UA75789C2 - Appliance for cryo-conservation of biological objects of plant and animal origin - Google Patents

Abstract

This invention relates to cryo-biology, in particular to development of unit for cryo-conservation of biological objects of animal and plant origin, and can be used in bio-technology of stock-raising and plant cultivation. This appliance has Dewar vessel and thermo-block installed in its mouth with possibility of change of depth of immersion with respect to the upper edge of the mouth of the Dewar vessel. The thermo-block has disc holding the vessel for placing the containers with bio-objects, thermo-sensor and metal cylindrical body arranged with possibility of displacement in vertical direction and fixation with respect to the vessel. At that the vessel for placing bio-objects is arranged as a continuous metal, for instance copper cylindrical cup, at its inner side thermo-sensor is placed. This invention makes it possible to provide optimal mode of freezing of bio-objects and gives possibility to use different types of containers for biological objects.

Description

Description of the invention

The invention relates to cryobiology, namely to devices for cryopreservation of biological objects 2 of animal and plant origin and can be applied in the biotechnology of livestock and plant breeding.

A well-known device for freezing embryos of cattle (cattle) (Pat. Mo1802700 of the USSR, IPC 5

Ab1019/00. Installation for freezing embryos / F.Y. Ostashko, N.D. Bezuglii, E.G. Valigura, Gorbunov

L.V./ application Mo4922296/14 Appl. 29.03.91. Publ. 15.03.93. Bul. Mo10, 1993), consisting of a vessel

A dewar, in the neck of which a thermal block is rigidly fixed with fasteners, which is a container in the form of a 70 cylinder, made in the form of a grid with a polymer plug and holes for packages with biomaterial (plastic straws), a bracket, a threaded sleeve, nuts and a thermocouple.

However, the use of this device allows you to realize only a fixed freezing speed of 0.32C/min. for cattle embryos, which excludes the possibility of choosing optimal cryopreservation modes for other /5 types of bioobjects, the cooling rates of which differ (for rabbit embryos - 1 gC/min, for wild boar spermatozoa - 102C/min, etc.).

The installation for freezing biological objects is also known (Pat. Mo 60161 A Ukraine, IPC 7 g2503/10.

Installation for freezing biological objects and the method of initiation of crystal formation in a cryoprotective medium/ Gorbunov L.V., Kabachny V.I. Antonenko T.S., Gorbunova N.I., To-marovska 7T.O0./ application

Mo2003021337. Application 14.02.2003. Publ. 15.09.2003, Bull. Me9, 2003), consisting of a Dewar vessel and a thermal block, and the latter contains a cylindrical body made of stainless steel, a disk with holes for placing packages with biomaterial, limiting plates for supporting said packages, which are rigidly connected to the disk using stainless steel rods became In the known installation, the thermal block is fixed at a given depth in the Dewar vessel by means of a hollow sleeve with a thread connected to the disc, through which a rod can be immersed in liquid nitrogen to initiate crystal formation in a cryoprotective environment. In addition, the housing of the thermoblock can be vertically moved relative to the disk. Inside the case there are pins o) for fixing the case in a certain position. The thermoblock contains a thermosensor in the form of a thermocouple or a thermoresistor. To ensure different cooling modes, the presence of a removable bottom of the thermoblock of the known installation is also provided. But the use of this device does not allow freezing cell suspensions with a volume of more than 0.2 ml due to a large temperature gradient (approximately 22C/mm), which is observed in it during the freezing process. With the help of this device, it is possible to carry out cryopreservation of the medium containing the biological object only in containers in the form of plastic straws and Ulengut tubes, which excludes the possibility of cryopreservation of other types of biological objects, such as suspensions of cells and tissues, in addition to oocytes and embryos of mammals from animals and plants, for example: fish ejaculate with a volume of 0.5 72.5 ml; petioles, meristem tissues, seeds and pollen of plants.

