RU1794236C - Device for freezing biological objects - Google Patents

Description

contact with the refrigerant. Containers for biological objects are inserted in the nest.

The freezer is made of a light material with a low coefficient of thermal conductivity.

Owing to the good thermal conductivity of metal rods and nests, biological objects in containers are frozen at an almost constant rate.

However, a sufficiently large modulus of the temperature gradient in the gas medium of the refrigerant arises in the volume of the freezing zone (since the upper part of the containers is in refrigerant vapor, and part of the container is in contact with liquid refrigerant through heat-conducting rods), which reduces the safety of biological objects.

In the known device, there is no possibility of initiating ice formation in the extracellular medium, which also reduces the safety of biological objects.

The cooling rate of the biological material in the known device is controlled by changing rods of different diameters, i.e. only discrete adjustment of the cooling rate is allowed, and not smooth.

The aim of the present invention is to increase the safety of biological objects during the freezing process by reducing the temperature gradient modulus in the freezing zone and providing a smooth adjustment of the cooling rate of objects with the initiation of ice formation in the extracellular medium to reduce initial supercooling.

This object is achieved in that in a device for freezing biological objects, comprising a body made of a material with a low coefficient of thermal conductivity, containers for a biological object and installed in the body with the possibility of placing on the surface of the liquid refrigerant a container holder including a disk with container slots, according to the invention, a holder equipped with a cover mounted on the disk with the formation between them of a cavity located along the vertical axis of the disk, a pipe with reinforced n and its ends with radiators and a control valve installed in the hole of the upper end of the pipe. The disk is thickened and has a central vertical channel around the pipe and a chamber for generating refrigerant vapor located at the lower base of the disk and communicates with the channel in the disk and with the working volume of the housing below the expected level of refrigerant in it. The disc channel is connected by means of capillary tubes to the zones of each container for supplying gaseous refrigerant to their walls.

The upper radiator of the pipe is located in the holder cavity above the containers, and the lower one is in the chamber to form refrigerant vapor.

Due to the design of the container holder in the form of a disk with a lid, a cavity is formed for freezing the biological object in containers with an adjustable valve by supplying refrigerant vapor through the central channel pipe and mixing in this

5 cavity refrigerant vapor upper radiator. As a result, the temperature gradient modulus in the entire cavity and, accordingly, in the entire volume of each of the containers with biological

0 material.

A decrease in the temperature gradient modulus in the zone of each container with biological objects increases their safety during cryopreservation according to

5 compared with the prototype.

The execution of capillaries from the central channel to each container allows refrigerant vapors to be brought directly to the wall of the container and to initiate ice formation inside the container in an extracellular medium. The resulting ice crystals are seeded to crystallize all the water in the container. Moreover, in the entire contents of the container

5 eliminates hypothermia, which also contributes to the preservation of biological objects during cryopreservation, which was not achieved with the prototype. The lower radiator on the pipe in the widened part of the central channel at the base of the container holder disk facilitates the evaporation of liquid refrigerant by transferring heat from the freezing zone from the containers and the upper radiator.

5 The amount of refrigerant vapor supplied to the cavity of the container holder is continuously controlled by a valve in the upper part of the pipe.

When searching the patent and scientific and technical literature, no objects were found with signs of the claimed technical solution, on the basis of which it can be concluded that it meets the criterion of significant differences.

5 The drawing shows a vertical sectional view of a general view of a device for freezing biological objects.

The device comprises a housing 1 with a cover 2, filled with liquid refrigerant 3, for example, nitrogen. Inside the housing 1, on the surface of the refrigerant 3, there is a holder in the form of a disk A with a thickened body 5 and a cover 6 forming a cavity located along the vertical axis of the disk 4.

Sockets 7 are made in the body 5 of the disk 4, with containers 8 for biological objects installed therein.

At least one vertical central channel 9 is formed in the body 5 of the disk 4 with an expanded part at the lower base of the disk 4. forming a chamber 10.

