JP2889654B2 - Ice plant freezing equipment - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an ice-plant freezing apparatus for freezing biological materials such as animal and plant cells, embryos, and fertilized eggs in order to cryopreserve the biological materials.

[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, a straw, or the like. And the liquid phase is cooled by a refrigerator.

When freezing biological materials in such an apparatus, simply cooling the materials and the preservation solution causes a large supercooling in the material freezing process, and freezing after the supercooling causes rapid ice crystal growth. There is a problem in that physical damage, induction of freezing in cells, rapid change in temperature due to heat of solidification, and the like occur, thereby reducing the survival rate of the biological material.

In order to prevent such supercooling, generally, ice seeding is performed.
Gives a shock, immerses a needle cooled with liquid nitrogen or the like in a preservation liquid, makes tweezers cooled with liquid nitrogen or the like contact the outer wall of the container, a passage for liquid nitrogen or the like in the container part which is exposed in the gas phase part of the cryopreservation device There is known a method of contacting an ice planting plate provided with a cooling device or an ice planting plate equipped with a cooling element utilizing the Peltier effect to a portion of the freezing apparatus that is exposed to the gas phase.

However, the method of applying the vibration or the impact requires time and effort to apply the vibration or the impact, and there is no guarantee of reliable and uniform ice implantation for all containers.

Further, in the method using the cooled needles or tweezers, since it is necessary to carry out the ice-planting operation for each individual container, it is troublesome, and there is a time difference between the beginning and the end of the ice-planting operation, and the freezing condition of the material is increased. Will vary. Furthermore, in the case of ice-planting, the container housing chamber of the apparatus must be once opened during the freezing operation, so that the room temperature rises and the sample cannot be frozen immediately.

In the case of an ice-planting plate through which liquid nitrogen or the like flows, there is an advantage that the ice-planting operation can be started simultaneously for all the containers, but liquid nitrogen and the like are expensive and management of the liquid nitrogen and the like is troublesome.

In the case of an ice planting plate employing a cooling element based on the Peltier effect, the cooling element is expensive.

In order to solve such a problem, a freezing apparatus shown in FIGS. 2 to 4 has recently been developed. FIG. 2 is a plan view showing a state in which the upper lid is removed, and FIG. 3 is a sectional view taken along line 3-3 in FIG.
The figure is a sectional view taken along the line 4-4 in FIG. 3 showing the top cover removed.

In this apparatus, an inner tank 82 is arranged in an outer tank 81, brine is put in the outer tank and the inner tank, and the outer tank brine B1 is cooled by a cooler 83 provided at a lower level of the outer tank, and the temperature is controlled by a heater 84. The inner tank brine B1 is cooled through the inner tank wall by the outer tank brine B1, while the temperature is controlled by a heater 85 provided in the inner tank.The outer tank brine B1 and the inner tank brine B2 are each stirred by an agitator 86. , 87 so that stirring is possible. The upper opening of the outer tank 81 can be opened and closed by a glass cover 89. The inner tank 82
The one side wall 88 is detachable by elevating and lowering, and has a plurality of holes 88a for supporting the straw S containing the biological material in a horizontal posture.

According to this device, a lid 89 is provided, a straw S containing a biological material together with a preservation solution is inserted into a hole 88a of an inner tank side wall 88 taken out, and the biological material containing portion is placed in the inner tank and the other portion is removed. The side wall is set in the inner tank 82 so as to be arranged in the tank. Next, the lid 89 is closed, and the outer tank cooler 83 and the heater 84 are closed.
Is operated to cool the outer tank brine B1 to a predetermined temperature and to cool the inner tank brine B2 to a predetermined temperature while controlling the temperature with the inner tank heater 85. During this time, the outer tank and inner tank brine B
1, B2 is stirred by stirrers 86 and 87. The portion of the preservation solution in each straw S that protrudes into the outer tub 81 is frozen first, and then the portion inside the inner tub 82 is frozen starting from the frozen portion, and the biological material of each straw is frozen.

