JPH0524420B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial field of application The present invention relates to cryobiology and cryomedicine.
More particularly, it relates to a freezing device for biological objects. The present invention finds greatest utility when applied to the cryopreservation of living organisms such as suspensions, liquids or solid tissues and organs as applicable in clinical practice. In addition, the device can be applied in cryogenic freezing of living organisms in agriculture, especially in livestock farming. The invention may also find application for the cryopreservation of organisms of the type whose vitality must be preserved at low temperatures for long periods of time. The present invention can be utilized whenever a significant reduction in the freezing time of an organism is required, which cannot be achieved by other methods. The increased requirements of public health services and national economies in high quality cryopreserved biological materials have led to further improvements in cryopreservability by increasing the efficiency of freezers and simplifying their construction. . Attempts to cryopreserve organisms by rapid freezing by direct immersion in liquid nitrogen have failed to produce satisfactory results, while existing freezers based on the occurrence of crystallization processes Since the operating principle causes considerable damage to the cells to be frozen, it is essential to develop a structurally simple and practically efficient cryofreezer that would be useful in achieving a vitrification effect in the organism to be frozen. The need for production arose. BACKGROUND OF THE INVENTION Virtually all cryogenic freezing methods are based on the principle of programmed freezing. To insure against negative representations of the crystallization process, freezing is carried out in a cryopreservation medium, while cryoprotectors that form part of the cryopreservation composition are used to protect biological structures. unrelated to, but somewhat toxic, producing adverse effects on biological macromolecules, especially when the concentration of cryoprotectants is increased (this is related to freezing of water and becoming pure ice) Causes adverse effects on biopolymers. The eutectic concentration of such cryoprotectants is equal to about 60-70%. High precision freezing of embryos is carried out using a programmed freezing and thawing apparatus for animal embryos (SUA No. 989272). The device includes an insulated tank containing a container with embryos, a holder for the container, a device for stirring the intermediate heating medium, and a system for controlling the freezing process. The tank has a cover, while the device for maintaining the temperature of the intermediate heating medium is made as a pipe coil placed on the inner surface of the tank such that the coil pitch increases towards the bottom of the tank, and the holder of the container has a cover. It is fixed inside. Low-temperature preservation of embryos during low-temperature preservation using the above device,
It is worth noting that the occurrence of the crystallization process is carried out by a crystallization initiation technique that results in dehydration of the cells to be frozen. This makes it possible to avoid intracellular crystallization when transporting the embryos to liquid nitrogen for storage there. The need for gradual dehydration of cells causes them to freeze at such slow rates that the crystallization process occurs only too slowly.
The biological structure in question is exposed to the effects of the extreme factor for a long period of time, such as 1.5 to 2 hours. The factors are related to water freezing and increase in electrolyte concentration, changes in PH value, growth of ice crystals, etc. Such cryo-storage conditions create the need for high (1-3M) concentrations of cryoprotectants. This results in toxic and quasi-toxic effects on the cells under conditions of slow crystallization development and thus prolonged exposure of the cells to the liquid phase. In addition, the device is characterized by a too narrow range of freezing rates, namely 0.3-1°C/min.
This limits the capabilities of cryopreservation methods, thus forcing the use of widely standard cryopreservatives whose compositions preclude optimization of existing methods. This disadvantage is so necessary that conventional methods of cryopreservation of embryos fail to provide adequately high and stable survival rates.
This has a negative impact on the use of embryos in clinical practices seeking to reduce infertility, strain stock breeding, and transplant outcomes in public health services. Additionally, the devices are complex in construction and generally incorporate expensive components that are not fitted with practical equipment. The device requires a skilled operator who should be well versed not only in cryopreservation techniques but also in the configuration techniques of such devices. It is common knowledge that the freezing of various materials, for example foodstuffs, can be carried out by immersion in liquid nitrogen, freon or nitrogen oxide [Vitsug Soviet Encyclopedia, 3rd edition, volume 9,
1972, Sovetskaya Encyclopedia Publishers, p. 327 (USSR)] and quite a large number of devices have been proposed for that purpose. However, a device suitable for carrying out a method of rapid freezing of biological structures based on direct immersion of the container holding the biological material into a liquid refrigerant is
Failure to achieve freezing rates above 400 or 500° C./min (which is quite low to avoid the occurrence of crystallization processes and elicit vitrification even in very small biological specimens). Therefore, in such cases one should resort to incorporating high concentrations of cryoprotectants (50 or 60%) into the cryoprotectant. This makes it possible to achieve the vitrification process even at said freezing rates. However, the application of cryoprotectants at said concentrations results in severe damage to the biological structures during the cryopreservant addition step or the cryopreservant removal step after thawing. Such a low freezing rate of biological specimens in the case of direct immersion in liquid nitrogen is due to the fact that the refrigerant, e.g.
