JPH01277485A - Method for freezing and preserving cell - Google Patents

Abstract

PURPOSE:To prevent growth of ice in a cell and enhance survival rate of the cell, by charging the cell into a metallic tube having shorter inside diameter than cell to be frozen and preserved and cooling the cell using a cooling medium. CONSTITUTION:A metallic tube having good thermal conductivity and having smaller inside diameter than diameter of a cell is used as a tube for preserving the cell and the cell is charged into the tube while transforming the cell to directly bring into contact with inside wall of the tube. Then whole tube is cooled to freeze and preserve the cell. When the charge is carried out, a part of inside diameter becoming smaller is further formed in the metallic tube and the cell can be charged in state trapping the cell at the part. By the above- mentioned method, charge of the cell to the metallic tube is made easy, since a change in the pressure of suction of feed occurs when the cell is trapped in the part having smaller inside diameter.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for freezing and preserving cells that have undergone treatments such as cell fusion and gene transfer, as well as valuable cells such as fertilized eggs. be.

(Conventional technology) When freezing cells, the water contained inside the cells crystallizes as they are gradually cooled, and the crystals grow and destroy the nucleus and cell membrane, causing the cells to die. . Therefore, when freezing cells, it is important to cool them rapidly to prevent the growth of intracellular water crystals.

Conventionally, cells are sucked into a plastic tube with a large inner diameter compared to the size of the cells, along with a solution such as physiological saline, and then the plastic tube is placed in a refrigerant such as liquid nitrogen for cryopreservation.

(Problem to be solved by the invention) When cells are placed in a thick plastic tube and cooled,
Due to the presence of solution around the cells, the heat transfer coefficient between the inner wall of the tube and the solution is low, resulting in poor heat transfer and a large heat capacity, which slows down the freezing rate of cells and prevents ice growth. There is a problem that the survival rate of cells becomes low because it cannot be prevented.

An object of the present invention is to provide a method for rapidly cooling cells to prevent the growth of ice within the cells, thereby increasing the survival rate of the cells.

(Means for Solving the Problems) In the present invention, a metal tube with good thermal conductivity is used as a tube for storing cells. Further, the metal tube used has an inner diameter smaller than the diameter of the cells, and the cells are loaded into the tube while being deformed, so that the cells are brought into direct contact with the inner wall of the tube.

The present invention further includes forming a portion with a reduced inner diameter in the metal tube, and traps and loads cells in that portion.

(Function) Since it is a metal tube, heat conduction is good, and the inside of the metal tube is rapidly cooled by the refrigerant.

Since the cells are in direct contact with the inner wall of the metal tube, heat transfer efficiency is high and the cells are rapidly cooled through the metal tube.

When cells are trapped in the reduced inner diameter, a change in aspiration or injection pressure occurs, making it easier to load cells into the metal tube.

(Example) 1 to 4 show one embodiment.

In FIG. 1, 1 is a cell to be cryopreserved. A tube 2 is made of thin metal to improve heat conduction, and its inner diameter is smaller than the diameter of the cell 1.

One end of the metal tube 2 is fitted into the tip 4 of the suction mechanism 5, and the tip of the metal tube 2 is open to facilitate suction of the cells 1.

When inhaling the cells 1 into the metal tube 2, the tip 3 of the metal tube 2 is brought close to the cells 1, and the cells 1 are inhaled together with the solution 6.

Cells 1 are aspirated into a metal tube 2 as shown in FIG. Since the inner diameter of the metal tube 2 is smaller than the diameter of the cell 1, the cell 1 enters the metal tube 2 while being deformed. As a result, the cells 1 come into direct contact with the inner wall surface of the metal tube 2 over a wide area 8.

Next, this tube 2 is removed from the suction mechanism 5, and as shown in FIG. 3, the end of the metal tube 2 is cooled to freeze the solution in that part and the metal tube 2 Seal one end of.

The metal tube 2 is then placed in a refrigerant 10, such as liquid nitrogen, with a high velocity flow 9, as shown in FIG. As a result, the metal tube 2 is rapidly cooled, and since the cells 1 are in direct contact with each other over a wide area within the metal tube 2, the heat within the cells 1 is rapidly released by thermal conduction. As a result, cell 1 is rapidly cooled and no ice crystals grow.

FIG. 5 shows a metal tube 2 with a constriction 11 and a cell 1.
This shows an embodiment in which the constricted portion 11 is prevented from coming out on the suction side.