[Problems to be solved by the invention]

According to the apparatus described above, the above-mentioned problem can be solved, but there is 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 mutual communication between the inner and outer tank brines.

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 decrease, and the biological material cannot be frozen quickly.

Since the inner tank side wall 88 also serves as a straw rack and is attached and detached, when the inner tank side wall 88 is removed for attachment and detachment of the straw, the inner and outer brines are mixed and a uniform temperature is attained. Therefore, it is difficult to lower the outer tank brine B1 to a considerably low temperature in advance and gradually lower the temperature of the inner tank brine B2 while controlling the temperature with the heater 85.

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

[Means for solving the problem]

The present invention has an outer tank, an inner tank accommodated in the outer tank, an outer tank having a heater for brine temperature control, and a brine circulating device. A brine supply port and a brine overflow port, and a rack portion that can be disposed in the inner tank with a portion of the biological material storage container being exposed to the outer tank. The brine that is supplied from the tank to the inner tank and overflows from the inner tank to the outer tank is positioned lower than the portion of the container that projects the brine level in the outer tank to the outer tank supported by the rack portion. It is configured to return to the external tank so as to maintain the same, and the outer tank is provided with a cooler in a liquid phase portion and a gas phase portion, and provides an ice plant freezing apparatus.

The brine supply port and the brine overflow of the inner tank may be provided separately or commonly on the inner tank wall or the like, but in order to simplify the apparatus, the upper end of the inner tank is opened and the brine supply port and It may be used for brine overflow. In the case where the rack supporting the biological material container is disposed at the upper end of the inner tank, an opening or a cutout provided in the rack may be used as a brine supply port or a brine overflow.

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

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

[Action]

According to the ice plant freezing apparatus of the present invention, the solution containing the biological material together with the preservation solution is supported by the rack, and the portion containing the biological material is arranged in the inner tank, and a part of the solution is stored in the outer tank. It is arranged in the gas phase.

The brine is cooled to a predetermined temperature by a cooler in a liquid tank section in the outer tank, and is supplied to the inner tank by a circulation device from the outer tank while being temperature-controlled by a heater in the outer tank, and overflows from the inner tank. The discharged matter is circulated from the outer tank 1 to the outer tank 4 again.

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

Thus, the preservation solution in the portion of the container disposed in the gas phase portion of the outer tank first starts freezing, and then the preservation solution and the biological material in the portion of the container disposed in the inner tank with the frozen portion as a starting point. Freezes without being supercooled.

In the case where the rack portion of the container includes a horizontal hole provided in the upper portion of the inner tank side wall, and in a case where there are a plurality of the horizontal holes, the container provided for freezing is less than the number of holes. If the container is filled in order from the lower hole, the upper hole does not necessarily need to be plugged, and even if it is open, the container is immersed in the brine supplied to the inner tank, and The brine that has risen to spills out of the open hole above.

〔Example〕

 Hereinafter, an embodiment of the present invention will be described with reference to FIG.

The ice plant freezing apparatus includes two outer tanks 1 and 10, an inner tank 2 in the outer tank 1, and an inner tank 20 in the outer tank 10.

The inner tank 2 is placed on the table 12 in the outer tank 1, and the outer tank lid
By removing 11, it can be attached to and detached from outer tank 1. The inner tank 20 is placed on a table 102 in the outer tank 10, and can be attached to and detached from the outer tank 10 by removing the outer tank lid 101.

The outer tank 1 has an upper-stage cooler 71 and a lower-stage cooler 72 therein. The inner tank 2 is provided with a rack portion 6, and the rack portion has a plurality of lateral holes 61 through which straws S can be liquid-tightly inserted into the inner tank 2 while leaving a part of the straw S in the outer tank 1. And a supporting member 62 fixed to the inner tank 2 and provided with a plurality of holes for supporting a straw portion protruding from the outer tank 1.