It concerns the fact that an insulating shield is formed around the container holding the biological specimens, resulting from the temperature difference between liquid nitrogen and liquid nitrogen, which has a temperature of 196 °C. The presence of such shielding slows the rate of freezing of the organism to such a rate that the crystallization process occurs and involves the use of cryopreservatives formulated with high concentrations of cryoprotectants to inhibit said process. Problem to be Solved It is an object of the present invention to provide a cryofreezer for living organisms having a structure that would promote the occurrence of vitrification. Means for Solving the Problem The object is a refrigerating apparatus for biological bodies, which includes an insulated tank for refrigerant and a container for biological bodies,
A container made of a highly thermally conductive material is provided;
This is achieved due to the fact that the apparatus for freezing living organisms is characterized in that the container is housed in a tank and is filled with a highly thermally diffusive powdered material in which the container with the living organisms is immersed. Ru. The proposed method makes it possible to prevent the formation of an adiabatic shield of nitrogen vapor around the container with the organisms, which makes the freezing rate of the organisms more dependent on the type of powdered material, its degree of dispersion and their particles. Depending on the shape of the surface, it is possible to increase the temperature up to tens of thousands of degrees per minute. As a result, the water molecules do not have enough time to become arranged in the crystal lattice, and therefore water freezing occurs due to vitrification, which means that the crystallization process occurs and completely exclude or minimize the negative effects that occur on biological structures due to polar factors associated with running, and this negative impact is caused by mechanical damage to biological macromolecules caused by ice crystals.
It is manifested in the denaturing effect of ultra-concentrated salt solutions and in the osmotic effect, in the excessive dehydration of biological structures resulting in irreversible damage to said structures. In a preferred embodiment of the invention, the metal is used as a highly thermally diffusive powder material. Advantageously, metal oxides are used as highly thermally diffusive powder materials. According to one of the other aspects of the invention, the metal alloy is used as a highly thermally diffusive powder material. According to yet another aspect of the invention, mixtures comprising metals, metal oxides and metal alloys in various proportions to each other are used as highly thermally diffusive powdered materials. The selection of a suitable powdered material depends on the time required to produce vitrification within the organism to be frozen using a cryopreservative of corresponding formulation. Further objects and advantages of the present invention will become more apparent from the following detailed description of certain embodiments, taken in conjunction with the accompanying drawings. Embodiments The device of the invention comprises a tank 1 (FIG. 1) made of a thermally insulating material, for example foamed plastic, and suitable for filling with a refrigerant 2. Tank 1 is
Made from a highly thermally conductive material, e.g. metal2
Containers 3 and 4 are accommodated. The container 3 is suitable for filling with a highly thermally diffusive powdered material 5, while the container 4 is suitable for filling with a refrigerant 2. A stirrer 6 with an electric drive 7 is installed inside the container 3. The tank 1 has two through holes 9 arranged on the respective containers 3 and 4,
A cover 8 having a diameter of 10 is provided. The device comprises a mechanism 11 for gripping a container 12 holding the organism to be frozen, and drives 13, 14 for imparting horizontal and vertical linear movements to the container 12. Container 12 can be made from aluminum foil, for example. As highly thermally diffusive materials 5 metals, metal oxides, taken individually or in various proportions to each other;
Or metal alloys can be used. The device of the invention operates as follows. Before operation, the cover 8 is removed and the tank 1 and the container 4 are filled with a refrigerant 2, for example liquid nitrogen having a temperature of -196 DEG C., while the container 3 is filled with a highly thermally diffusive powdered material 5. The drive 7 of the stirrer 6 is then engaged to balance the temperature of the material 5 in the container 3, while the tank 1 is closed with the cover 8. Once the material 5 has cooled to refrigerant temperature, the container 12 filled with biological material is transferred to the mechanism 11 (FIG. 1).
and introduced into the container 3 through the hole 9 using the drives 13, 14 (Figs. 2 and 3).
), immersed in the powdered material 5 held in its container. In 5 to 10 seconds, the container 12 with frozen organisms is evacuated from the tank 1 by means of the drives 13, 14 (FIG. 4) and advanced into the hole 10, during which time the container is removed for short-term storage. (For several days) Insert into container 4 (FIG. 5) through the hole. In order to confirm the practicality of the proposed device, −
Freezing and freezing of water samples to a temperature of 50 °C was carried out. A container 12 having a capacity of 0.1 ml and made of food foil with a thickness of 0.03 mm is filled with water and coolant 2
It is immersed in a container 3 filled with powdered alumina (Al ₂ O ₃ ) cooled to a temperature of . As a result, -50℃
temperature is achieved in 0.95 seconds. A control biological specimen is placed in a container 4 filled with liquid nitrogen, and the time for achieving a temperature of -50°C is equal to 1.87 seconds. Results obtained using other powdered materials are displayed below.

