RU2611166C2 - Self-contained device for vitrification of biological objects using cryogenic coolant - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: invention relates to devices for vitrification of biological objects. The self-contained device for vitrification of biological objects using a cryogenic coolant has a coaxial design, which includes a cylindrical housing, inside of which there is a cylindrical coaxially extensible support, the end of which is connected to a collet clamp which provides fixation of the tube of a container with biological objects, a starts spring which interacts with a cylindrical support and provides movement of the container with biological objects into a vessel with cryogenic coolant through its mouth, a cylindrical heat insulation screen with a movable cover, a protective disc screen fixed on outer protrusions of the cylindrical housing, and a control button arranged in the upper part of the device.

EFFECT: invention improves reliability and reduces the weight of the device, increases its axial length when releasing the container with biological objects into a Dewar vessel and increases cooling rate thereof.

9 cl 18 dwg

Description

The invention relates to devices for vitrification of biological systems (viruses, cells, tissues, organs, integral living organisms, as well as parts and components of the above systems, for example, cell organelles or vital products of biological systems and their individual components or fragments, for example antigens, antibodies, hormones), hereinafter for brevity of “bioobjects”, using liquid cryogenic refrigerant, in which the container with bioobjects is cooled in one cryogenic vessel, for example measures in the Dewar vessel, after which the operation of moving the container into a portable cryogenic vessel is carried out for further research on the storage of biological objects.

In cryobiology, vitrification is a method of ultrafast freezing (cryopreservation) of biological objects without the formation of ice crystals, which are harmful to many types of biological objects. It is possible to provide vitrification in existing technologies with the help of high concentrations of special substances - cryoprotectants. However, cryoprotectants themselves negatively affect biological objects. The critical cooling rate required for vitrification is inversely proportional (a function close to the inverse negative logarithm) of the cooling rate. Therefore, the higher the cooling rate of these bioobjects, the less cryoprotectant is needed to ensure the vitrification process, which means that as a result this leads to better quality of the biomaterial after thawing (heating) of the bioobject.

One way to increase the cooling rate of a container with bioobjects, and thus reduce the cryoprotectant, is to intensively move the container in the volume of liquid cryogenic refrigerant.

A self-contained device for vitrification of bioobjects is known, having a coaxial design, including a cylindrical body, inside which a cylindrical axially extendable support is installed, the end of which is connected to a collet clamp that secures the container tube with bioobjects, a trigger spring that interacts with the cylindrical support and allows the container to move with biological objects into a vessel with cryogenic refrigerant through its neck, a cylindrical heat-insulating screen with a movable cover, a protective disk screen fixed on the outer protrusions of the cylindrical body, and a control button [1] located in the upper part of the device.

This device has an electric motor with a power source, which provides rotation of the container with bioobjects. However, to provide the necessary power to overcome the forces of resistance to rotation of the container in a cryogenic liquid, the dimensions of the electric motor and the power source should be significant, which increases the weight and dimensions of the entire device.

When a container with bioobjects is quickly moved from room temperature to a cryogenic liquid, such as liquid nitrogen, a so-called “steam jacket” (Leidenfrost effect) is formed at the boundary of their interaction, which has heat-insulating properties and prevents the container from cooling rapidly. If the container is rotated by an electric motor in one place, as occurs in a known device, a significant (close to soybean rotation) part of the cryocontainer is always in this “steam jacket” and its cooling rate is low. In addition, this creates a significant gradient in the cooling rate along the radius of rotation, which can lead to heterogeneity as a defrostable biomaterial inside the container, which is a serious technological disadvantage.

In addition, the device cannot provide an increase in the axial length of the support by more than 2 times, which complicates its practical use in the presence of a deep Dewar vessel or a low level of cryogenic liquid.

The device is inharmonious and unreliable due to the presence of several control buttons, each of which carries out only one function.

In the known device, the cylindrical screen is suspended on thin wires - guide elements, and its cover is controlled by thin wires - movable rods, which makes the design unreliable. The connection of the lid with a cylindrical screen in a small area also contributes to this.

