesterol, which is used as a substrate and is applied to the surface of cryovials, tubes, etc. After thawing, the larvae frozen in this cryoprotective mixture during the first week lagged behind the development of control unfrozen larvae, and after 35 days the control and thawed larvae had the same dimensional characteristics.

The disadvantage of this method is that it was tested only on mollusk larvae and there is no data on the percentage of surviving larvae after freezing and thawing. Based on the biological material obtained after thawing, it is practically impossible to quantify the effectiveness of this cryoprotectant, and, as a result, it is impossible to talk about the degree of preservation of larval cells.

The objective of the invention is to increase the degree of preservation of cells of marine invertebrates without disturbing their functional activity after freezing and thawing by maintaining the integrity and stability of their cell membranes and the possibility of quantitatively assessing the safety of cells.

The problem is solved in that in the known method of cryopreservation of biological material, including processing biological material with a cryoprotective mixture containing a solution of sugars and lipids, embryonic cells of marine invertebrates are taken as biological material, and lipid extracts of marine hydrobionts with low temperatures are used as lipids phase transitions that are pre-treated with ultrasound. To prepare the cryoprotective mixture, a saline solution is used, into which antioxidants are additionally added. The cryoprotective mixture is used in the following ratio of components in wt.%:

Sugar solution 1.0-10.0 Lipid extracts 0.08-0.15 Antioxidants 0.3-1.0 Saline solution rest

An additional 5-10% dimethyl sulfoxide (DMSO) can be added to the cryoprotective mixture.

The achievement of the claimed technical result is ensured by using as an object of study the embryonic cells of marine invertebrates obtained by dissociation of larvae of different stages of development. The use of cells rather than whole embryos and larvae as a model system allows one to quickly (within 24 hours) and quantify the effect of various cryoprotectors on the integrity and functional activity of cells. In the method, in particular, embryonic cells of mollusks and sea urchins of various species can be used.

As lipid extracts, lipid extracts of marine hydrobionts with low crystal-liquid crystal phase transition temperatures are used, in particular lipid extracts of mussel, green ulva algae or brown sargassum algae, which are characterized by a large amount of lipids with unsaturated fatty acids. Most of the thermograms of these lipid extracts are localized at a temperature below 0 ° C, which probably determines the special behavior of these lipids during cryopreservation and ensures the achievement of the claimed technical result.

However, when more than 0.15% lipid extract is introduced into the cryoprotective mixture, toxic effects may occur due to the presence of methanol and chloroform in the lipid extracts, with which lipids are secreted, and when using less than 0.08% lipid extract, the effectiveness of this cryoprotective mixture starts decrease and does not provide an increase in the degree of preservation of cells.

It is advisable to pre-treat the lipid extracts of marine aquatic organisms with low temperatures of phase transitions with ultrasound (22 kHz, 1-2 min). This allows you to get a stable microemulsion. Its stability, which is controlled by the size of emulsified particles (for example, on a particle counter from Malvern, England), is maintained for 3-4 days.

The introduction of lipid extracts in the form of an emulsion into the cryoprotective mixture helps to restore the destruction area, and also prevents membrane damage during freezing-thawing, thereby ensuring the achievement of the claimed technical result - increasing the degree of preservation of embryonic cells without impairing their functional activity.

It should be noted that the lipid extracts of marine hydrobionts with low phase transition temperatures, introduced into the composition of the cryoprotective mixture, perform a function different from that performed by the lipid in the prototype. In the prototype, cholesterol (lipid) is used as a substrate, which is applied to the surface of cryovials, tubes, etc. In the claimed technical solution, lipid extracts are an essential component of the cryoprotective mixture and contribute to increasing the degree of preservation of cells.

For the preparation of a cryoprotective mixture, a nutrient medium (with osmosis equal to sea water), or artificial sea water, or sterilized natural sea water can be used as a saline solution.

A 1.0-10.0% sugar solution, in particular trehalose, which acts as a membrane stabilizer, is introduced into the cryoprotective mixture. In practice, a 0.1-10 M sugar solution is usually used. The introduction of less than 1.0% trehalose does not increase the degree of preservation of embryonic cells of marine invertebrates, and the addition of more than 10.0% trehalose to the cryoprotective mixture reduces the viability of marine invertebrate cells after freezing.

0.3-1.0% of antioxidants (natural or synthetic, such as α-tocopherol, ascorbic acid or echinochrome) are introduced as an additional component of the cryoprotective mixture. The introduction of antioxidants into the cryoprotective mixture apparently prevents oxidative stress during freezing of stem cells, embryos and larvae. Adding antioxidants at a concentration of less than 0.3% does not increase cell viability after freezing and thawing compared to cryoprotective mixtures without antioxidants, and at a concentration of more than 1%, toxic effects of antioxidants can be observed.

