JPH0426601A - Freezing apparatus for implantation in ice - Google Patents

Abstract

PURPOSE:To obtain the subject apparatus by providing an outer tank, an inner tank, an exterior tank having a heater for the control of brine temperature and a brine- circulation apparatus and placing a bio-specimen holding vessel in the inner tank in a state to expose a part of the vessel to the space in the outer tank. CONSTITUTION:The objective freezing apparatus for the implantation in ice is provided with two outer tanks 1,10, inner tanks 2, 20 placed in respective outer tanks, an exterior tank 4 having a heater 3 for controlling the brine temperature and a brine- circulation apparatus composed of pipes 51, 52, 53 and shut-off valves V1 to V4. In the case of freezing a bio-specimen in the outer tank 1 and the inner tank 2, the valves V1 and V2 are opened and the valves V3 and V4 are closed. The inner tank 2 is taken out of the outer tank together with a rack part 6, a straw S containing a bio-specimen and a preservation liquid is set to the inner tank and the whole assembly is placed in the outer tank 1. Brine is circulated by operating a pump 50 to cool the gaseous phase part and a liquid phase part in the outer tank with coolers 71, 72 and propagate the freezing from the storage liquid in each straw protruded into the outer tank successively to the inner bio-specimen without causing super-cooling of the liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an ice-plant freezing device for freezing biological materials such as cells, embryos, and fertilized eggs of animals and plants, in order to preserve them by cryopreservation.

[Conventional technology]

There are various types of devices for freezing biological materials, one of which is to store the material together with a preservation solution in a container called a tube, straw, etc. There is a method in which the liquid phase is soaked in a liquid phase and the liquid phase is cooled using a refrigerator.

When freezing biological material in such equipment,
Simply cooling the material and storage solution will result in large supercooling during the material freezing process, and freezing after long periods of supercooling will cause physical damage and induction of intracellular freezing due to rapid acid content of ice crystals. ■There is a problem that rapid temperature changes occur due to heat of solidification, which reduces the survival rate of biological materials.

In order to prevent such supercooling, trees are generally planted, but these planting methods include: (1) mechanically vibrating or impacting the container; (2) immersing a needle cooled with liquid nitrogen, etc. in a preservation solution. , ■ Bringing a bottle set cooled with liquid nitrogen, etc. into contact with the outer wall of the container, ■ Bringing a plant plate with a path for liquid nitrogen, etc. into contact with the part of the container that is exposed to the gas phase of the cryopreservation device, A known method is to bring a planting plate equipped with a cooling element that utilizes the Peltier effect into contact with the part exposed to the gas phase.

However, in the method of applying vibration or impact, it is time-consuming to apply the vibration or impact, and there is no guarantee that plants will be reliably and uniformly planted in all containers.

In addition, with the method of using cooled needles or tweezers, the planting work must be carried out for each individual container, which is time-consuming and creates a time lag between the start and end of the planting work, resulting in variations in the freezing conditions of the material. occurs. Furthermore, when planting trees, the container storage chamber of the apparatus must be opened once during the freezing operation, and as a result, the room temperature rises, making it impossible to freeze the sample quickly.

In the case of a planting plate in which liquid nitrogen or the like is poured, there is an advantage that planting work can be started on all containers at the same time, but liquid nitrogen and the like are expensive and management of the liquid nitrogen and the like is troublesome.

In the case of a planting plate that employs a cooling element based on the Peltier effect, the cooling element is expensive.

In order to solve such problems, freezing devices shown in FIGS. 2 to 4 have recently been developed.

Fig. 2 is a plan view with the top cover removed, Fig. 3 is a sectional view taken along line 3-3 in Fig. 2 with the top cover attached, and Fig. 4 is a plan view with the top cover removed. FIG. 4 is a sectional view taken along line 4-4 in FIG. 3;

In this device, an inner tank 82 is arranged inside an outer tank 81, brine is put into the outer tank and the inner tank, and the outer tank brine B1 is cooled by a cooler 83 installed at the lower part of the outer tank, and the temperature is controlled by a heater 84. The temperature of the inner tank brine B2 is controlled by a heater 85 provided in the inner tank, and the outer tank brine B1 is cooled through the inner tank wall. It is configured so that it can be stirred at a speed of .87 cm. The upper opening of the outer tank 81 is a glass lid 8.
Can be opened and closed at 9. The inner tank 82 has a side wall 88 that is removable by moving up and down, and has a plurality of holes 88a that support the straw 8 containing biological material in a horizontal position.

