JPH0512691Y2 - - Google Patents

Description

[Detailed Description of the Invention] <<Industrial Application Field>> The present invention relates to an apparatus for freezing living cells such as lymphocytes, bone marrow cells, sperm, and fertilized eggs using helium gas.

《Prior art》 When freezing living cells for long-term preservation, it is most important to increase their survival rate, and the survival rate of frozen cells depends on the cooling rate of the cells. The relationship between these factors is shown in FIG. 2.

As shown in the figure, there are two ranges of cooling rates that are advantageous for improving the survival rate, one of which is 1°C ~
One is a slow cooling region A of several degrees Celsius per minute, and the other is an extremely rapid cooling region B of 10,000 degrees Celsius per minute or more.

In addition, the appropriate cooling rate and inappropriate cooling rate are determined depending on the degree of salt concentration, dehydration, intracellular ice crystal formation, etc. of the cell fluid, and especially ultra-rapid cooling of 10,000 degrees Celsius or more per minute. In region B, cells are said not to suffer from freezing damage because intracellular water freezes in an amorphous state (so-called glass-like state), or even if ice crystals are formed, they are extremely minute. .

However, conventional methods for freezing living cells include
Cells are stored in a tube-like storage tube such as a straw tube with a buffer solution containing a cryoprotectant, and then frozen by cooling the cells with a low-temperature liquefied gas such as liquefied nitrogen or a refrigerator. , therefore, the cooling area is the slow cooling area A.

In the conventional method described above, the cells that are directly targeted for freezing exist in a suspended state in an extremely large amount of buffer compared to this method, so the surrounding environment of the storage tube must be carefully adjusted under conditions for rapid freezing. Even in this case, the buffer solution surrounding the cells provides an extremely large resistance to the cooling of the cells, so it is not possible to increase the cooling rate of the cells to the ultra-rapid cooling area B, and in the end, the cooling rate of the cells cannot be increased to the ultra-rapid cooling area A. I had no choice but to depend on it.

As a result, the following problems are pointed out in the conventional method described above.

(b) A high survival rate can be obtained in the slow cooling region A, but this is only under the condition that a frost damage inhibitor is added, so when using the conventional method, the addition of a frost damage prevention agent is Not only is this essential, but even under the above conditions, in the slow cooling region A, depending on the type of cell, an impractically low survival rate of about 20% can be obtained.

(b) When adding the above-mentioned cryoprotectant, if the concentration is suddenly increased to a certain level, the negative effect on the cells will be very large, so the concentration must be increased step by step over 4 to 5 times. This work is extremely tedious and time-consuming, and even with such effort, one cannot escape from some negative effects.

(c) Furthermore, in order to prevent adverse effects on living cells, it is necessary to wash and remove the antifreeze agent when thawing the cells, and furthermore, there is a loss due to washing away of the cells.

(d) Since cooling must be performed in a relatively narrow area of 1 to several degrees Celsius per minute, the cooling rate must be strictly controlled, requiring advanced control equipment such as a program controller, and It will take 2 to 3 hours.

(e) Pretreatment such as addition of a cryoprotectant takes time, and the freezing time is also long, so it takes a long time overall to freeze the cells, so the activity of the cells tends to decrease.

Therefore, a means for freezing living cells in the ultra-rapid cooling region B has been proposed, and the use of low-temperature helium vapor has already been proposed.

In this case, of course, if the object to be frozen is immersed in liquid helium, ultra-quick freezing is possible, but in practice it is extremely difficult to store liquid helium in an open container and use it. Even if the object to be frozen is immersed, it is a so-called boiling heat transfer, and since the outer periphery of the object to be frozen is helium vapor, the cooling rate will not be particularly fast just because it is a liquid.

In addition, the latent heat of vaporization of helium is small and the sensible heat of vapor is extremely large, but when immersed in liquid, most of this large sensible heat is wasted, and the cooling efficiency of expensive liquid helium is extremely poor. It becomes difficult to dispose of the remaining liquid helium in the container,
For reasons such as the fact that a lot of things end up being wasted, we use helium vapor instead of liquid helium itself. This makes it possible to adjust the temperature by adjusting the amount of helium vapor sprayed. It has also been proposed to use only as much liquid helium as necessary to make use of sensible heat as effectively as possible, greatly improving economic efficiency.