The introduction of the cryoprotective mixture of 5-10% DMSO, the most commonly used cryoprotectant for cryopreservation of biological material, also allows to achieve the claimed technical result. However, its use is not preferred, since at positive temperatures it exhibits a toxic effect.

As a result of a patent search, information sources were found that individually use each of the components of the claimed cryoprotective mixture, as noted in the description. However, the use of a combination of the three most important components as cryoprotectants has significantly reduced the loss of biological activity of marine invertebrate cells due to the synergistic action of all components of the used cryoprotective mixture. Each of the components affects the integrity of cell membranes. However, we found that in this case, not the total effect of all three cryoprotectants is observed, but their cumulative effect. Antioxidants (α-tocopherol, ascorbic acid and echinochrome) were only effective when used together with lipid extracts.

The method is as follows.

Marine invertebrate embryonic cells are obtained by methods known in the art. So, in particular, sea urchins (Strongylocentrotus intermedius, St. nudus and Echinorachnius mirabilis) are induced to spawn by electric shock or by injection into the animal cavity of a 0.5 M KCl solution. Fertilized eggs are grown to the desired stage of development, the larvae dissociate into cells using 0.125% collagenase solution for 30-120 minutes at room temperature. Then the cell suspension (10-30 × 10 ⁶ cells / ml) is transferred into polypropylene cryovials and a cryoprotective mixture is added. Next, cryovials are kept for 20 minutes in an ice bath, after which they are placed in a glycerol-alcohol bath in a laboratory setup for freezing at a temperature of -25-30 ° C (approximate freezing rate of 5-6 ° / min). After 15-20 minutes, frozen samples are transferred to a container with liquid nitrogen. Thawing is carried out in a water bath with vigorous stirring at a temperature of 20-22 ° C. Non-frozen cells are used as a control. The effectiveness of the method is evaluated using the well-known method of determining viability by directly counting the number of cells in the Goryaev’s chamber in the presence of the trypan blue vital dye, and the physiological activity of the cells is assessed by the level of RNA synthesis (Odintsova NA, Belogortseva NI, Ermak AV, Molchanova VI, Luk'yanov PA Adhesive and growth properties of lectin from the ascidian Didemnum ternatanum on cultivated marine inverte invertebrate cells. Biochem. Biophys. Acta. 1999. V.1448: 381-389).

Example 1

Spawning of flat sea urchins Echinarachnius mirabilis, induced by electric shock. Artificially fertilized eggs are grown to the gastrula stage (24 hours after fertilization at 17 ° C) and dissociated into cells by treatment with a 0.1% collagenase solution for 20-30 minutes at 15 ° C. The resulting cell suspension is washed twice in sterile sea water, containing penicillin 500 units / ml and gentamicin 40 μg / ml.

0.33 ml suspension of dissociated cells (10-15 × 10 ⁶ cells / ml) is placed in polypropylene cryovials (2 ml) and 0.67 ml of the cryoprotective mixture, the composition of which is indicated in the table, is added. Natural echinochrome is introduced into the cryoprotective mixture as an antioxidant, lipid extract from the tissues of mussel Crenomytilus grayanus (pretreated with ultrasound) - MBE is used as a lipid extract, and Leibovich nutrient medium is used as a saline solution. Thus, the cryoprotective mixture includes 1% trehalose, 0.08% MBE, 1% echinochrome, and 97.92% Leibovich culture medium.

The mixture is frozen as described above. Then the cells are thawed in a water bath with vigorous stirring at a temperature of 20-22 ° C. A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

The results of determining the viability of dissociated gastrula cells of a flat sea urchin and RNA synthesis after cryo-freezing in various cryoprotective mixtures are presented in Table 1. The viability of cells frozen in the cryoprotective mixture proposed in this example is almost 14 times higher than the viability of cells frozen without cryoprotectants and the level of RNA synthesis almost reaches the level of RNA synthesis of cells before freezing (per 1 million living cells).

Example 2

Take sea urchins Strongylocentrotus intermedius. Spawning of sea urchins is induced by injecting 0.2-0.4 ml of a 0.5 M KCl solution into the animal cavity. Artificially fertilized eggs are grown to the blastula stage, placing them for 12 hours in vessels with sterile sea water at 17 ° C. Blastula dissociate into cells by treatment with 0.1% collagenase solution for 20-30 minutes at 15 ° C. The resulting cell suspension is washed twice in sterile seawater containing penicillin 500 units / ml and gentamicin 40 μg / ml.