On the other hand, the outer tank 10 includes an upper cooler 710 and a lower cooler 720 therein. At the upper end of the inner tank 20, a rack unit 60 is arranged. The rack portion 60 is formed of a plate-like member, has a plurality of holes 601 through which a part of the tube T can be inserted into the inner tank while leaving the tube T in the outer tank, and also has openings 602, 603 through which brine can flow.

An external tank 4 is provided outside the outer tanks 1 and 10, and a brine temperature controlling heater 3 is provided in the tank.

The apparatus further includes a brine circulation device 5.
The apparatus 5 includes a pump 50 attached to the external tank 4 and pipes 51 and 52 for supplying the brine B in the tank 4 to the internal tanks 2 and 20, and the brine B overflowing from the internal tank to the external tanks 1 and 10. 4 is provided with an on-off valve V ₁ in the pipe 51, and an on-off valve V _{2 in a} passage returning from the outer tank 1 to the tank 4.
But the pipe 52 off valve V _3, on-off valve V ₄ is a path back from the outer tub 10 to the tank 4, respectively.

In the apparatus described above, when the outer tub 1 and the inner tank 2 to freeze the biological material, as well as open the valve V ₁ and V ₂ in the circulation device 5, close the valve V ₃ and V _4.

Then, the lid 11 of the outer tub 1 is opened, the inner tub 2 is taken out from the inside together with the rack 6, and a straw S is set in the rack 6. Each straw to be set contains a preservative solution together with the biological material. Each of the straws S is set so that the biological material accommodating portion is disposed in the inner tank 2 with a part of the straw S protruding out of the inner tank 2.

Thus, the inner tank 2 and the straw S are placed in the outer tank 1,
It is placed on the table 12 and the outer tank lid 11 is closed.

In this state, the pump 50 in the circulation device 5 is operated.
Thus, the brine in the outer tank 4 is supplied from the upper end opening of the inner tank 2 to the inner tank 2 through the pipe 51, and the biological material storage portion of the straw S is immersed in the brine B. The brine B overflowing from the inner tank 2 is piped from the bottom of the outer tank 1
Circulating again to the external tank 4 through 53.

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

While the brine circulates in this manner, the gas phase in the outer tank 1 and the brine are gradually cooled, and the outer tank 4 is cooled.
The brine supplied to the inner tank 2 from the heater 4 gradually decreases while the temperature is controlled by the heater 3 in the tank 4. In addition,
The temperature of the brine supplied to the outer tank gas phase and the inner tank may be reduced to some extent in advance.

Thus, first, the preservation solution in the portion of each straw that protrudes into the outer tub 1 starts to freeze, and then the preservation solution and biological material in the portion in the outer tub 2 are supercooled starting from the frozen portion in the outer tub 1. Freezing without being done. In other words, the coolers 71 and 72 and the heater 3 are controlled as such.

On the other hand, in the case of frozen operation by utilizing the portion on the left side in the figure, with open the valve V ₃ and V ₄ in the circulation system,
Close the valve V ₁ and V _2. 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. In each tube, a biological material is stored together with a preservation solution, and a part of the biological material is protruded into the outer tank 10 so that the biological material storing part is
Place in 20. After closing the outer tank lid 101, the pump 50 is operated to supply the brine in the outer tank 4 from the opening 603 of the rack member 60 to the inner tank 20, and the biological material storage portion in each tube T is replaced with the brine B. Soak in Inner tank
The brine B overflowing to 20 is the opening 602, 603 of the rack member 60.
From the outer tank 10 and circulates from the lower part of the outer tank to return to the tank 4 again.

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

Thus, in the outer tank 10, the gas phase is cooled by the cooler 710, the liquid tank is cooled by the cooler 720, and the brine in the outer tank 4 is cooled while the temperature is controlled by the heater 3. In addition, the temperature of the brine supplied to the gas phase portion of the outer tank and the inner tank may be reduced to some extent in advance.