However, in the case of conventional equipment that attempts to achieve ultra-quick freezing as described above, when freezing living cells with helium vapor, heat enters from the outside, making it insufficient to realize economical ultra-quick freezing. In addition, problems such as frozen living cells being thawed involuntarily are likely to occur.
Furthermore, when transferring the frozen cells to a storage container, the temperature rises due to contact with the outside air and contamination occurs, and the frozen cells die during the transfer process. , satisfactory measures have not been taken.

《Problems to be solved by the invention》 Therefore, the present invention provides a new freezing device that achieves freezing of living cells by ultra-rapid cooling of over 10,000°C per minute. It eliminates the defects of conventional methods, is extremely easy to operate, has a high survival rate of frozen cells, maintains a high activity level, and has almost no recovery rate of frozen cells.
The goal is to improve your performance to near 100%.

<Means for Solving the Problems> In order to achieve the above-mentioned purpose, the present invention has _two LN containers containing liquid nitrogen, a nitrogen shield outer cylinder that protrudes upward from the LN _two containers, and the nitrogen shield. A cryopreservation inner cylinder that is inserted through the upper opening of the outer cylinder until its lower end is immersed in the liquid nitrogen and placed on the outer cylinder to form a nitrogen passage between the outer cylinder and the outer cylinder. This cryopreservation inner cylinder is equipped with a bottomed helium shield cylinder with an exhaust port at the top,
The freezing tube is inserted into the helium shield tube and fixed detachably, and forms a helium passage communicating with the exhaust port between the helium shield tube and the helium shield tube, and the freezing tube is connected to the upper closed tube. and an aeration recovery container part connected thereto, and a cell supply part to which biological cells are supplied together with a buffer solution, and a closing lid provided at the upper end opening of the freezing tube. The present invention aims to provide a method for freezing living cells using helium gas, which has a helium gas supply section installed therethrough.

《Operation》 According to the present invention, when the device is operated, biological cells with a buffer added thereto are dropped into the closed cylinder part of the cryopreservation inner cylinder by the cell supply part, and this is then released from the helium gas supply part. The helium gas supplied to the closed cylinder section performs freezing in the ultra-rapid cooling zone B, whereby the frozen living cells are collected into the ventilation collection container section connected to the closed cylinder section. be done.

At the same time, the helium gas passes through the ventilation recovery container section, flows into the next helium passage, and is further discharged to the outside air from the exhaust port, so the outer periphery of the freezing tube is covered with helium gas. As a result, intrusion of heat from the outside is blocked, and living cells are quickly frozen.

Furthermore, in this invention, not only does the helium gas have an insulating effect, but also the liquid nitrogen in the LN ₂ container is vaporized and the nitrogen vapor is released to the outside air through the nitrogen passage, so the freezing tube is able to absorb the helium gas and nitrogen vapor. It is double shielded by this, and can block heat intrusion from the outside air.

Moreover, since the ventilation collection container section is placed in a state where it can be cooled by liquid nitrogen, the frozen biological cells are surrounded by helium gas and liquid nitrogen around the helium gas. There is no situation where frozen living cells are inadvertently thawed.

In addition, after the frozen living cells are obtained,
If you want to transfer this to a separately prepared storage LN ₂ container (not shown) and store it, remove the cryopreservation inner cylinder from the LN ₂ container and protect the living cells from temperature rise and contamination caused by outside air. can be protected.

The present invention will be described in detail with reference to the illustrated embodiment. As shown in FIG. 1, an _LN2 container 2 containing liquid nitrogen _LN2 and having an opening 1 for insertion has an insulating layer 3 such as vacuum insulation.

Next, the illustrated nitrogen shield cylinder 4 is loosely fitted from the opening 1 and placed on the bottom surface of the LN ₂ container 2, thereby protruding from the LN ₂ container 2, and the nitrogen shield cylinder 4 is fitted loosely through the opening 1 at the top end. The lower edge of the cylinder is curved with a mounting peripheral edge 6, and a desired number of notches 7, 7... are cut out, so that the liquid nitrogen LN ₂ in the LN ₂ container 2 is It is also possible to enter into the nitrogen shield cylinder 4.

Furthermore, on the inner peripheral surface of the nitrogen shield cylinder 4,
A lower steady rest protrusion 8 with a vent hole 8a and a receiving edge 10 with a vent hole 9 are installed horizontally.