0.33 ml of a suspension of dissociated cells (10-15 × 10 ⁶ cells / ml) are placed in polypropylene cryovials (2 ml) and 0.67 ml of cryoprotective mixture is added. The composition of the cryoprotective mixture includes 0.5% natural echinochrome; 0.15% lipid extract from the tissues of the green algae Ulva fenestrat (ULE), pre-treated with ultrasound; 10% trehalose; the remaining percentage is saline, which is used as sterilized marine natural water. The mixture is frozen as described above. Then the cells are thawed in a water bath with vigorous stirring at a temperature of 20-22 ° C. A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

Table 1 presents the results of determining the viability of dissociated sea urchin blastula cells and the synthesis of RNA in them after cryogenic freezing. Blastula cells are less resistant to various damaging effects, therefore, the difference in the viability of these cells frozen in the presence of the proposed cryoprotective mixture is 8 times less than the viability of cells frozen without cryoprotectants, and the level of RNA synthesis does not reach the level of RNA synthesis of cells before freezing - it is approximately 60% of that, but 20 times higher than that in cells frozen without cryoprotectants.

Example 3

The embryos of sea urchins S. intermedius from the blastula stage are taken, as described in example 2. All subsequent procedures are the same as in example 2, except that the composition of the cryoprotective mixture consists of: 0.5% natural echinochrome; 0.15% lipid extract from the tissues of the green alga Ulva fenestrata, pre-treated with ultrasound (ULE); 8% trehalose solution; the remaining percentage is saline, which is used as sterilized marine natural water, in which dimethyl sulfoxide (DMSO) is present in an amount of 5%.

A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

Table 1 presents the results of determining the viability of sea urchin cells after cryogenic freezing. As in the previous example, the viability of these cells frozen in the presence of the proposed cryoprotective mixture is 8 times higher than the viability of cells frozen without cryoprotectants. However, the level of RNA synthesis is less than in cells frozen in such a mixture, but without DMSO, about two times, but it is 10 times higher than that in cells frozen without cryoprotectants.

Example 4

Spawning of flat sea urchins Echinarachnius mirabilis is induced by electric shock. Artificially fertilized eggs are grown to the gastrula stage (24 hours after fertilization at 17 ° C) and dissociated into cells, as described in example 1. All subsequent procedures are the same as in example 1, except that the composition of the cryoprotective mixture consists of : 1% synthetic echinochrome; 0.08% lipid extract from the tissues of the mussel C. grayanus (MBE), (pre-treated with ultrasound); 10% trehalose; the remaining percentage is the saline solution, which is used as a modified Leibovich culture medium (Odintsova et al., 1999. V.1448: 381-389), in which dimethyl sulfoxide (5% DMSO) is present.

A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

Table 1 presents the results of determining the viability of dissociated gastrula cells of a flat sea urchin and the synthesis of RNA in them after cryogenic freezing.

Gastrula cells are more resistant to various damaging effects, therefore, the difference in viability of these cells frozen in the presence of the proposed cryoprotective mixture is 1.5 times less than cells frozen simply in a solution containing DMSO and trehalose, but the level of RNA synthesis in their cells increased by more than 7 times (based on 1 million living cells). These data, as in Example 1, prove the cumulative effect of the used components of cryoprotective mixtures, such as a membrane stabilizer (10% trehalose), exogenous lipid extract (0.08% MBE) and antioxidant (1% natural echinochrome) in the presence of DMSO .

The viability of gastrula cells of a flat sea urchin frozen in the presence of the inventive cryoprotective mixture is almost 14 times higher than the viability of cells frozen without cryoprotectants, and the level of RNA synthesis almost reaches the level of RNA synthesis of cells before freezing and is approximately equal to that in cells frozen in a similar cryoprotective mixture, but in the presence of natural echinochrome.

Example 5

Take mussels Mytilus trossulus. Spawning is induced by thermal shock (from 10 to 20 ° C). Artificially fertilized eggs are grown to the trochophore stage (24 hours after fertilization at 17 ° C) and dissociated into cells, as described in example 1. All subsequent procedures are similar to examples 1-4. The composition of the cryoprotective mixture: alpha-tocopherol (vitamin E) - 0.3%, 0.15% lipid extract from mussel tissue (MBE), pre-treated with ultrasound; 10% trehalose; the remaining percentage is saline, which is used as a modified Leibovich nutrient medium in which dimethyl sulfoxide (10% DMSO) is present.

A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

Table 1 presents the results of determining the viability of the trochophore mussel cells after cryogenic freezing. Despite the fact that mollusk cells are more sensitive to damage after freezing-thawing, the viability of mussel embryonic cells frozen in the presence of the proposed cryoprotective mixture increased by 7.3 times compared to cells frozen simply in a solution containing 10% DMSO and trehalose.

