JPH0129761B2 - - 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, osteopathic cells, sperm, and fertilized eggs.

<<Conventional technology>> When freezing living cells for the purpose of long-term preservation, the most important point is to increase their survival rate, and the survival rate of frozen cells depends on the cooling rate of the cells. The relationship is shown in Figure 4.

As shown in the figure, there are two regions of advantageous cooling rates for improving survival rate, one of which is 1°C to several degrees Celsius/
One is a slow cooling region A, which is a cooling rate of 10,000° C./minute, and the other is an ultra-rapid cooling region B, which is a very rapid cooling of 10,000° C./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, intracellular water is frozen in an amorphous state (so-called glass-like state), or even if ice crystals form, they are extremely fine, so that cells are not subject to freezing damage.

However, conventional methods for freezing living cells include
Cells are stored in a tube-like storage tube such as a straw tube along with a buffer solution containing a cryoprotectant, and this is cooled using a low-temperature liquefied gas such as liquefied nitrogen or a refrigerator to freeze the cells. , 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 achieved in the slow cooling region A, but this is only under the conditions where a frost damage inhibitor is added, so when using the conventional method, the addition of a frost damage inhibitor is not possible. Not only is this essential, but even under the above conditions, in the slow cooling region A, depending on the type of cell, only 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°C to several degrees per minute, the cooling rate must be strictly controlled, requiring advanced control equipment such as a program controller, and freezing 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.

<Problems to be Solved by the Invention> In order to solve the above-mentioned problems, the present invention provides a novel freezing device that freezes living cells by ultra-rapid cooling of 10,000°C or more per minute. The obtained product has a simple structure, is extremely easy to operate, has a high survival rate of frozen cells, maintains a high activity level, and has a very low recovery rate of frozen cells.
The goal is to improve your performance to near 100%.

<<Means for Solving the Problems>> That is, the present invention has a tube-shaped sample holder that is removably attached to the bottom of an insulated chamber and connected to a mesh recovery container for recovering frozen cells. A transfer tube for supplying liquid helium vapor, which is airtightly penetrated from the outside, and a microtube for supplying biological cells in the smallest possible unit, wrapped in culture medium, are arranged in the upper part of the tube, respectively. This configuration solves the above problems.

<<Example>> Hereinafter, an example of the present invention will be described in detail based on the drawings. As shown in FIG.
A vacuum insulation layer 2 is formed between the two cylinders 1a and 1b by combining the chambers 1a and 1b at an appropriate interval. A cylindrical sample holder 4 made of a water-repellent material such as Teflon (trade name: Teflon) or polyimide is inserted, and the holder flange 5 provided on the outer periphery of the upper end is fixed around the opening 3 of the chamber 1. The sample holder 4 is clamped and fixed between the provided flange 6 and a holding member 8 which is fixed on the flange 6 in an overlapping state with bolts 7, and the sample holder 4 is removed and attached to the approximate center of the chamber 1. It can be hung freely.

At the bottom of the sample holder 4, a frozen cell collection container 9 with a bottom made of a mesh smaller than the frozen fine particles to be generated, preferably a mesh material of sterile filter grade, is adhered, or a heat-shrinkable tube is shrunk. It has been frozen using other means.

Further, on the upper part of the sample holder 4, there is a transfer tube 10 for supplying liquid helium vapor into the sample holder 4, and a microtube for supplying living cells together with a culture medium, also inside the sample holder 4. 11 is the pressing member 8
The sample holder 4 is opened to the upper space inside the sample holder 4 by passing through the sample holder 4 in an airtight manner.

The outer end of the transfer tube 10 is connected to a liquid helium vapor generating container (not shown), while the outer end of the microtube 11 is connected to a liquid helium vapor generating container (not shown).
It is connected via a pump P to a cell container 14 containing living cells 13 dispersed in the microtube 1 for supplying the living cells.
At least the open end 11a opened in the sample holder 4 of 1 is preferably filled with living cells.
It is made of, for example, a glass capillary tube having an inner diameter small enough to allow only a small number of particles to pass through.

In the figure, reference numeral 15 indicates an exhaust pipe provided at the bottom of the chamber 1.

The configuration of the device of the present invention has been described above, and its operation will be explained next.

First, liquid helium is supplied in the form of vapor or mist to the internal space of the sample holder 4 through the transfer tube 10, preferably at a rate of 20〓(-
The cells are pre-cooled to an extremely low temperature below 235 degrees Celsius, and while supplying liquid helium to maintain the temperature within that temperature range, the cells are placed in the same sample holder 4 from the microtube 11 while being wrapped in microdroplets of culture medium. Drip into.

It is desirable that the fine droplets contain as few biological cells as possible, such as one to several, and therefore, when the fine tube 11 is subjected to minute vibrations with a vibrator, the droplets drop. The droplets become smaller, making it easier to form droplets containing only one biological cell.

In this way, the biological cells dropped from the top of the sample holder 4 are kept at an extremely low temperature of 20°C or less.
In addition, due to the atmosphere of liquid helium vapor having an extremely large heat capacity, the cells are cooled and frozen at an extremely rapid rate of 10,000° C./min or more during the fall, and are deposited in the collection container 9 at the bottom of the sample holder 4 as frozen cells.

