JPH0417921B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to cryobiology, and relates to a method for cryopreservation of mammalian embryos. INDUSTRIAL FIELD OF APPLICATION The present invention is most advantageously applied with respect to deep freezing of embryos for the purpose of establishing cryobanks of embryos of endangered and rare animal species; , theriogenology, pharmacology, immunology, virology and experimentation - vitally needed in maintaining genetically valuable breeds of mice that are advantageous to use for further advances in clinical medicine. It is. The invention may also find application in several other cryobiological applications, such as in public health services, where the resulting successful embryo transfer into a female patient is the most fundamental anti-infertility measure. However, ovarian tissue transplantation is an effective method for improving the overall hormonal status of patients with respect to several diseases of various etiologies. Moreover, perfusion of embryonic bone marrow with reduced immunogenicity is a promising tool in the treatment of hematopoietic suppression in female patients. The therapeutic measures described above require sufficient quantities of the appropriate biological substance, which can only be obtained if long-term storage is carried out in deep-frozen conditions. The present invention is useful in animal husbandry, commercial animal husbandry, where cryopreserved embryos have wide application in breeding new breeds of domestic animals, and in fish farming, in particular for the cryopreservation of fish embryos of sturgeon and carp. can also be applied. The applications of such low temperatures and especially deep freezing for embryo storage after isolation from the living body extend to an even wider range of fields, including human economic activity and public health maintenance. At present, the trend of the times to establish cryogenic banks for genetic resource storage has become the most widespread development, especially regarding cryogenic banks for embryo storage of commercially useful farmed animals carried out to promote animal husbandry. has been achieved. The large-scale use of mammalian embryos in medicine and animal husbandry has made the need for implantable materials even more pressing, the quality of which can combat infertility and foster high-productivity breeding animals. It is important to promote the final outcome of the necessary transplantation operation. However, methods developed to date for cryopreservation of embryos do not allow high levels of preservation of frozen cells, resulting in large losses of valuable biological material. Experiments on embryo cryopreservation have revealed that their optimization is related to the possibility of vitrification formation in biological samples frozen in one step of ultrafast cooling. Prior Art What is currently known in the art is to start crystallization at -3.5°C and to -40°C at a rate of 0.2-2.0°C/min.
There is a method of freezing mouse embryos in a 1.0 M dimethyl sulfoxide (DMSO) medium, which is characterized by cooling to a temperature of 100% and then immersing it in a refrigerant, namely liquid nitrogen. The survival rate of the embryos after thawing is 65% [Experimental Cell Research, Vol. 89, No. 1, 1974, S.P. Raybo, P.M. Mazur, S.C. Jiakowski (Experimental Cell Research, v.
No.1, 1974, SPLeibo, PMMazur, SC
Jackowsky, "Factors Affecting Survival of Mouse Embryos During Freezing and Thawing," pp. 79-89]. The disadvantage of this method is the low survival rate of cryopreserved embryos, and the freezing results are dependent on even the most insignificant differences from the optimal values for the freeze-thaw process parameters. It was a bad influence. Yet another prior art method for the cryopreservation of mouse embryos in phosphate buffered saline (PBS) containing 1.45 M DMSO is known to be available to date. According to that method, the cryoprotectant is introduced dropwise into the embryo and removed from it, the cooling rate is 0.3 °C/min, and the survival rate is 67-68 °C.
%. The method suffers from low survival rates of the embryos involved, poor reproducibility of the method, manual control of the cooling operation, and movable components in the equipment that complicate the synchronization of crystallization process control and adversely affect the reproducibility of the results. The problem is that there are too many. Other disadvantages of the method include the long duration of the cryopreservation cycle, as well as the complexity and high labor consumption of the work performed [Cryobiology, Vol. 16,
1979, G.H.Railmaker, C.