The objective of the invention is to increase the reliability and reduce the mass of the device, increase its axial length when starting the container with biological objects in the Dewar vessel and increase its cooling rate.

The problem is solved due to the fact that in the well-known stand-alone device for vitrification of biological objects using a cryogenic refrigerant having a coaxial design, including a cylindrical body, inside which there is a cylindrical axially elongated support, the end of which is connected to a collet clamp that secures the container tube with bioobjects to be fixed, trigger spring, which interacts with a cylindrical support and provides movement of the container with biological objects in a vessel with cryogenic through its mouth, a cylindrical heat-insulating screen with a movable cover, a protective disk screen fixed on the outer protrusions of the cylindrical body, and a control button located in the upper part of the device, in accordance with the invention, both the body and the telescopic type support comprise an outer and an inner section, between which at least one intermediate section is located, the outer section of the housing is connected to the disk base, and its inner section is connected to the cylindrical a heat-insulating screen using an outer ring, with a fixing spring installed between the disk base and the outer ring, the outer section of the support is connected to a coaxially mounted control button, and its inner section is connected to the collet with the inner ring, the outer and inner rings are in the same plane that in the internal grooves of the outer ring are the outer teeth of the inner ring with the possibility of axial movement of the inner ring relative to the outer rings, the movable cover of the cylindrical heat-insulating screen is made in the form of a diaphragm with rotary tabs that regulate the diameter of the hole, the control button is located above the disk base and connected to it using a return spring, which is covered by a number of protective walls located with the possibility of movement in the holes of the disk base a spring installed inside the support and connected to the control button and the inner ring is held in a compressed state by changing its radial p dimensions with an elastic retainer, which is connected to the starting handle located in the recess of the control button, the protective disk screen is mounted on the outer section of the housing and is made of transparent heat-insulating material.

In addition, the disk base is made with an expanded part designed to be gripped by the fingers of the operator’s hand.

In addition, the protective disk screen is made of organic glass.

In addition, the outer diameter of the protective disk screen exceeds the diameter of the neck of the Dewar vessel with cryogenic refrigerant.

In addition, the springs are made of cylindrical screw.

In addition, the elastic retainer is connected to the starter handle using a cable, which is located inside the Central tube.

In addition, the latch wall facing the launch handle is located at an angle of about 90 ° to the axis, and its wall facing the inner ring is located at an angle of about 45 ° to the axis.

In addition, the launch handle is rotatable in a plane perpendicular and parallel to the axis of the device.

In addition, liquid nitrogen is used as a cryogenic refrigerant.

The lack of an electric motor and power supply increases reliability and reduces the weight of the device.

The execution of both the housing and supports of the telescopic type allows you to multiply at least 3 times to increase their axial length due to the sequential location of the outer and inner sections, between which at least one intermediate section is located.

The device is controlled using one control button, which ensures the fixation of the container with biological objects, its linear reciprocating movement in a cryogenic liquid, and the opening of the cylindrical screen opening. The reciprocating movement of the container increases its cooling rate, since it constantly moves from the area from the “steam jacket” to the liquid that cools it.

The reliability of the device is enhanced by: the absence of thin wires (guide elements and rods), a fixed design of a cylindrical heat-insulating screen and the design of its cover in the form of a rotary diaphragm.

The implementation of the protective disk screen of a transparent heat-insulating material, for example of organic glass, the outer diameter of which exceeds the diameter of the neck of the Dewar vessel with cryogenic refrigerant, allows you to install it on the neck of the vessel and observe the cooling process of the container with bioobjects.

The trigger handle that controls the elastic lock can be pulled out if necessary and hidden in the recess of the control button due to the possibility of rotation in a plane perpendicular and parallel to the axis of the device.

Due to the fact that the latch wall facing the starter grip is located at an angle of about 90 ° to the axis, and its wall facing the inner ring is located at an angle of about 45 ° to the axis, the compressed start spring is fixed and it is free to move from expanded to compressed state.