According to this device, the lid 89 is opened and the straw 3 containing the biological material is inserted into the hole 88a of the side wall 88 of the inner tank taken out, and the part containing the biological material is placed in the inner tank and the other part is placed in the inner tank. The side wall is set in the inner tank 82 so as to be placed in the outer tank. Next, the lid 89 is closed, and the outer tank cooler 83 and heater 84 are operated to cool the outer tank brine B1 to a predetermined temperature, while the inner tank brine B2 is cooled to a predetermined temperature while controlling the temperature with the inner tank heater 85. During this time, the outer tank and inner tank brines B1, B2 are stirred with stirrers 86,87. The part of the storage liquid in each straw 8 that protrudes into the outer tank 81 is frozen first, and then the part inside the inner tank 82 is frozen starting from that frozen part, and the biological material in each straw is frozen. Ru.

[Problem to be solved by the invention]

According to the apparatus described above, the above-mentioned problems are solved, but there is still the following problem.

That is, when all the holes 88a of the inner tank side wall 88 are not used, the unused holes must be plugged to prevent the inner and outer tank brine from communicating with each other.

Since the biological material in the inner tank must be frozen through the inner tank wall, there is a limit to the rate of temperature drop, and the biological material cannot be frozen as quickly.

The inner tank side wall 88 also serves as a straw rack and can be attached and detached, so when the inner tank side wall 88 is removed for attaching and detaching the straw, the inside and outside brine mix and reach a different temperature, resulting in a short freezing time. For the purpose of shortening the temperature, the outer tank brine B1 is lowered to a considerably low temperature in advance, and the temperature of the inner tank brine B2 is lowered by heating the heater 8.
5, it becomes difficult to gradually lower it while controlling it.

Therefore, it is an object of the present invention to solve the problems in the conventional device.

[Means to solve the problem]

In order to solve the above problems, the present invention includes an outer tank, an inner tank housed in the outer tank, an outer tank having a heater for controlling brine temperature, and a brine circulation device, and the inner tank has an upper part. The brine circulation device has a rack portion having a brine supply port and a brine overflow port, and a rack portion that can be placed in the inner tank with a part of the biological material storage container exposed to the outer tank. Brine is supplied from the tank to the inner tank and overflows from the inner tank to the outer tank, so that the brine liquid level in the outer tank is lower than the part of the container that is supported by the rack part and protrudes into the outer tank. The present invention provides an ice planting freezing device which is configured to return ice to the external tank so as to maintain the frozen ice, and the outer tank is equipped with coolers in a liquid phase portion and a gas phase portion.

The brine supply port and brine overflow port of the inner tank may be provided separately or in common on the inner tank wall, etc.
To simplify the apparatus, the upper end of the inner tank may be opened and used as a brine supply port and a brine overflow port. Further, when a rack part supporting a biological material storage container is disposed at the upper end of the inner tank, an opening or a notch provided in the rack part may be used as a brine supply port or a brine overflow port.

The rack part may be formed using an upper side wall of the inner tank. In this case, it is preferable to form a horizontal hole in the upper part of the side wall of the inner tank so that the container can be inserted into the inner tank in a liquid-tight manner. In this type of rack, the container is usually a straw.

Further, the rack portion may be constituted by a member having a vertical hole portion into which the container can be inserted into the inner tank. In this case, the container may be a straw or a tube.

[For production]

According to the planting ice freezing apparatus of the present invention, the container containing the biological material together with the preservation solution is supported by the rack part, and the part containing the biological material is disposed in the inner tank, and the part containing the biological material is arranged in the outer tank. Placed in the gas phase.

The brine is cooled down to a predetermined temperature by a cooler in the liquid tank section of the outer tank, and is then supplied from the outer tank to the inner tank by a circulation device while the temperature is controlled by a heater in the outer tank, and the brine overflows from the inner tank. The discharged material is circulated from the outer tank 1 back to the outer tank 4.

On the other hand, the gas phase portion of the outer tank is cooled by a cooler placed there.

Thus, the storage solution in the portion of the container placed in the gas phase section of the outer tank first starts freezing, and then, starting from the frozen portion, the storage solution and biological materials in the portion of the container placed in the inner tank start to freeze. freezes without becoming supercooled.

If the rack part of the container includes a horizontal hole provided in the upper part of the inner tank side wall, and there are a plurality of horizontal holes, and the number of containers to be frozen is less than the number of holes, If the holes in the lower tier are filled with the container sequentially, the holes in the upper tier do not necessarily need to be plugged, and even if they are left open, the container will be soaked by the brine supplied to the inner tank, and the holes in the upper tier than the container will be filled with brine. The brine that has risen to overflows through the open hole at the top.