The task of the invention is to create a device for cryopreservation of biological objects of animal and plant origin, in which, thanks to the new constructive solution of the thermoblock, the temperature gradient in the device is reduced to 1C/mm, a change in freezing rates is ensured in the range from 0.1 to 52 oC/min. , which "enables the implementation of optimal freezing regimes, in addition, the new device allows the use of containers of different designs and sizes for biological objects. y The task is solved in such a way that in a device for cryopreservation of biological objects "of animal and plant origin, consisting of a Dewar vessel and a thermoblock installed in its neck with the possibility of changing the depth of immersion relative to the upper edge of the Dewar vessel neck, and the thermoblock contains a disk that holds a container for placing containers with bioobjects, a temperature sensor and a metal cylindrical body, made with the possibility of vertical movement and fixation relative to the disk, it is additionally provided that the container for placing containers with biological objects is made in the form of a solid cylindrical metal , for example, a copper cup rigidly connected to the disk with the help of metal rods, and the temperature sensor is fixed on the inner side of the metal cup.

The technical essence and principle of operation of the proposed device is explained by the drawing on which

Fig. 1 - scheme of the device for cryopreservation of biological objects of animal and plant origin; (Che) Fig. 2 - diagram of the thermal block of the device for cryopreservation of biological objects of animal and plant origin.

The device consists of a Dewar vessel 1, a thermal block 2, which in turn contains a disk C with criss-crossing rods 4, a container 5 for placing containers with biomaterial 6, a metal case 7, a thermal sensor 8.

Fastening of the thermal block 2 in the neck of the Dewar vessel 1 is carried out with the help of a riser 9 connected to

Ф) threaded sleeve 10 attached to disk 3. Fixation of the immersion depth of the thermoblock 2 relative to the upper edge of the neck of the Dewar vessel 1 is achieved using the fastening nut 11.

The container 5 for placing containers with biomaterial 6 is made in the form of a solid metal glass because it is cylindrical in shape, made, for example, of copper. Fixation of the metal cylindrical body 7 relative to the container 5 is ensured, for example, by a pin 12.

The claimed device is used as follows. The first option. Biomaterial in the form of cells, buds, cuttings, etc. is placed in container b, which is placed in container 5. Envelopes made of aluminum foil and polyethylene, Ulengut straws, etc., can be used as containers. The speed 65 of cooling the containers depends on the location of the body relative to the glass ХО - 0 0030 (І) cm (І-15 - the height of the cylindrical body), and the final freezing temperature depends on the depth of immersion of the thermoblock G. in the neck of the Dewar vessel relative to its upper level (Fig. 1). H and Y are set according to predefined freezing modes depending on the type of biological object. The value of H is adjusted using the fastening nut 11, and Y - using the pin 12. With the immersion of the thermoblock 2 in the Dewar vessel to a given depth, the required speed and final temperature of the bioobject cooling are achieved, after which the thermoblock 2 is immersed in liquid nitrogen for storage for a given period time

The second option. Containers with biomaterial b are mixed into the pre-cooled thermoblock 2. To pre-cool the thermoblock, it is placed at a depth І to reach the required final temperature. The location of the container 5 relative to the metal body 7 and the immersion of the thermoblock 2 relative to 7/0. The neck of the vessel is dewared as in the first method. The thermoblock pre-cooling mode lasts from 0 to 90 minutes, depending on the set cooling speed. Then containers with biomaterial are placed in the thermoblock cooled in this way and kept for the time required to dehydrate the bioobject. After reaching the required final temperature in the containers, they are transferred to liquid nitrogen.

The invention is illustrated by examples.

Example 1

As an option, the device for cryopreservation of biological objects of animal and plant origin consists of a Dewar X-34A vessel (with a volume of 35 l and an inner diameter of the neck of b cm). If it is necessary to transport, or when freezing in the field, it is advisable to use a Dewar vessel X-5 (with a volume of 5 l and an inner diameter of the neck of 6 cm). Thermoblock glass 5 cm high

Made of copper. The thickness of the walls of the glass is 0.5 cm. The use of a copper glass provides a temperature gradient of 0.12C/mm. Polyethylene or aluminum foil envelopes, plastic straws and cryotubes are used as containers for biomaterial. The housing of the thermoblock is represented by a hollow cylinder 15 cm high, made of stainless steel, the housing relative to the glass can be moved both up and down with fixation in the desired position. When moving the body relative to the glass for every centimeter s, the temperature difference between the copper glass and the body changes by approximately 5 C, due to which the cooling rate of the copper glass changes. at

When the body is lowered relative to the glass, the cooling rate increases, and vice versa. The reproducibility of the freezing mode and the accuracy of determining the final temperature of cooling of biological objects are determined by the spread of the temperature distribution in the neck of the Dewar X-34A vessel, which is -2.59C, that is, it does not depend significantly. Ф) from the degree of filling of the vessel with nitrogen, changes in ambient temperature and atmospheric pressure under conditions of compliance with the requirements for laboratory premises and passport data of Dewar vessels. co

The spread of temperature with respect to the average value in vessel X-5 (when simulating transportation conditions) is 596.