This metal tube 2 is attached to a suction mechanism in the same manner as shown in FIG. 1, and the cells 1 are sucked together with the solution 6 from the tip 3. The inhaled cells 1 stop at the constriction 11 and a pressure change occurs in the suction mechanism 5, so that it can be easily detected that the cells 1 are loaded at a predetermined position.

In order to seal one end of the metal tube 2 loaded with cells 1, instead of freezing a portion as shown in FIG. 3, for example, a lid may be attached to one end of the metal tube 2.

The refrigerant is not limited to liquid nitrogen, and for example, cryogenic helium gas or the like can also be used.

In FIG. 5, a constricted portion 11 is provided in order to reduce the inner diameter of a part of the metal tube 2, but the metal tube 2 may be bent. At the bend, the inner diameter of the metal tube 2 becomes smaller, and the inhaled cells 1 are trapped.

6 to 9 show a method of injecting and loading cells 1 into a metal tube having a constricted portion 11.

Syringe 21 is used to load cells 1 into the tube. The syringe 21 has almost the same structure as a syringe, but a threaded portion 23, for example, is provided at the part where the metal tube 2a is attached so that the metal tube 2a will not come off even if the internal pressure becomes somewhat high.

First, as shown in FIG. 6, a tube 27 having an inner diameter larger than the diameter of the cell 1 is attached to the syringe 21. Although the material of the tube 27 is not limited, the tube 27 is screwed into the syringe 21 as shown in an enlarged view in FIG.

The cells 1 are sucked up into the syringe 21 together with the solution 6 from the container 24 containing the cells 1 to be stored.

After the cells 1 are placed in the syringe 21, the tube is replaced with a metal tube 2a for storage, as shown in FIG. The metal tube 2a has an inner diameter smaller than the diameter of the cell 1, and a constriction 11 is formed in a part.

As shown in an enlarged view in FIG.
It can be attached to the tube using the tube attachment sleeve 29.

In this state, internal pressure is applied to the syringe 21. This internal pressure is set to a constant value determined by the viscosity of the solution 6 and other factors. As a result, the solution 6 gradually flows out from the metal tube 2a, but
Eventually, the cell 1 enters the metal tube 2a and enters the constriction 11.
When caught in the cell 1, the cell 1 acts as a stopper and stops the outflow of the solution 6. At that point, the cells 1 have been loaded into the metal tube 2a.

The mechanism for attaching the tubes 2a and 27 to the syringe 21 is as follows:
It does not necessarily have to have a screw structure as shown in the embodiments, and may have a structure that can be simply inserted like a general syringe.

Even when the metal tube 2a is provided with a bent portion instead of the constricted portion 11, the cells 1 can be easily loaded into the metal tube 2a in the same manner.

A manual syringe 21 is shown in the figure, which includes aspiration of cells 1 from container 24 and injection of cells 1 into storage metal tube 2a.
The injection may be performed automatically.

(Effects of the Invention) In the present invention, a metal tube is used as a tube for storing cells, and the inner diameter of the tube is made smaller than the diameter of the cells. Therefore, when cells are loaded into the tube and placed in a refrigerant, the cells are rapidly cooled. This prevents the growth of ice within the cells and increases cell survival.

Furthermore, by providing the tube with a portion with a small inner diameter for trapping cells, the cell loading operation can be performed reliably.

[Brief explanation of the drawing]

Fig. 1 is a schematic end view showing the state before cells are absorbed in one embodiment, Fig. 2 is a schematic end view showing the state after cells are inhaled in the same embodiment, and Fig. 3 is a schematic end view showing the state in which cells are inhaled in the same embodiment. FIG. 4 is a schematic end view showing the tube in a sealed state; FIG. 5 is a schematic end view showing the state in which cells are inhaled in another embodiment; FIG. 6 is a schematic end view showing the tube in a refrigerant state; FIG. 7 is an enlarged sectional view of section A in FIG. 6, and FIG.
The figure is a sectional view showing a state in which cells are injected into a metal tube in the same embodiment, and FIG. 9 is an enlarged sectional view of section B in FIG. 8. 1...Cell, 2,2a...Metal tube, 5
...Suction mechanism, 6...Solution, 10...
Refrigerant, 11... Constriction, 21... Injector. Patent applicant: Shimadzu Corporation

Claims (2)

[Claims] (1) A method of cryopreserving cells by loading cells into a metal tube with an inner diameter smaller than that of the cells to be cryopreserved, bringing the cell membrane into direct contact with the inner wall of the tube, and cooling the entire tube with a refrigerant. (2) The method according to claim 1, wherein a portion with a reduced inner diameter is formed in the metal tube, and the inhaled or injected cells are trapped and loaded in the portion with a smaller inner diameter.