MPM Belharm (Cryobiology,
v.16, 1979, GHReilmaker, CMPM
Verhamme), “A Simplified Method for Freezing Mouse Embryos”, pp. 6-10]. Another method of cryopreservation of mouse embryos in a medium of 2.0 M glycerol + 0.5 M sutucarose is currently known in the art. After 15 minutes of incubation, the embryos were placed in a 4 mm inner diameter polystyrene tube, hermetically sealed, and then immersed in liquid nitrogen. The survival rate of embryos treated in this way was 84%. The disadvantages of this method are the formation of crystals and the high osmotic activity that leads to potential damage to the cells that may disadvantageously proceed in the medium after thawing. Too low cooling rates (approximately 20 °C/min), which are associated with the formation of an insulating shell around the tube, can accelerate the crystallization process and affect the possibility of vitrification in the liquid part of the cryogenic biological system. [Theriogenology, Volume 23, No. 1, 1985
, T.G.A. Williams, S.E.
Tauntson (Theriogenology, v, 23, No. 1,
(1985, TJ Williams, SETohnson), “Rapid freezing of 4-day-old mouse embryos”, p. 235]. Yet another method of cryopreservation of mouse embryos in a medium of 3.5M glycerol mixed with 0.5M sutucarose is currently known in the art. If the cryopreservative is added fractionally into the cells, the latter is placed in a polystyrene tube;
After being hermetically sealed, they were immersed in liquid nitrogen, and the survival rate of embryos treated this way is 85%. The disadvantages of the above methods are that the concentration of permeable substances in the cryopreservation medium is too high, the cooling rate of the embryos is low, the latter being responsible for potential damage to the cell membranes, and the medium after thawing. [Theriogenology, Vol. 25, No. 1, 1986, K. A. Barley, G. E. Seder Giuniah, R.
P. Elsden (Theriogenology, v.25,
No.1, 1986, KABiery, GESeddel Jr, RP
Elsden), “Cryopreservation of mouse embryos by direct immersion in liquid nitrogen,” p. 140]. Another embryo cryopreservation method is known in which embryos isolated from living mice are placed in polystyrene tubes containing a medium of 1.4 M glycerol and 1.1 M sutucarose, and the tubes are hermetically sealed at both ends. cells are placed in the tube.
Leave for 25-30 minutes [Theriogenology, No. 23]
Volume, No. 1, 1985, K.T. Club, I.A. Kohler, R.Double Light (Theriogenology, v.23, No.1, 1985, K.T.
Krag, IM Koehler, RWWright), “Method for freezing early mouse embryos by direct immersion in liquid nitrogen,” p. 199]. The tube was then immersed in liquid nitrogen. Thawing was performed at 37°C. The tube contents were poured into Petri dishes and washed three times with aliquots of 0.5M sutucarose, after which they were cultured in nutrient medium. Between 40 and 67% of thawed embryos developed to the blastocyst stage. Highly concentrated cryoprotectants, such as glycerol, which penetrates very little into the embryo, and sutucarose, which penetrates completely into the embryo, are recommended for rapid cooling (approximately 20
The dehydration of the cells is aimed at establishing the background for their vitrification after (rate of 1°C/min). The method in question yields poor results due to too high a cryoprotectant concentration in the cryoprotectant medium, and even when added to the embryo in three stages, the cryoprotectant is They are acting the cause of damage. The toxic effects of cryoprotectant compounds on embryos are also a concern when the total concentration of cryopreservative used is 48.6%. In addition to all of the above, the cooling rate of embryos obtained by their direct immersion in liquid nitrogen is not high enough for vitrification to proceed, even with high concentrations of cryopreservatives. , because the heat-resistant shell formed between the tube and liquid nitrogen and consisting of nitrogen vapor prevents rapid heat release from the embryo being processed. Problems to be Solved The present invention aims to provide a method for cooling embryos, in which the cooling process is sufficiently high for the progression of vitrification of the majority of water, both extracellular and intracellular. Recrystallization during thawing would occur at a faster rate and would cause adverse effects on the biological structure of the embryo to occur to a lesser extent. Means for Solving the Problems The object is to provide a method for cryopreservation of embryos comprising cooling to a refrigerant temperature in a buffered saline solution containing glycerol as an anti-freeze agent. due to the fact that the buffer saline further contains dimethyl sulfoxide (DMSO) and one of the dihydric alcohol derivatives as an antifreeze agent. achieved based on The method of cryopreservation of embryos proposed here contributes to an even higher survival rate of the embryos involved due to their cooling with the aid of a high thermodiffusive powder substance, which is heated at temperatures of several thousand degrees per minute. It makes it possible to achieve high cooling rates so that the vitrification process proceeds in the embryos being treated. Moreover, according to the method proposed in this specification,