To determine the possibilities of the proposed device, the selection of freezing modes was empirically carried out on samples of glycerin solution. Envelopes with a volume of M-2.5 ml are placed in a thermal block with a different location of the body relative to a copper glass, which was immersed to different depths in the neck of the vessel -

Dewar X-Z4A.

The speed of freezing varies depending on the degree of heat dissipation of the metal cup, which is provided by the different location of the cylindrical body of the thermoblock relative to it. To realize low freezing speeds, the heat is removed from the copper cup due to the elevation of the cylindrical body upwards. At the same time, the value H has a "-" sign. If the heat removal from the copper cup is carried out due to - lowering the body down, then the value H has the sign "-". h» For each freezing rate B and the final cooling temperature T, the values of H and Y were determined "experimentally. Since the cooling mode of the thermoblock is implemented according to an exponential dependence, the freezing rate was determined by the formula of the weighted average value in the temperature range from 09C to the final freezing temperature. - Data of the experiment are given in Table 1. o Table 1 - !And !!

Dependence of the freezing rate (V, gS/v..) on the immersion depth of the thermoblock (I, cm) in the Dewar vessel and the location of the body (Fe) 20 of the thermoblock relative to the glass (Н, cm) at different final cooling temperatures (Т Кк, С) for glycerol solutions in polyethylene cooling envelopes, cylinder location (H, cm) and different immersion depths (І.,, cm) 5 in 00004163 08 52063808 52 o yu 60

Thus, the change in the degree of location of the thermoblock body relative to the copper cup H (from "Z to -10 cm) thereby determines the cooling rate of containers with biological objects in a wide range from 0.1 to 30.72С/min. in the temperature range from 20 to -7096.

Example 2 65 The proposed device makes it possible to obtain different characteristics of changes in freezing rates of containers with biological material. For this purpose, the thermal block of the device is pre-cooled by placing it at a depth І to reach the required final temperature. The pre-cooling mode of the thermoblock lasts from 0 to 60 min., depending on the set freezing speed. Then, envelopes made of polyethylene or foil containing the biological object are placed in the thermoblock cooled in this way, kept for several minutes (to dehydrate the cells), after which the envelopes are transferred to liquid nitrogen for storage.

Using the proposed device, the main parameters and characteristics of which are presented in example 1, the following results were obtained (Table 2).

Table 2

Dependence of the freezing rate (V, C/min) on the pre-cooling time of the thermoblock (in, min) at a given position of the thermoblock body relative to the H-O cup see 51105118 tyu v8010zh01v61 vv 11111111 y y

Thus, the use of the proposed device allows you to increase the freezing speed up to 522C/min., to realize different characteristics of the change in the freezing speed.

Example C

The proposed device makes it possible to implement optimal modes of freezing of various types of biological objects using Dewar xX-5, X-34 and X-35 vessels in laboratory and production conditions.

With the help of this device, carp sperm were cryopreserved (Fig. 3, mode a) at optimal (o) freezing rates (Methodical manual for cryopreservation of sperm! carp, salmon and sturgeon species of fish: All-Russian Research Institute of Pond Fisheries. - Moscow, 1997-10 p.). The location of the cylindrical housing of the thermoblock relative to the copper cup corresponded to the position of H-0. Fo

The freezing process included the following stages: 1) Cooling of the thermoblock to 52.5 "С (this is the initial temperature of each mode of freezing (2.0) of fish sperm) by immersing it in the neck of the Dewar X-5 vessel to a depth of I -2 cm. 2) Immersion of the thermal block in the neck of the Dewar vessel to a depth at which the final cooling temperature of -15--2.5 C is realized. For cryopreservation of carp sperm at a speed of B.4-1 5 2 "C/min. Shcheo depth,

