JPH0240467A - Refrigerating freezer for organism - Google Patents

Abstract

PURPOSE: To ensure refrigeration and freezing of living things for use in clinical practice, by dipping a container containing living things therein in a container made from a high heat conductivity material in which a high heat diffusion powder material, and solidifying water in the living things in a short time. CONSTITUTION: A tank 1, which is formed with a heat insulating material and in which a refrigerant is filled accommodates two containers 3, 4 formed with a high heat conductive material. The container 3 is filled with a high heat diffusion powdered material 5 and in contrast the container 4 is filled with a refrigerant 4. A container 12 for holding a living thing to be frozen is dipped in the container 3 with the aid of container movement drivers 13, 14 to solidify water in the living thing in a short time, and thereafter to insert the same into the refrigerant 2 in the container 4 for freeze and storage. As a result, solidification time of the water in the living thing can be shortened so that the apparatus is applicable also for freezing of a living thing in clinical practice.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field of Application The present invention relates to cryobiology and cryomedicine, and more particularly to the field of cryobiology and cryomedicine.
) related to freezing equipment.

The present invention finds greatest utility when applied to the cryopreservation of living organisms such as suspensions, liquids or solid tissues and organs that can be applied 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 cryopreservation methods by increasing the efficiency of freezers and simplifying their construction. Attempts to cryopreserve living organisms by rapid freezing by direct immersion in liquid nitrogen have failed to produce satisfactory results, while the operating principles of existing freezers based on the occurrence of a crystallization process causes considerable damage to the cells to be frozen, leading to the painstaking construction of a structurally simple and practically efficient cryo-freezing device that would be useful in achieving the vitrification effect in the organism to be frozen. need has arisen.

BACKGROUND OF THE INVENTION Virtually all cryogenic freezing methods are based on the principle of programmed freezing. In order to guarantee against negative representations of the crystallization process, freezing is carried out in a medium of cryogenic preservation, while
A cryoprotectant (cryoprotectant) that forms part of the cryopreservation composition
oprotectors) are unrelated to biological structures, but are somewhat toxic and produce adverse effects on biomacromolecules, especially when the concentration of cryoprotectants is increased (this is due to the freezing of water and pure It has an adverse effect on biopolymers (related to ice formation). The eutectic concentration of such cryoprotectants is equal to about 60-70%.

High-precision freezing of embryos is carried out by means of a programmed freezing and thawing apparatus for animal embryos (SUA No. 989.272). This device consists of an insulated tank that houses a container containing embryos, a holder for the container,
It includes 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 is It is fixed inside the cover.

It is worth noting that the cryopreservation of embryos during cryopreservation with said device is carried out by crystallization initiation techniques such that the occurrence of the crystallization process 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. Said factors are related to water freezing and increase in electrolyte concentration, changes in pH value, growth of ice crystals, etc. Under such low temperature storage conditions, high levels of cryoprotectant (1-3M)
The need for six degrees arises. 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 has a too narrow range of freezing rates, i.e. 0.3 to cryopreservatives are used.

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 bractis to breed stock stocks and transplant outcomes in public health services in an effort to reduce infertility.

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 freezing of various materials, for example foodstuffs, can be carried out by immersion in liquid nitrogen, freon or nitrogen oxide [Big Soviet Encyclopedia, 3rd edition, volume 9, 1972] Sovetskaya Encyclopedia Publisher, page 327 (USSR)] and quite a large number of devices have been proposed for that purpose.

However, equipment 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 limited to 400 or 500 °C.
Fail to achieve a freezing rate of more than 1/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, high concentrations of cryoprotectants (
50 or 60%) should be resorted to incorporating cryopreservatives. 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 temperature between the uncooled container and the liquid nitrogen consists of vapor of a refrigerant, e.g. The difference relates to the fact that an insulating shield is formed around the container holding the biological specimen.

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.

OBJECTS TO BE SOLVED It is an object of the present invention to provide a cryofreezing device for living organisms having a structure that would promote the occurrence of vitrification.