The degree of adverse effects caused by recrystallization on the biological structure of the embryo during thawing can be reduced by the use of DMSO and dihydric alcohol derivatives as cryoprotectants in the cryopreservation treatment. Both act as anti-salt buffers that prevent crystal migration and impede crystal size increase that aids in the hydration shell around biomacromolecule and protein catalytic centers. The three types of cryoprotectants used (in glycerol, DMSO or one of the dihydric alcohol derivatives) with different cell membrane permeability can cause permeation impairment or No toxic progression occurs, at the same time promoting vitrification and comprehensively protecting the embryo during cryoprotection and thawing. Moreover, the cryopreservative mixture used is recovered without damaging the cells. According to the proposed method, the process of cryopreservation of embryos can be substantially simplified, since it consists only of the smallest combination of the simplest operations, which does not require any special expertise on the part of the operator. . Moreover, this method allows to significantly reduce the time required to carry out a complete cycle of cryopreservation and the time spent on each of these operations due to the reduced number of operations, so that the method It becomes even more efficient and cheaper. In the present invention, zinc is used as a highly thermally diffusive powder material. The selection of such a powder material is made with respect to the higher thermal conductivity of a comparison of several priced powder materials, which can provide a high cooling rate to the embryo. 1,2-propanediol is used as one of the dihydric alcohol derivatives, and DMSO is a substantial cryoprotectant that most easily penetrates into embryonic cells and can protect intracellular biological structures during freezing and thawing. Due to the fact that its mixture with DMSO is advantageously selected. The use of 1,2-propanediol can successfully protect not only the intracellular structures of the embryo but also its membranous organs, compared to other cryoprotectants used in this method. This is due to the average permeability of alcohol in the case. According to one aspect of the invention when liquid nitrogen is used as the refrigerant, each of the three antifreeze agents is
Used at concentrations within the range of 4.75-5.25%. When used at the above concentrations, cryoprotectants express their antifreeze action most effectively and are characterized by virtually non-toxicity and low permeability. According to another aspect of the invention, each antifreeze agent is used in a 1:1 ratio. In the case of such a ratio, it can be expected that an equivalent cryoprotective effect can be given to the intracellular structures and membrane organs of the embryo based on the uniform distribution of the cryoprotectant in the region of the cryobiological system. However, this is due to the different intracellular penetration abilities of the cryoprotectants. In a preferred embodiment of the invention, the buffered salt solution contains a biologically active substance. The use of such substances greatly contributes to cell viability by protecting the cytoplasmic membrane from temperature shocks and "melting effects" during the phase transformation to ice. Biologically active substances can stabilize the natural structure of cell membranes and thus prevent their damage during the crystallization process. It is highly desirable that choline chloride solution be used as a biologically active substance, as it is involved in the natural methylation process of biological structures, stabilizing the natural membrane structure by the presence of methyl groups. ing. According to another embodiment of the invention, the choline chloride solution is used at a concentration of 