The use of liquid nitrogen as a cryogenic refrigerant allows for a high cooling rate and high safety.

In FIG. 1 shows a container impregnated with a cryoprotectant (which in this case is permeable to cryoprotectants and water) for biological objects;

in FIG. 2 - a container filled with bioobjects;

in FIG. 3 - stand-alone device for vitrification of biological objects using cryogenic refrigerant (hereinafter referred to as the "device") in its original state;

in FIG. 4 - part of the device at the time of releasing the starting spring;

in FIG. 5 - the device is in the working position, in which the container with bioobjects is inside the Dewar vessel with cryogenic refrigerant;

in FIG. 6 - the device of FIG. 5 without a Dewar flask and cryogenic refrigerant;

in FIG. 7 - the device of FIG. 6 at the time of lowering the container with bioobjects down;

in FIG. 8 - a device taken out of a Dewar vessel with a cryogenic refrigerant;

in FIG. 9 - a device in a position in which a container with bioobjects is in a portable cryogenic vessel;

in FIG. 10 - a container with biological objects in a closed portable cryogenic vessel;

in FIG. 11 is a view A in FIG. 7;

in FIG. 12 is a view C in FIG. 3;

in FIG. 13 is a view B in FIG. 6.

in FIG. 14 is a view B in FIG. 3;

in FIG. 15 is a view D in FIG. 8 with a slightly ajar cover of the cylindrical screen;

in FIG. 16 is a section Α-A in FIG. 7;

in FIG. 17 - position of the starting spring and the elastic retainer in FIG. 3 enlarged in the initial state;

in FIG. 18 is a view of FIG. 17 when the starting spring is open in working condition.

A stand-alone device for vitrification of bioobjects using a cryogenic refrigerant (hereinafter referred to as “the device”) consists of a container 1 impregnated with a cryoprotectant, which is connected to a tube 2 designed to move the container and fill it with bioobjects 3.

The device has a coaxial design. The cylindrical body of the telescopic device 4 includes an outer section 4a and an inner section 4b, between which one, for example 4c, or several intermediate sections can be located. The telescopic design allows you to reduce or increase the length of the outer casing by moving the inner section 4b inside the intermediate section 4c, and the latter inside the outer section 4a.

The outer section 4a of the casing is connected to the disk base 5, in which there is an extended part 5a, designed to be captured by the fingers of the hands of an operator who works with the device (not shown in Fig.). The inner section 4b of the housing is connected to the cylindrical heat-insulating screen 6 using the outer ring 7.

Inside the cylindrical housing 4 is a cylindrical support 8 of a telescopic type, which contains an outer section 8a and an inner section 8b, between which one, for example 8c, or several intermediate sections can be located. The telescopic structure allows you to reduce or increase the length of the support 8 by moving the inner section 8b inside the intermediate section 8c, and the latter inside the outer section 8a.

The outer section 8a of the support 8 is connected to the control button 9, and the inner section 8b of the support using the inner ring 10 is connected to the collet clamp 11, which allows you to grab and fix the tube 2 of the container 1. The grips (not shown) of the collet clamp 11 conical extension at the end. This allows, applying the axial pressing force, insert the tube 2 into them and fix the container 1 in the collet clamp 11.

In the initial position, the outer 7 and inner 10 rings are located in the same plane (Fig. 3). The outer ring 7 is made with orderly arranged inner grooves 7a, in which the outer teeth 10a of the inner ring 10 are located (Fig. 13). The teeth 10a are located in the grooves 7a with the possibility of free axial movement of the inner ring 10 relative to the outer ring 7.

The cover 6a of the cylindrical screen 6 is made in the form of a diaphragm, the rotary petals 6b of which allow you to adjust the diameter of the hole 6B from the maximum (Fig. 14) to zero. (The rotary aperture is structurally similar to the optical aperture of the camera.)

On the outer section 4a of the cylindrical body, protrusions 12 are made into which a protective disk screen 13 is inserted. The protective screen 13 is made of a transparent material. It is designed to monitor the process of cooling the container in a cryogenic refrigerant while protecting the operator’s hands from cryogenic refrigerant.