From the immersion of the thermoblock into the neck of the Dewar vessel X-5 was I -4.9 cm h- 3) Immersion of the thermoblock into the neck of the Dewar vessel to the depth at which the final cooling temperature is realized -70 2.590. For cryopreservation of carp sperm at a speed of 15 5 20 oC/min. the depth of immersion of the thermal block into the neck of the Dewar X-5 vessel was 1 -15 cm. 4) Transfer of packages with sperm into liquid nitrogen. "

At the same time, the level of preservation of carp sperm was 4095, which coincides with the level when using existing technologies. And with the help of the proposed device, spermatozoa were also cryopreserved according to the above "" stages with a freezing speed of 302С/min. to a temperature of -70-1С with subsequent immersion in liquid nitrogen (Fig. 3, mode b). Optimal freezing speeds ( Kharkiv technology of aseptic sampling and

KkCryopreservation of sperm! bykov-proizvodiets: Methodical recommendations under the editorship. Prof. F.I. Ostashko - I - Kharkiv. - 1990 - 47c) were provided with the help of a thermal block, which is placed in the neck of the vessel

Dewar Kh-34 at a depth of immersion of I-16 cm. Location of the cylindrical body relative to the copper cup and Nae-10. - I The level of preservation of deconserved sperm was 5095, which is 1095 higher than when frozen on traditional installations.

Me Example 4. Using the proposed device, cherry petioles were frozen at a rate of 12C/hour in the temperature range from -5 to -309C with exposure of biological material at this final temperature for 48 hours and subsequent immersion in liquid nitrogen. The location of the cylindrical body relative to the copper beaker N-Z cm. The optimal modes of freezing biological objects were implemented using a Dewar X-35 vessel (with a volume of 35 l and an inner diameter of the neck of 12 cm). Due to the increase in the internal diameter of the Dewar vessel by two times, the volume of the thermoblock increased four times at the same height of the copper cup (5 cm) and 8 times at the height of the copper cup 10 cm. The thermoblock was immersed in the neck of the Dewar vessel X-35 to a depth of I -11 cm. The level of preservation of deconserved cuttings was 2890, which coincides with the data obtained during freezing with the help of traditional devices (Tom! //

Stuo-IeNegv. - 1999, - Mine. 20.- Mo4.-R. 215-222).

Thus, the use of the proposed device in comparison with the existing ones has the following advantages: - the temperature gradient in the device is reduced to 1 ""С/mm thanks to the design of the thermoblock, optimal freezing modes are implemented due to the change in freezing speeds in the range from 0.165 to 522С /xMin. - the use of containers of different designs and sizes expands the possibilities of freezing various types of biological objects.

The cost of manufacturing the proposed device and the freezing process itself for animal sperm is 10 times lower than when using traditional technologies, and for embryos it is similar to known devices.

The developed device allows obtaining different characteristics of changes in cooling rates and carrying out freezing in both laboratory and production conditions. Varying the characteristic heat exchange time of the thermoblock allows its use for freezing various types of biological objects: sperm of mammals and fish, meristem tissues and petioles of plants, etc.

Modes of freezing animal sperm using a thermoblock placed in the neck of a vessel 7/0 Dewar X-5: a - bull sperm at Н-А-10; 6 - carp sperm H-O; I - the depth of immersion of the thermoblock of the device into the neck of the Dewar vessel |cm; B is the value of the freezing rate (eC/min.

Claims (1)

The formula of the invention What is the device for the cryopreservation of biological objects of animal and plant origin, consisting of a Dewar vessel and a thermoblock installed in its neck with the possibility of changing the depth of immersion relative to the upper edge of the Dewar vessel neck, and the thermoblock contains a disc that holds a container for placing containers with bioobjects, a temperature sensor and a metal cylindrical body made with the possibility of vertical movement and fixation relative to the container, which is characterized by the fact that the container for placing containers with biological objects is made in the form of a solid cylindrical metal, for example, a copper cup, rigid connected to the disk with the help of metal rods, and the thermistor is fixed on the inner side of the metal cup. c schi 6) (o) (ee) u iv) and - - . and? -i 1 -i (ee) 3e) ime) 60 b5