Example 6

Perform similarly to Example 5 except that a mixture of vitamins E and C is introduced into the composition of the cryoprotective mixture as an antioxidant - 0.5% each.

A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells are used, as well as cells frozen without the addition of a cryoprotective mixture.

Table 1 presents the results of determining the viability of the trochophore mussel cells after cryogenic freezing. It is noteworthy that the best cryoprotective effect is observed when using a mixture of two vitamins - vitamin E and C. as antioxidants.

As can be seen from the results presented in table 1, the claimed method of cryopreservation of marine invertebrate cells allows very quickly to obtain marine invertebrate cells with high functional activity after freezing-thawing.

Example 7

Take sea urchins Strongylocentrotus intermedius. Spawning of sea urchins is induced by injecting 0.2-0.4 ml of a 0.5 M KCl solution into the animal cavity. Artificially fertilized eggs are grown to the blastula stage, placing them for 12 hours in vessels with sterile sea water at 17 ° C. Blastula dissociate into cells by treatment with 0.1% collagenase solution for 20-30 minutes at 15 ° C. The resulting cell suspension is washed twice in sterile seawater containing penicillin 500 units / ml and gentamicin 40 μg / ml.

0.33 ml of a suspension of dissociated cells (10-15 × 10 ⁶ cells / ml) are placed in polypropylene cryovials (2 ml) and 0.67 ml of cryoprotective mixture is added. For more clear evidence of the manifestation of a synergistic effect in this example, cryoprotectants of various compositions are used, which are shown in the table (Examples 7b-7d).

The mixture is frozen as described above. Then the cells are thawed in a water bath with vigorous stirring (20-24 ° C).

A quantitative degree of preservation of cells is carried out by assessing cell viability and the level of RNA synthesis. As control, unfrozen cells and cells frozen without cryoprotectant are used.

Table 2 presents the results of the influence of the composition of the components of the cryoprotective mixture on the viability of the dissociated cells of the sea urchin blastula and the synthesis of RNA in them after cryogenic freezing.

The viability of the blastula cells of sea urchins frozen in the presence of the inventive cryoprotective mixture increased 1.75 times compared with cells frozen simply in a solution containing 10% DMSO and trehalose, and the level of RNA synthesis in their cells increased by more than 4 times. The use of a combination of three major components as cryoprotectants has significantly reduced the loss of biological activity of marine invertebrate cells due to the synergistic action of all components of the used cryoprotective mixture. Each of the components affects the integrity of cell membranes. However, we found that in this case, not the total effect of all three cryoprotectants is observed, but their cumulative effect. It is noteworthy that for the cells of sea urchins, the cumulative effect of the used components of cryoprotective mixtures was high both in the absence and in the presence of dimethyl sulfoxide (DMSO).

table 2 Example No. Object control cells The composition of the cryoprotective mixture Cell viability RNA synthesis level, pulses / 10 ⁶ cells Living cells,% Dead cells,% Destroyed cells,% one 2 3 four 5 6 7 Example 7. Sea urchin Strongylocentrotus intermedius blastula cells unfrozen cells (positive control) 99.2 ± 0.2 0.8 ± 0.11 - 22000 ± 500 7a cells frozen without cryoprotective mixture (negative control) 2.6 ± 0.6 17.2 ± 0.2 80.2 ± 0.6 600 ± 200 7b 10% DMSO + 90% arts, sea water 9.6 ± 0.6 17.8 ± 0.97 72.6 ± 2.15 1800 ± 200 7c 10% DMSO + 1% trehalose + 89% arts, sea water 16.5 ± 1.2 21.9 ± 1.1 61.6 ± 1.8 2100 ± 200 7g 10% DMSO + 1% trehalose + 0.15% MBE + 88.85% arts, sea water 20.4 ± 0.5 18.0 ± 0.1 61.6 ± 0.6 5000 ± 200 7d according to the invention 10% DMSO + 1% trehalose + 0.15% MBE + 0.3% echinochrome + 88.55% arts, sea water 29.1 ± 0.5 25.9 ± 0.1 45.0 ± 0.4 9700 ± 300

Claims (2)

1. A method of cryopreservation of biological material, including treating the biological material with a cryoprotective mixture containing a solution of sugars and lipids, characterized in that embryonic cells of marine invertebrates are taken as biological material, lipid extracts of marine hydrobionts with low phase transition temperatures, pre-treated, are used as lipids ultrasound, and as a solution using a saline solution, which is additionally injected with antioxidants, in the following ootnoshenii, wt.%: Trehalose 1.0-10.0 Lipid extracts 0.08-0.15 Antioxidants 0.3-1.0 Saline solution Rest 2. The method according to claim 1, characterized in that 5-10% of dimethyl sulfoxide is added to the composition of the saline solution in a cryoprotective mixture.

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