Here, the nitrogen shield cylinder 4 mentioned above is the LN ₂ container 2.
The LN 2 may be placed in the LN ₂ container 2 without being inserted into the LN 2 container 2. In other words, the nitrogen vapor from the liquid nitrogen LN ₂ enters the nitrogen shield cylinder 4 through the opening 1. That's fine.

In the present invention, a cryopreservation inner cylinder 11 is further provided, which is connected to the upper end opening 5 of the nitrogen shield cylinder 4.
The lower end 11a is immersed in the liquid nitrogen LN ₂ of the LN ₂ container 2, and in this state, the mounting edge 12 protruding from the inner cylinder 11 is connected to the receiving edge 1.
0, the cryopreservation inner cylinder 11
A nitrogen passage 13 is formed between the nitrogen shield tube 4 and the nitrogen shield cylinder 4, and as a result, nitrogen vapor from the liquid nitrogen LN ₂ is discharged from the upper end opening 5 to the outside air through the vent 9.

The cryopreservation inner cylinder 11 is inserted through a bottomed helium shield cylinder 15 with an exhaust port 14 at the top and an opening 15b having a receiving flange 15a of the shield cylinder 15, and placed in the lid opening 16a. A freezing tube 1 whose flange 16b is placed on the receiving flange 15a and removably fixed with screws etc.
6, and a helium passage 17 communicating with the exhaust port 14 is formed between the helium shield tube 15 and the freezing tube 16.

Further, the freezing tube 16 has an upper closing tube portion 16
c and a lower ventilation recovery container part 16d, and the closed cylinder part 16c is made of metal such as stainless steel,
Preferably, a polymeric material having low temperature resistance and water repellency, such as tetrafluoroethylene (Teflon) or polyimide, is selected to prevent biological cells from adhering to it.

Of course, the ventilation collection container part 16d should be of a size that does not allow living cells to pass through (5 microns or less).
It can be formed by a mesh plate having pores 16e, and a commercially available polymer product such as polyester or polypropylene can be used for this. It is not necessary to use a water-repellent material like the forming cylinder part 16c, and the closing cylinder part 16c and the ventilation recovery container part 1
6d can be connected by adhesion or by using a heat shrink tube for the latter.

Note that any material can be used for the helium shield tube 15 as long as it has low temperature resistance, and for example, stainless steel, copper alloy, or polymeric material (Teflon, nylon) can be used.

Next, the closing opening 16a of the closing cylinder portion 16c is
The closing lid 18 is removably closed by screws or the like, and a cell supply section 19 and a helium vapor supply section 20 are provided through the closure lid 18 .

The cell supply unit 19 is composed of a cell container 19a in which biological cells 22 are stored together with a buffer solution 21, and a liquid delivery pipe 19e connected to this and having a pump 19b, an on-off valve 19c, and a dripping nozzle 19d. On the other hand, the helium gas supply section 20 is equipped with a gas pipe 20c having an on-off valve 20a and a supply nozzle 20b, and the pipe 20c is filled with helium vapor evaporated from liquid helium (not shown) or cooled by a refrigerator or the like. Helium gas will be sent.

To use the freezing apparatus having the above-mentioned configuration, in the state shown in FIG. 1, the on-off valve 20a of the helium gas supply unit 20 is opened to supply helium gas into the closed cylinder portion 16c, thereby precooling the inside until it reaches a predetermined temperature (e.g., 20K). Then, the pump 19b of the cell supply unit 19 is operated and the on-off valve 19 is closed.
By opening the closed cylindrical portion 16c, the biological cells 22 enclosed in the buffer solution 21 in the cell container 19a are dropped into the closed cylindrical portion 16c.

As a result, the dropped material D comes into contact with the helium gas having a low temperature and large heat capacity, is cooled extremely rapidly, freezes while falling, and becomes frozen cells E in the ventilation recovery container section 16d.
It will be captured and deposited.

On the other hand, the helium gas that has completed the heat exchange with the dripping material D as described above is heated slightly (nearly 60K), passes through the pore 16e of the ventilation recovery container section 16d, and then passes through the helium passage 17 to the exhaust port. 14 to the outside air, and therefore the closed cylinder part 16c
is helium gas in the helium passage 17, and
The nitrogen vapor (around 77 K) in the nitrogen passage 13 prevents heat from entering from the outside air, and ultra-rapid cooling can be guaranteed with less consumption of liquid helium.