SUMMARY OF THE INVENTION The objective is to provide a refrigerating apparatus for living organisms, comprising an insulated tank for a refrigerant and a container for living organisms, the container being made of a highly thermally conductive material, the container being a tank. This is achieved due to the fact that the cryofreezer for living organisms is characterized in that the containers housed within and containing the living organisms are filled with a powdered material of high thermal diffusivity for immersion.

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 by vitrification, which means that the crystallization process occurs and runs completely eliminate or minimize the negative effects occurring on biological structures due to polar factors associated with the mechanical damage to biological macromolecules by ice crystals, in the denaturing effects of ultra-concentrated salt solutions and in the osmotic effects. is manifested in 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, a mixture comprising a metal, a metal oxide, and a metal alloy in various proportions to each other comprises:
Used as a highly thermally diffusive powder material.

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 made of an insulating material, for example foamed plastic, and suitable for filling with a refrigerant 2.
(Figure 1). The tank 1 contains two containers 3.4 made of a highly thermally conductive material, for example metal. 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. Tank 1 contains each container 3
．． It comprises a cover 8 with two through holes 9,10 arranged on top of the cover 4.

The device comprises a mechanism 11 for gripping the container 12 holding the organism to be frozen and a drive 13.14 for imparting horizontal and vertical linear movements to the container 12.

Container 12 can be made from aluminum foil, if desired.

As highly thermally diffusive material 5 metals, metal oxides or metal alloys, taken individually or in various proportions to each other, can be used.

The device of the invention operates as follows.

Before operation, remove cover 8 and close tank 1 and container 4.
is filled with a refrigerant 2, for example liquid nitrogen having a temperature of -196° 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
Close with cover 8. - Once the material 5 has cooled to the refrigerant temperature, the container 12 filled with living organisms is transferred to the mechanism 11 (first
(Fig.) and using the drive device 13.14,
It is introduced into the container 3 through the hole 9 (FIGS. 2 and 3) and immersed in the powdered material 5 held in the container. In 5-10 seconds, the container 12 with the frozen organism is moved to the drive device 13.
14 (FIG. 4) and advanced into hole 10, during which time the container is placed for short-term storage (for a few days).
Insert into the 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. O, 1m
A container 12 made from a food cylinder having a volume of 18 and a thickness of 0.03 mm is filled with water and immersed in a container 3 filled with powdered alumina (A 1203 ) cooled to the temperature of the refrigerant 2. do.

As a result, a temperature of -50°C is achieved in 0.95 seconds. A control biological test piece was placed in a container 4 filled with liquid nitrogen, and the time for achieving a temperature of -50°C was 1.
Equals 87 seconds. Results obtained using other powdered materials are displayed below.

The results obtained show that immersing a container 3 filled with powdered material 5 in a refrigerant (said powdered material serves as a refrigerant for biological organisms in container 12) reduces the solidification time by a factor of 2 to 15. This demonstrates 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. Figure 1 is a diagram of the apparatus in Figure 1, 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, and Figure 4 is a diagram of the apparatus in Figure 1. Figure 5 shows the apparatus of Figure 1 at the moment when the container holding the biological body is about to be evacuated from the container filled with the highly thermally diffusive powdered material; 2 shows the apparatus of FIG. 1 at the moment of introduction into a container filled with refrigerant; FIG. DESCRIPTION OF SYMBOLS 1...Tank, 2...Refrigerant, 3...Container for high heat diffusivity powder material, 4...Refrigerant container, 5...High heat diffusivity powder material, 6... Stirrer, 7... Stirrer drive device, 8... Cover, 9... Hole, 10...
Hole, 11... Container gripping mechanism, 12... Living body container, 13... Container horizontal movement drive device,
14... Container vertical movement drive device.

Claims (1)

[Claims] 1. A refrigerating apparatus for living organisms, including an insulated tank (1) for a refrigerant (2), and a container (12) for living organisms, comprising:
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 containing a biological body. (12) A refrigerating device for biological bodies, which is suitable for accommodating. 2. Device according to claim 1, characterized in that a metal is used as the highly thermally diffusive powder material (5). 3. Device according to claim 1, characterized in that a metal oxide is used as the highly thermally diffusive powder material (5). 4. Device according to claim 1, characterized in that a metal alloy is used as the highly thermally diffusive powder material (5). 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.