The control button 9 is connected to the disk base 5 by means of a return spring 14, which is enveloped externally by a number of protective walls 9a, arranged in a tangential direction (Fig. 16). Protective walls 9a, designed to prevent contact between the operator’s hands and the return spring 14, are located in the holes 5b of the disk base 5. The protective walls 9a of the button are made with the possibility of free tangential movement inside the holes 5b when the button 9 is rotated relative to the central axis 15 of the device.

Inside the cylindrical support 8, a starting coil spring 16 is installed, which is connected to the control button 9 and the inner ring 10. In the initial compressed state, the starting spring 16 is held by an elastic retainer 17 (Fig. 3). The latch 17 is connected to the starter handle 18 using a cable 19, which is located inside the Central tube 20 (Fig. 17). The wall 17a of the latch 17 facing the launch handle 18 is located at an angle of about 90 ° to the axis 15, and its wall 17b facing the inner ring 10 is located at an angle of about 45 ° to the axis 15.

The start handle 18 is located in the recess 21 of the control button 9 and is made to rotate into a plane perpendicular to the axis 15 (the handle is recessed in the button) (Fig. 11) and into a plane parallel to the axis 15 (the handle is ready for operation) (Fig. 12 )

When pulling the handle 18 upward from the recess 21 of the control button 9, the cable 19 pulls the elastic retainer 17 into the central tube 20, so that its radial dimensions are reduced (Fig. 18).

For vitrification of biological objects, a Dewar vessel 22 with liquid cryogenic refrigerant 23, for example liquid nitrogen, and a portable cryogenic vessel 24 with cryogenic refrigerant 23, closed by a lid 25, are used.

The outer diameter of the protective disk screen 13 is larger than the diameter of the neck 22a of the Dewar vessel 22 (Fig. 5).

Between the disk base 5 and the outer ring 7 has a fixing spring 26.

The cylindrical body 4 and the telescopic support 8 are made of durable, poorly conductive metal, for example stainless steel. The protective disk screen 13 is made of transparent plastic, for example of organic glass. The cylindrical heat-insulating screen 6 is made of a heat-insulating material, such as plastic. The control button 9, the start handle 18 and the disk base 5 are made of decorative plastic.

Stand-alone device for vitrification of biological objects using cryogenic refrigerant works as follows.

Before starting work, prepare the device as follows.

On the outer section 4a of the cylindrical body, a protective disk screen 13 is inserted and fixed into the protrusions 12.

The cover 6a of the cylindrical screen 6 is mounted with a maximum opening 6c. For this, fixing the disk base 5, turn the control button 9 around the axis 15. At the same time, the telescopic support 8 rotates relative to the housing 4. Inner ring 10 with its teeth 10a through the grooves 7a rotates the outer ring 7 (Fig. 13) and the rotary petals 6b of the cover 6a cylindrical screen 6 open the hole 6 in the diaphragm.

The control button 9 is pressed downward relative to the disk base 5, slightly rotated about the axis 15 and released. In this case, the outer teeth 10a of the inner ring 10 come out of the inner grooves 7a of the outer ring 7. The inner ring 10 is installed below the outer ring 7, abutting against it with its teeth 10a.

After this, an axially compressive force is applied between the disk base 5 and the cylindrical screen 6. Compressing the starting spring 16 and the fixing spring 2b, fold the housing 4 and the support 8. In this case, the turns of the starting spring 16 easily pass through the wall 17b of the elastic retainer 17. After the starting the spring 16 is completely compressed, its coils are held in a compressed state by the wall 17a of the elastic retainer 17. In the telescopic support 8, the inner section 8b is placed inside the intermediate section 8c, which, in turn, is located inside the outer section 8a. In the telescopic housing 4, inside the outer section 4a, an intermediate section 4b is located, inside which the inner section 4b is placed.