〔Example〕

Embodiments of the present invention will be described below with reference to FIG.

This ice planting freezing device is equipped with two outer tanks 1.1o, an inner tank 2 is located inside the outer tank 1, and an inner tank 20 is located inside the outer tank 10.
Each is equipped with

The inner tank 2 is placed on a stand 12 inside the outer tank 1, and can be attached to and removed from the outer tank 1 by removing the outer tank lid 11. The inner tank 20 is placed on a stand 102 inside the outer tank 10, and can be attached to and removed from the outer tank 10 by removing the outer tank lid 101.

The outer tank 1 includes an upper cooler 71 and a lower cooler 72 inside. The inner tank 2 includes a rack part 6, which has a side wall of the inner tank with a plurality of horizontal holes 61 through which the straw 8 can be inserted into the inner tank 2 in a fluid-tight manner while leaving a portion of the straw 8 in the outer tank 1. It consists of an upper part and a support member 62 fixed to the inner tank 2 and provided with a plurality of holes for supporting a portion of the straw protruding into the outer tank 1.

On the other hand, the outer tank 10 includes an upper cooler 710 and a lower cooler 720 inside. A rack section 60 is arranged at the upper end of the inner tank 20. The rack section 60 is made of a plate-like member, and includes a plurality of holes 601 through which the tubes T can be inserted into the inner tank while leaving a portion in the outer tank, and openings 602 and 603 through which brine can flow.

An external tank 4 is provided outside the outer tank 1.10, and a heater 3 for controlling the brine temperature is provided inside the tank.

The apparatus further includes a brine circulation device 5. The device 5 includes a pump 50 attached to the external tank 4 and a pipe 51.5 that supplies brine B in the tank 4 to the internal tank 2.20.
2 and a pipe 53 that guides the brine B overflowing from the inner tank to the outer tank 1.10 to the outer tank 4 again. An on-off valve V2 is provided on the pipe 52, an on-off valve V3 is provided on the pipe 52, and an on-off valve (4) is provided on the path returning from the outer tank 10 to the tank 4.

In the apparatus described above, when freezing biological materials in the outer tank 1 and the inner tank 2, valves V and 2 in the circulation device 5 are opened, and valves 3 and 4 are closed.

Then, open the lid 11 of the outer tank 1, take out the inner tank 2 together with the rack part 6, and insert the straw into the rack part 6.
Set 8. Each straw to be set contains a preservation solution along with biological material. Each straw 3 is set so that a portion of the straw 3 protrudes outside the inner tank 2 and the biological material storage portion is disposed within the inner tank 2.

In this way, the inner tank 2 and the straw 3 are placed in the outer tank, placed on the stand 12, and the outer tank lid 11 is closed.

In this state, the pump 50 in the circulation device 5 is operated.

In this way, the brine in the external tank 4 is supplied to the inner tank 2 from the upper end opening of the inner tank 2 through the pipe 51, and the brine is supplied to the inner tank 2 through the pipe 51.
The biological material storage section of is soaked by said brine B.

The brine B overflowing from the inner tank 2 is circulated from the bottom of the outer tank 1 through the pipe 53 to the outer tank 4 again.

During this time, the gas phase portion in the outer tank 1 is cooled by the upper stage cooler 71. The liquid tank section is always located below the straw 3 and the upper cooler 71, and is cooled to a predetermined temperature by the lower cooler 72.

While the brine circulates in this way, the gas phase and brine in the outer tank 1 are gradually cooled, and the brine supplied from the outer tank 4 to the inner tank 2 is also
The temperature gradually decreases while being controlled by Note that the temperature of the brine supplied to the outer tank gas phase portion and the inner tank may be lowered to some extent in advance.

Thus, first, the storage solution in the part of each straw that protrudes into the outer tank 1 starts to freeze, and then the storage solution and biological material in the part inside the outer tank 2 start from the frozen part in the outer tank 1. As a result, it freezes without becoming supercooled. In other words, the coolers 71, 72 and the heaters 3 are controlled accordingly.

On the other hand, when performing a freezing operation using the left side portion in the figure, valves V3 and ■4 in the circulation system are opened, and valves ■1 and ■2 are closed. Then, the outer tank lid 101 is opened and the tube T is inserted into the rack member 60 at the upper end of the inner tank. A biological material is stored in each tube together with a preservation solution, and the biological material storage part is placed in the inner tank 20 so that a part thereof protrudes into the outer tank 10. After closing the outer tank lid 101, by operating the pump 50, the outer tank 4
The brine inside is supplied to the inner tank 20 from the opening 603 of the rack member 60, and the biological material storage portion of each tube T is immersed in the brine B. The brine B overflowing into the inner tank 20 overflows into the outer tank 10 from the openings 602 and 603 of the rack member 60 and circulates back to the tank 4 from the lower part of the outer tank.