On the other hand, the liquid helium vapor supplied into the sample holder 4 heats up somewhat due to heat exchange with the living cells, passes through the mesh of the collection container 9, and enters the chamber 1.
It is exhausted to the atmosphere from the exhaust pipe 15.

When a predetermined amount of living cells have been frozen as described above, the supply of liquid helium vapor and cells is stopped, the bolt 7 and the holding member 8 are removed, the sample holder 4 is taken out from the chamber 1, and the sample holder 4 is removed from the chamber 1 to avoid contamination. In order to prevent foreign matter from entering the sample holder, a separately prepared cap for the sample holder 4 (not shown) should be attached to the top opening of the sample holder 4 by means such as screwing, or the top opening of the sample holder 4 should be sealed by melt sealing. The sample holder 4 is then immersed and held in a low-temperature liquefied gas such as liquid nitrogen.

In this way, the frozen cells can be collected into the collection container 9 and the sample holder 4 can be used as a holder, which saves the trouble of collecting the frozen cells and also reduces the work of transferring the frozen cells to another storage container. Further, there is no need to recover frozen cells, and the time of exposure to the outside air after freezing can be minimized, so it is unlikely that the frozen cells will be thawed carelessly.

In addition, the mesh recovery container 9 serves as an exhaust part for liquid helium vapor from the sample holder 4 during freezing, and also serves as an inlet or outlet for liquid nitrogen when immersed in or taken out of liquid nitrogen. It's convenient.

Furthermore, if the mesh in the collection container 9 is made of a fine mesh similar to that of a sterilization filter, in the process using this device, there is no opportunity for exposure to the outside air during cell transfer, etc., so recontamination of living cells can be completely prevented. is possible.

Furthermore, if the sample holder 4 is made of a water-repellent material or coated with the same material, biological cells will not adhere to the inner wall surface during freezing, and the recovery rate can be increased.

FIGS. 2 and 3 show other embodiments in which the sample holder 4 is placed approximately in the center of the chamber 1. FIG.

As shown in the figure, the bottom 1c of the chamber 1 is formed as a separate body, and the bottom 1c is removably formed using flanges 16, 17 and means such as bolts (not shown).
A pin 18 is erected at the center of the inner surface of the bottom 1c, and a bush 19 that fits the pin 18 is fixed downward on the bottom of the collection container 9 connected to the bottom of the sample holder 4. , the bush 19 and the pin 18 are engaged with each other, a short tube 20 is suspended from the upper end opening 3 of the chamber 1, and the short tube 20 is formed to fit into the upper end opening of the sample holder 4. ing.

With such a configuration, the sample holder 4 can also be fixed to a substantially central portion within the chamber 1, and the sample holder 4 can also be easily attached and detached.

<<Effects of the Invention>> As explained above, since the living cell freezing apparatus according to the present invention is configured, the unit to be cooled and frozen is almost one living cell or a small number of units close to this. Moreover, since it can be cooled in an atmosphere of liquid helium vapor that has a large heat capacity at an extremely low temperature, biological cells can be cooled at an ultra-high speed of over 10,000 degrees Celsius per minute, so there is no need for antifreeze agents and compared to conventional methods. Living cells can be frozen with a relatively high survival rate, and there is also a sample holder 4 in the chamber 1.
Since the sample holder 4 is arranged to form a double tube, it is easy to obtain a low temperature especially inside the sample holder 4, and the refrigerant can be used efficiently.
It has a cell collection container 9 with a mesh at the bottom of the cell,
Since the sample holder 4 can be detachably attached to the chamber 1 and can also be used as a storage holder, there is no need for the trouble of recovering frozen cells, the recovery rate is high, and there is no need to transfer the frozen cells to a storage container, etc. It is possible to minimize the opportunity for the liquid nitrogen to be exposed to outside temperature, and the mesh serves as a convenient entrance and exit for the liquid nitrogen when putting it in and out of the liquid nitrogen for storage.
Furthermore, since the structure is simple, it is extremely easy to operate and can be manufactured at low cost.

[Brief explanation of drawings]

FIG. 1 is a longitudinal sectional front view of one embodiment of the living cell freezing device according to the present invention in use. FIGS. 2 and 3 are partially enlarged views of other embodiments of the same freezing device. The longitudinal front view and FIG. 4 are graphs showing the relationship between the survival rate of frozen cells and the cooling rate. 1...Chamber, 4...Sample holder,
9... Frozen cell collection container, 10... Transfer tube, 11... Microtube.

Claims (1)

[Claims] 1. A tube-shaped sample holder connected to the bottom of the insulated chamber with a mesh recovery container for frozen cell recovery is removably housed inside the chamber, and an airtight tube is attached to the top of the sample holder to ensure airtight access from the outside. A transfer tube for supplying liquid helium vapor and a microtube for supplying biological cells in the smallest possible unit in a state surrounded by a culture medium are each arranged with openings. Living cell freezing device. 2 The sample holder is made of polytetrafluoroethylene,
2. The biological cell freezing device according to claim 1, which is made of a water-repellent material such as polyimide or a material coated with the same material. 3. The biological cell freezing device according to claim 1, wherein the frozen cell collection container is formed of a sterilization filter.