Container 1 is placed in a vitrified solution (not shown in FIG.) At room temperature for a certain time, for example, 1 hour, allowing the container to “soak”, i.e. penetrate the middle. After that, using the tube 2, the container 1 is transferred to a cold zone with a temperature of 0 ° C.

After 5-10 minutes, a cryoprotectant is added to bioobjects 3 outside the container. In this case, cryoprotector is saturated with bioobjects.

After that, through the tube 2 load the container 1 with bioobjects 3 (Fig. 2).

Insert the end of the tube 2 into the cone-shaped extension of the grips of the collet clamp 11 and, applying axial force, insert the tube into the grips that fix the container 1 with bioobjects 3 in the collet clamp 11 (Fig. 3).

After that, the device is installed opposite the neck 22a of the Dewar vessel 22. Moreover, the protective transparent screen 13 can be fixed on this neck and observe the subsequent process from the outside, without fear of thermal damage to the operator’s hands.

The operator pulls up the start handle 18. In this case, the cable 19 connected to the handle 18 pulls the elastic retainer 17 into the central tube 20, so that its outer diameter decreases (Fig. 18). The trigger spring 16 disengages the elastic retainer 17 and rapidly unclenches. In this case, the starting spring 16 pushes the inner section 8b of the telescopic support 8, and the fixing spring 2b pushes the inner section 4b of the telescopic housing 4.

Thus, the axial lengths of the support 8 and the housing 4 are increased, and the container 1 with the heat-insulating screen 6 is in the cryogenic refrigerant 23 of the Dewar vessel 22 (Fig. 5).

After that, the control button 9 is rotated around the axis 15 so that the outer teeth 10a of the inner ring 10 coincide with the inner grooves 7a of the outer ring 7.

After that, the operator with two fingers of one hand (not shown in FIG.), Holding the expanded part 5a of the disk base 5, intensively presses the control button 9 with his free finger (Fig. 7). In this case, the return spring 14 is compressed, and the protective walls 9a of the button go into the holes 5b of the disk base 5. The telescopic support 8 together with the container 1 moves relative to the housing 4 and falls down, which ensures its intensive cooling by cryogenic refrigerant 23.

After lowering the button 9 down to the stop (the coil of the spring is in contact with each other), the operator immediately releases the button 9. Under the action of the return spring 14, the support 8 together with the container 1 rise up to the initial position (Fig. 6). After that, the operator again presses the control button 9 and the process is repeated until the cooling process of the container 1 with biological objects 3 is completed.

The cyclic reciprocating axial movement of the container 1 relative to the cryogenic refrigerant 23 helps to reduce the Leidenfrost effect and helps to intensify cooling. In the process of cooling around the container 1, intense vaporization is observed, followed by the drilling of the cryogenic refrigerant 23. After the container 1 is completely cooled, the drilling stops. The operator controls the entire cooling process through a transparent protective screen 13.

After cooling the container 1 with bioobjects 3, holding the disk base 5, turn the control button 9 around the axis 15. At the same time, the telescopic support 8 together with the container 1 rotates relative to the housing 4. Inner ring 10 rotates the outer ring 7 through grooves 7a (Fig. . 13), and the rotary lobes 6b of the cover 6a of the cylindrical screen 6 close the aperture 6b. Thus, part of the cryogenic refrigerant 23 is located inside the cylindrical screen 6, forming a closed tank.

After that, the device with the cryogenic refrigerant 23 is removed from the Dewar vessel 22 and transferred to another place, for example, to a sterile room or under a laminar box (not shown in Fig.). Since the container is located in cryogenic refrigerant 23 (Fig. 8), its temperature is maintained for a long time, which is important for a relatively long transfer process.

After that, the device is lowered into a portable cryogenic vessel 24 with cryogenic refrigerant 23 (Fig. 9). Holding the disk base 5, turn the control button 9 around the axis 15 in the opposite direction to the previous one. In this case, the telescopic support 8 together with the container 1 rotates relative to the housing 4. The inner ring 10 rotates the outer ring 7 through its grooves 10a through the grooves 7a, and the rotary petals 6b of the cover 6a of the cylindrical screen 6 open the aperture 6b (Fig. 14).