While the brine is circulated in this state, in each tube, the preservation solution in the portion protruding into the outer tank 10 first starts freezing, and then the preservation solution and the biological solution in the inner tank 20 start from the frozen portion. The material freezes without being supercooled. In other words, the coolers 710, 720 and the heater 3 control as such.

In performing freezing operation using the right and left portions at the same time, it is sufficient to remain open all valves V ₁ ~V _4.

If there are not as many straws S as the number of the straw insertion holes 61 in the upper part of the side wall in the inner tank 2 on the right side, the straws S may be inserted sequentially from the lowermost hole 61 toward the upper hole. By inserting in this manner, all the supported straws S are immersed in the brine supplied from the tank 4, and the brine that has reached a higher level than the straws S overflows into the outer tub 1 also from the opening 61 in the upper stage.

If one or more of the holes 61 at the same level as the uppermost straw among the straws set in the inner tank 2 are not filled with the straw S, the holes may be plugged. Since the hole of the straw S is small, by increasing the amount of brine supplied from the tank 4 by the circulation device 5 to a certain extent, the liquid level in the inner tank can be higher than that of the straw S even if the hole is left open. It is not necessary to plug the hole.

On the other hand, in the left part, a vertical hole 601 of the rack member 60 is provided.
Is not filled with the tube T, there is no problem even if the opened hole is left as it is.

In addition, the coolers 71 and 72 of the outer tub 1 may be provided separately or may be connected to one. Cooler 71 for outer tank 10
The same applies to 0 and 720.

〔The invention's effect〕

As described above, according to the ice plant freezing apparatus of the present invention, the container for storing the biological material can be easily used, whether it is a straw type or a tube type. Even when the number of container insertion holes is less than the number of holes, it is not always necessary to plug the holes, so that it is easy to handle, and the temperature of the gas phase of the outer tank and the temperature of the brine supplied to the inner tank are individually controlled. Thus, the ice-freezing operation can be performed promptly without fear of overcooling.

[Brief description of the drawings]

1 is a sectional view showing an embodiment of the present invention, FIGS. 2 to 4 are views showing a conventional example, FIG. 2 is a plan view with a cover removed, and FIG. 3 is a view with a cover attached. FIG. 3 is a sectional view taken along line 3-3 of FIG. 2;
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 ... upper end opening of inner tank 2 ... heater 4 ... external tank 5 ... brine circulation device 6, 60 ... rack part 61 ... sideways Hole 601… Vertical hole 602, 603… Rack opening 71, 72, 710, 720… Cooler S… Straw T… Tube

──────────────────────────────────────────────────続 き Continuation of front page (58) Field surveyed (Int. Cl. ⁶ , DB name) A01N 1/00 A01N 3/00 F25D 13/00 A F25D 17/02 301

Claims (3)

(57) [Claims] 1. 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, wherein the inner tank has a brine supply port at an upper part thereof, A rack having a brine overflow port and a rack portion capable of disposing a biological material container in the inner tank while partially exposing the biological material container to the outer tank, wherein the brine circulating device moves brine from the outer tank to the inner tank. And the brine that has overflowed from the inner tank to the outer tank so that the brine level in the outer tank is maintained at a position lower than the portion of the container that is supported by the rack portion and protrudes into the outer tank. An ice plant freezing apparatus configured to return to the external tank, wherein the external tank includes a cooler in a liquid phase part and a gas phase part. 2. The ice plant freezing apparatus according to claim 1, wherein the rack portion includes an upper portion of the inner tank side wall having a lateral hole through which the container can be inserted into the inner tank. 3. The ice-plant freezing apparatus according to claim 1, wherein the rack portion includes a member having a vertical hole portion through which the container can be inserted into the inner tank and arranged at an upper portion of the inner tank.