[Table] The results obtained show that immersing the container 3 filled with the powdered material 5 in a refrigerant (said powdered material serves as a refrigerant for the biological body in the container 12) reduces the solidification time by 1/2 to 1/2. /15, thereby demonstrating that the device can be used in cryobiology and cryomedicine. Effects of the Invention The present invention finds application in the freezing and freezing of biological organisms, such as suspensions, liquids and solid tissues and organs, which can be applied in clinical practice. Furthermore, the device can be applied in agriculture, in particular in the freezing and freezing of living organisms in animal husbandry, fish breeding, and plant growth.

[Brief explanation of the drawing]

Fig. 1 is a schematic functional diagram of the device according to the present invention at the moment when the container holding the living organism is to be grasped, and Fig. 2 is the moment when the container holding the living organism is being transferred towards the refrigerant tank. The first in
Figure 3 is a diagram showing how a container holding an organism is immersed into a container filled with a highly thermally diffusive powdered material; Figure 4 is a diagram of the apparatus in which the organism is held. Figure 5 shows the apparatus of Figure 1 at the moment when the holding container is about to be evacuated from a container filled with a highly thermally diffusive powdered material, and Figure 5 is filled with a refrigerant for storing frozen organisms therein 2 shows the device of FIG. 1 at the moment of loading into a container in which the product has been prepared; FIG. 1... Tank, 2... Refrigerant, 3... Container for highly thermally diffusive powder material, 4... Container for refrigerant, 5...
High thermal diffusivity powder material, 6... Stirrer, 7...
Stirrer drive device, 8... Cover, 9... Hole, 10
... Hole, 11 ... Container gripping mechanism, 12 ...
Container for biological body, 13... Drive device for horizontal movement of the container, 14... Drive device for vertical movement of the container.

Claims (1)

[Scope of Claims] 1. A refrigerating apparatus for living organisms, including an insulated tank 1 for refrigerant 2 and a container 12 for living organisms,
A container 3 made of a highly thermally conductive material and housed in the tank 1 is provided, said container 3 being filled with a highly thermally diffusive powdered material 5 and suitable for accommodating a container 12 with a biological body. A freezing device for living organisms, characterized by: 2. The device according to claim 1, wherein a 2 metal is used as the highly thermally diffusive powder material. 3. The device according to claim 1, wherein a metal oxide is used as the highly thermally diffusive powder material. 4. The device according to claim 1, wherein a metal alloy is used as the highly thermally diffusive powder material. 5. Device according to claim 1, characterized in that mixtures of metals, metal oxides and metal alloys in various proportions to each other are used as highly thermally diffusive powder materials.