0.4%, since at high concentrations the membrane structure is disturbed due to the surfactant action of said substance; This is because when the concentration becomes low, the above-mentioned stabilizing effect is no longer exhibited. Other objects and advantages of the invention will become more apparent from the detailed description of some specific embodiments below. Embodiments The cryopreservation method for embryos is characterized by their cooling to the latter temperature with the aid of a refrigerant in a buffered saline solution. The cooling process takes place in a highly thermally diffusive powder material that has been previously cooled to refrigerant temperature. Buffered saline is a mixture of the following cryoprotectants: glycerol,
dimethyl sulfoxide (DMSO) and one of the dihydric alcohol derivatives, as well as biologically active substances,
For example, it contains a choline chloride solution with a concentration of 0.4%. Zinc can be used as a highly thermally diffusive powder material. 1,2-propanediol can be used as one of the dihydric alcohol derivatives. When liquid nitrogen is used as the refrigerant, each of the three antifreezes is used at a concentration of 4.75-5.25% and in a 1:1 ratio. The present invention was carried out regarding the cryopreservation of murine embryos that have reached blastomere stage 32. Example 1 A total of 60 embryos each consisting of 10 embryos (5 in each group)
(in the drop) divided into 6 groups. For each group, glycerol
10.5%, propylene glycol 10.6%,
DMSO 10.5% and choline chloride 0.8% (the concentration of each antifreeze is 5.25%, and the concentration of choline chloride is
Five drops of Dulbecco's phosphate solution medium containing 0.4%) were added. all operations 4
I did it at ℃. The embryos in cryoprotectant-containing solution were then transferred to food grade foil containers 1 x 1.5 cm, capacity 0.1 ml and wall thickness 0.03 mm. The free end of the container was flared and dipped for 10 seconds into a metal container filled with powdered zinc cooled to the temperature of liquid nitrogen. The containers were then left in liquid nitrogen for three weeks, after which they were placed in a water bath at 40°C.
I unzipped it. The survival rate of thawed embryos, as assessed by their growth in culture, was 94.2
It was %. To demonstrate the concentration of cryoprotectant, an experiment was conducted as in Example 1 with varying concentrations of cryoprotectant. The viability of the embryos was also investigated from data based on their growth in culture up to the blastocyst stage.
The experimental results are shown in Table 1. The growth of embryos during culture after cold storage is the first
In Table ~5, it is calculated relative to the growth of the same number of natural embryos.

TABLE It can be seen from Table 1 that the highest embryo survival rate is obtained when the cryoprotectant is used at a concentration of 4.75-5.25% (total concentration ranges from 14.25-15.75%). An experiment similar to Example 1 was conducted to demonstrate the ratio between the concentrations of cryoprotectants in the cryoprotectant medium, but their total concentration in the cryoprotectant ranged from 14.25 to 15.75%. It was used in various proportions as shown in the table below. The results are shown in Tables 2-4.

【table】

[Table] Grise Propylene Embryo survival rate
Roll Glycol DMSO%

Claims (1)

[Scope of Claims] 1. A method for cryopreservation of embryos comprising cooling to a refrigerant temperature in a buffered saline solution containing glycerol as a cryoprotectant, wherein the cooling comprises a highly thermodiffusible powder pre-cooled to the refrigerant temperature. A process carried out in the substance zinc, characterized in that the buffer saline further contains dimethyl sulfoxide and one of dihydric alcohol derivatives as antifreeze agents. 2 As one of the dihydric alcohol derivatives, 1,2-
2. The method according to claim 1, wherein propanediol is used. 3 When liquid nitrogen is used as a refrigerant, 3
2. The method of claim 1, wherein each of the seed cryoprotectants has a concentration of 4.75-5.25%. 4. The method of claim 1, wherein each of the three antifreeze agents is used in a 1:1 ratio. 5. The method of claim 1, wherein the buffered saline contains a biologically active substance. 6. The method according to claim 5, wherein a choline chloride solution is used as biologically active substance. 7. The method of claim 6, wherein the choline chloride solution has a concentration of 0.4%.