During this time, in the outer tank 1, the liquid tank section is connected to the upper cooler 710.
It is located further down and is cooled to a predetermined temperature by the lower cooler 720.

In this way, in the outer tank 10, the gas phase is the cooler 710.
The liquid tank section is cooled by the cooler 720, and the brine in the external tank 4 is cooled while its temperature is controlled by the heater 3.

Note that the temperature of the brine supplied to the gas phase portion of the outer tank and the inner tank may be lowered to some extent in advance.

While the brine is being circulated in this state, in each tube, the preservation solution in the part protruding into the outer tank 10 first starts freezing, and then the preservation solution and the organisms in the inner tank 20 start from this frozen part. The material freezes without being supercooled. In other words, the coolers 710, 720 and the heaters 3 are so controlled.

In addition, when performing a freezing operation using the left and right parts at the same time, all valves (1) to (4) may be left open.

If there are not as many straws 8 as there are straw insertion holes 61 in the upper part of the side wall in the inner tank 2 on the right side, insert the straws 3 sequentially from the bottom hole 61 toward the upper hole. Bye. By inserting like this,
The supported straws 3 are all immersed in brine supplied from the tank 4, and the brine that has reached the upper level of the straws 8 overflows into the outer tank 1 through the open hole 61 in the upper stage.

The highest straw among the straws set in inner tank 2
If one or more of the holes 61 on the same level as the straw S are not filled with straws 3, the holes may be plugged, but since the holes in the straw S are small, the circulation system 5, the amount of brine supplied from the tank 4 is increased to a certain extent (by this, the inner tank liquid level can be made higher than the straw 3 even if the hole is left open, so there is no need to plug the hole. do not have.

On the other hand, in the left part, the vertical hole 60 of the rack member 60
1 is not completely filled with the tube T, there is no problem even if the open hole is left open as it is.

Note that the coolers 71 and 72 of the outer tank 1 may be provided separately, but they may not be provided as one unit. Cooler 7 of outer tank 10
The same applies to 10.720.

〔Effect of the invention〕

As explained above, according to the ice planting freezing apparatus of the present invention, containers for storing biological materials can be easily used regardless of whether they are straw type or double type. Even if the number of insertion holes is less than the number of container insertion holes, it is not necessary to plug the holes, so handling is easy, and the temperature of the gas phase of the outer tank and the temperature of the brine supplied to the inner tank can be controlled respectively. There is no risk of overcooling (plant freezing operations can be carried out immediately).

[Brief explanation of drawings]

Fig. 1 is a sectional view showing an embodiment of the present invention, Figs. 2 to 4 show a conventional example, Fig. 2 is a plan view with the lid removed, and Fig. 3 is a plan view with the lid attached. FIG. 2 is a sectional view taken along line 3-3, and FIG. 4 is a sectional view taken along line 4-4 of FIG. 3 with the lid removed. 1.10...Outer tank 2.20...Inner tank 21...Top opening 3 of inner tank 2...Heater 4...Outer tank 5...Brine circulation device 6.60...・Rack part 61...Horizontal hole 601...Vertical hole 602.603...Lower between rack parts 1.72.710.720...Cooler S...Straw T...Tube

Claims (3)

[Claims] (1) An outer tank, an inner tank stored in the outer tank, an external tank having a heater for controlling brine temperature, and a brine circulation device, and the inner tank has a brine supply port and a brine overflow at the top. The brine circulation device has a rack part that has an outlet and can be placed in the inner tank with a part of the biological material storage container exposed to the outer tank, and the brine circulation device supplies brine from the outer tank to the inner tank. The brine overflowing from the inner tank to the outer tank is controlled by the outer tank so as to maintain the brine level in the outer tank at a lower level than the part of the container that is supported by the rack part and projects into the outer tank. 1. A planting ice freezing device configured to be returned to a tank, and characterized in that the outer tank is equipped with coolers in a liquid phase portion and a gas phase portion. (2) The ice planting freezing device according to claim 1, wherein the rack section includes an upper part of the side wall of the inner tank, which has a horizontal hole through which the container can be inserted into the inner tank. (3) The ice planting freezing device according to claim 1, wherein the rack section includes a member disposed above the inner tank and having a vertical hole section through which the container can be inserted into the inner tank.