Once the necessary living cell freezing work is completed, the supply of helium vapor will be stopped, but since nitrogen vapor will still pass through the nitrogen passage 13 at this time, the frozen cells E will not be thawed. .

Next, the lid plate 18 is removed from the mounting flange 16b, and the lid opening 16a is closed with a separately prepared blind flange for storage (not shown), and then the cryopreservation inner cylinder 11 is pulled out and placed in the LN ₂ (not shown). However, when performing such an operation, the ventilation recovery container part 16d is transferred with the helium passage 17 still provided, so that the frozen cells E are not directly exposed to the outside air and are therefore prevented from thawing due to temperature rise. Not only is there no contamination caused by bacteria, it is convenient.

Of course, if temperature rise and contamination are not a problem as mentioned above, remove the mounting flange 16b from the receiving flange 15a, pull out only the closing cylinder part 16c, and quickly charge the LN into a storage LN ₂ container. Also, the closed cylinder portion 16c can be closed by heat-sealing a sheet, instead of being closed by a blind flange for storage as described above.

<<Effects of the invention>> Since the present invention is constructed as described above, when living cells enclosed in a buffer solution are frozen by helium gas, the freezing atmosphere is divided by helium gas and nitrogen vapor. Heavy heat shielding makes it possible to achieve ideal ultra-rapid freezing of living cells with minimal consumption of expensive liquid helium.

Furthermore _, since the frozen cells E are kept in an atmosphere surrounded by liquid nitrogen _LN2 , there is no need to worry about inadvertent thawing or contamination. By using the helium gas inside, it is possible to prevent temperature rise and contamination of frozen cells due to outside air, and the freezing tube has an aeration collection container.
Since it can be used as a storage container as it is, there is no need to transfer the obtained frozen cells to another container, which not only makes the process more effective, but also makes it easier to thaw the cells by transferring them. It is possible to avoid the risk of cell loss.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal sectional front view of an embodiment of the apparatus for freezing living cells using helium gas according to the present invention, and FIG. 2 is a chart showing the survival rate of living cells with respect to the cooling rate. 2...LN ₂ containers, 4...Nitrogen shield outer cylinder, 5
...Top opening, 7...Cut, 9...Vent, 1
0... Receiving edge, 11... Cryopreservation inner cylinder, 11a...
... lower end, 12 ... mounting edge, 13 ... nitrogen passage,
14...Exhaust port, 15...Helium shield tube,
16...Freezing tube, 16a...Top opening, 16c...
...Closing cylinder part, 16d... Ventilation recovery container part, 17...
... Helium passageway, 18 ... Closed lid, 19 ... Cell supply section, 20 ... Helium gas supply section, 21 ... Buffer solution, 22 ... Living cells, LN ₂ ... Liquid nitrogen.

Claims (1)

[Scope of claim for utility model registration] (1) _Two LN containers containing liquid nitrogen, a nitrogen shield outer cylinder protruding upward from the _two LN containers,
Cryopreservation in which the nitrogen shield outer cylinder is charged through its upper opening until the lower end is immersed in the liquid nitrogen, and then placed on the outer cylinder to form a nitrogen passage between the outer cylinder and the outer cylinder. The cryopreservation inner cylinder includes a bottomed helium shield cylinder with an exhaust port at the top, and a helium shield cylinder that is inserted into the helium shield cylinder and fixed in a detachable manner, and is connected to the helium shield cylinder. It consists of a freezing tube that forms a helium passage communicating with the exhaust port in between, and the freezing tube is formed by an upper closed tube section and an aeration recovery container section connected thereto. A device for freezing biological cells using helium gas, in which a cell supply section for supplying biological cells together with a buffer solution and a helium gas supply section are installed through a closing lid provided at an upper end opening. (2) Utility model registration claim No. 1, in which the nitrogen shield outer cylinder is inserted and placed in an LN ₂ container, and the lower edge of the cylinder is formed with a cutout through which liquid nitrogen flows. A device for freezing living cells using helium gas as described in . (3) Practical use in which the helium shield cylinder of the cryopreservation inner cylinder is placed with the mounting rim provided on the outer periphery on the receiving rim with a vent provided on the upper inner periphery of the nitrogen shield outer cylinder. A device for freezing living cells using helium gas as claimed in claim 1.