Capillaries 11 extend from channel 9 to the area of each container 8.

Inside the channel 9 is a pipe 12, at the lower and upper ends of which are installed radiators 13 and 14, respectively. The lower radiator 13 is located in the chamber 10. The upper radiator 14 is located in the cavity of the holder above the containers 8.

In the upper part of the pipe 12 there is a control valve 15.

The pipe section 12 between the body 5 of the disk 4 and the radiator 14 is insulated with thermal insulation 16.

A ballast 17 with an opening 18, coaxial to the channel 9, is attached to the lower base of the disk 4 from the outside.

Case 1 with a cover 2, a disk 4 with a cover 6 and containers 8 are made of material with a low coefficient of thermal conductivity. Radiators 13 and 14, as well as the pipe 12 are made of highly conductive material, for example, aluminum, copper.

PRI me R 1. Used a cylindrical body 1 with an inner diameter of 190 mm and a height of 400 mm, in which three liters of liquid refrigerant 3 (nitrogen) were poured.

The cylindrical foam disk 4 had an outer diameter of 170 mm and a height of 150 mm. The cavity between the body 5 of the disk 4 and the cover 6, forming the working cooling zone, had dimensions: diameter 120 mm and height 80 mm.

Plastic containers 8 had an outer diameter of 15 mm with a wall thickness of 1.5 mm and a height of 80 mm.

To control the cooling rate and initial supercooling of the extracellular medium of the contents of the containers 8, we used a differential copper – copel thermocouple with the corresponding recording equipment (not shown in the drawing). The measuring junction of the thermocouples was placed in the center of the volume of one of the containers 8,

The freezing of biological objects in containers 8 was carried out at a rate of 1 bC / min to -40 ° C with further immersion in liquid nitrogen.

The role of the temperature in the working area in the cavity of the disk 4 was performed at six points also by thermocouples with the corresponding recording equipment (not shown in the drawing). Each of the six points was located at a distance of 10 mm from the surface of the wall of the container 8. In height, the measuring points were located at 1/3 and 3/4 of the height of the containers 8. In terms of points

alternated through 60 angular degrees.

8 ml of leuko-concentrate obtained from human donated blood, diluted in a 1: 1 ratio with a cryoprotector - 10% dimethyl sulfoxide solution were poured into containers 8. The containers 8, after filling them with a biological object, were hermetically sealed.

Seven such containers 8 with a positive initial temperature with the cover 6 and radiator 14 removed were installed in the slots 7 of the body 5 of the disk 4. Then, the radiator 14 was fixed, the cooling rate was set at 1 ° C / min with the control valve 15. After that, the lid 6 was closed and

the disk 4 was immersed in the housing 1 and closed its cover 2.

The heat from the containers 8, the gas medium in the working area and the radiator 14 was transferred through the pipe 12 and channel 9 to the chamber 10 to the radiator 13. Thanks to this heat, liquid nitrogen boiled in the chamber 10, and its vapor entered the pipe 12 through the control valve 15 into the working zone disk 4. Radiator 1.4 contributed to equalization of temperature throughout the volume of the cavity. At a given speed, the temperature in the containers 8 was reduced to cryoscopic. At the same time, nitrogen vapors from chamber 10 along channel 9 and capillaries 11 rose directly to the walls

containers 8. The local effects of nitrogen vapor on the walls of containers 8 causes inside them almost in a point volume a decrease in temperature relative to the entire volume of the contents of containers 8. B

the result was local cooling of the solution, sufficient to form ice crystals from the aqueous component of the solution, which were seed for crystallization of the aqueous

components of the entire bulk of the extracellular environment of a biological object during the transition of water to ice.

At the same time, overcooling was practically excluded in the entire contents of containers 8 (see the measurement results).

During the freezing process, when control temperature was reached at point No. 1, the temperature at points No. 2 was simultaneously measured; No. 3. No. 4, No. 5 and No. 6.

The results of temperature measurements at six points during the freezing period are given in Table 1 in the form of averaged values over three (p 3) tests. Measurements were carried out with an interval of time (10 ± 0.05) min.