Then, with the collet clamp 11, the tube 2 is released from the container 1. For this, the control button 9 is pressed down relative to the disk base 5, slightly rotated about the axis 15 and released. In this case, the outer teeth 10a of the inner ring 10 come out of the inner grooves 7a of the outer ring 7. The inner ring 10 is located below the outer ring 7, abutting against it with its teeth 10a. After that, the control button 9 is pulled upward relative to the disk base 5, by opening the grips of the collet clamp 11.

The tube 2 of the container 1 with bioobjects 3 is released and lowered to the bottom of the cryogenic vessel 24, and the device is removed from this vessel. The control button 9 is rotated around the axis 15 so that the outer teeth 10a of the inner ring 10 coincide with the inner grooves 7a of the outer ring 7. The cryogenic vessel 24 is closed with a lid 25 and sent for further storage or examination of the container 1 with bioobjects 3.

Information sources

1. US Patent Application Publication No.US 2015/0150241 A1. - Int. C1. A01N 1/02. Portable Device and Method for Cryopreservation of Cells Encapsulated in Immunoisolating Devices. - Provisional application No. 61/910,263, filed 01.12, 2014. - Pub. Date 06/04/2015.

Claims (18)

1. A stand-alone device for vitrification of bioobjects using a cryogenic refrigerant, having a coaxial design, including a cylindrical body, inside of which a cylindrical axially elongated support is installed, the end of which is connected to a collet clamp that secures the container tube with bioobjects, a trigger spring that interacts with the cylindrical support and ensures the movement of the container with bioobjects into the vessel with cryogenic refrigerant through its neck, a cylindrical thermal insulation Ion screen with a movable lid, a protective screen disc fixed to the shoulders of the cylindrical housing, and positioned in the upper part of the control button, characterized in that and the housing and the telescopic type support comprise an outer and an inner section, between which at least one intermediate section is located, the outer section of the casing is connected to the disk base, and its inner section is connected to the cylindrical heat shield using an outer ring, and a fixing spring is installed between the disk base and the outer ring, the outer section of the support is connected to a coaxially mounted control button, and its inner section is connected to the collet by means of an inner ring, the outer and inner rings are located in the same plane so that in the grooves of the outer ring there are outer teeth of the inner ring with the possibility of axial movement of the inner ring relative to the outer ring, the movable cover of the cylindrical heat-insulating screen is made in the form of a diaphragm with rotary petals that regulate the diameter of the hole, the control button is located above the disk base and connected to it by means of a return spring, which is covered by a number of protective walls located with the possibility of movement in the holes of the disk base, a trigger spring mounted inside the support and connected to the control button and the inner ring is held in a compressed state by an elastic retainer that changes its radial dimensions, which is connected to the trigger handle located in the recess of the control button, a protective disk screen is mounted on the outer section of the housing and is made of transparent heat-insulating material. 2. The stand-alone device according to claim 1, characterized in that the disk base is made with an expanded part intended to be gripped by the fingers of the operator’s hand. 3. The stand-alone device according to claim 1, characterized in that the protective disk screen is made of organic glass. 4. The stand-alone device according to claim 1, characterized in that the outer diameter of the protective disk screen exceeds the diameter of the neck of the Dewar vessel with cryogenic refrigerant. 5. The stand-alone device according to claim 1, characterized in that the springs are made of cylindrical screw. 6. The stand-alone device according to claim 1, characterized in that the elastic retainer is connected to the starting handle using a cable, which is located inside the Central tube. 7. The stand-alone device according to claim 5, characterized in that the retainer wall facing the trigger handle is located at an angle of about 90 ° to the axis, and its wall facing the inner ring is located at an angle of about 45 ° to the axis. 8. The stand-alone device according to claim 1, characterized in that the starting handle is rotatable in a plane perpendicular and parallel to the axis of the device. 9. The stand-alone device according to claim 1, characterized in that liquid nitrogen is used as the cryogenic refrigerant.

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