Table 2 presents the data on the initial hypothermia in the extracellular medium inside one of the containers 8.

Seven similar containers 8 with the same biological object were frozen in a prototype device with a cooling rate of 1 ° C / min to -40 ° C, followed by immersion in liquid nitrogen.

In the prototype device, the cooling rate of the cell suspension and the initial supercooling of the extracellular medium were controlled by a similar thermocouple, the measuring junction of which was located inside one of the containers.

Same. as in the inventive device, the temperature was controlled at six points in the working area of the freezer.

The measurement results are given in table 1 and 2.

In both cases, the cell suspension of the leukoconcentrate was thawed up to 0 ° C in a water bath with a temperature of 37 ° C.

In the native and thawed cell suspension, the total number of cells was counted and their viability after staining with vital dyes was determined.

Table 3 presents the results. Unreliability P of a quantitative assessment of cell viability is estimated by an experimental value of P 0.05.

From the analysis of the measurement results presented in Table 1, it follows that when a biological object is cooled in a prototype device, the temperature gradient modulus in the working zone varies over a wide range - from 0.4 ° C to 2b, 4 ° C, which leads to uneven conditions cooling each of the containers (top of them).

The cooling of the biological object in the inventive device allows to reduce the variation of the temperature gradient modulus in the working area in the disk cavity 4 to (0.5-3.2) ° C, which is about 10 times less than that of the prototype.

Table 2 shows that in the prototype device during freezing of a biological object, significant overcooling (about 6.3 ° C) of the contents of the container takes place, and in the proposed device practically no overcooling is observed, i.e. close to 0 ° C.

The uneven cooling of each container, as well as the large hypothermia of the aqueous component of the extracellular medium during the water-ice phase transition, reduces the viability of the biological object when it is frozen in a prototype device. The claimed device is devoid of these drawbacks, which is confirmed by clause 3. The viability of the cells of the deconserved leukocyte mass frozen in the inventive device is 14% higher compared to the prototype.

Example 2. In the claimed device and the prototype device, hematopoietic cells obtained from pig embryonic liver and diluted 1: 1 with a protective solution containing Hanks solution - 90% and dimethyl sulfoxide - 10% were frozen.

The prepared suspension was poured into 8 ml in plastic containers 8, sealed and placed in the freezer of the claimed device and prototype device.

Subsequently, freezing and heating of biological objects was carried out analogously to example 1, but without control

temperatures since previously calibrated devices were used.

The viability of native and thawed suspension cells was determined after staining with vital dye (Methylene Blue).

The number of cells in the suspension was counted in a Goreva chamber. The unreliability of the assessment of the parameters of the objects is evaluated, as before, by a value of P 0.05,

The results are presented in table 4.

From the data given in table 4, it is seen that the safety of cells frozen in the prototype device (about 14%).

Thus, the claimed device

for each freezing cycle (1.5-2 hours) allows you to save 14% more viable cells of biological objects, compared with the prototype device.

Claims (1)

SUMMARY OF THE INVENTION A device for freezing biological objects, comprising a body made of a material with a low coefficient of thermal conductivity, containers for a bioobject, and a container holder mounted on the surface of a liquid refrigerant, including a disk with container slots, characterized in that the holder is provided with a lid; mounted on the disk with the formation of a cavity between them, located along the vertical axis of the disk, with a pipe with a radiator fixed at its ends E and adjusting kla0 5 the pan installed in the hole of the upper end of the pipe, the disk is thickened and has a central vertical channel formed around the pipe, a chamber for generating refrigerant vapor located at the lower base of the disk, in communication with the channel and with the working volume of the housing below the expected level of refrigerant in it, the channel is connected by capillary tubes to the areas of each container for supplying gaseous refrigerant to their walls, the upper radiator is located in the holder cavity above the containers And the bottom - to form refrigerant vapor in the chamber. Table 1 table 2 Table 3 Table 4