JPH03251187A - Method for introducing and incorporating foreign material in organism or cell and apparatus therefor - Google Patents

Abstract

PURPOSE:To easily and stably introduce a foreign material into an organism, etc., by ejecting compressed gas and fine particles carrying foreign material from the tip end of a specific nozzle. CONSTITUTION:The nozzle 1 has a nozzle tip hole 4 having an inner diameter of 1.0mm and a length of 5mm and connected to a hole 2 for holding fine particles carrying foreign material. The hole 2 has an inner diameter of 5mm and a length of 18mm. Foreign material-carrying fine particles are produced by supporting about 1mug of a foreign material such as chlorella gene on about 10mg of tungsten fine particles having particle diameter of about 0.6mum and the particles are placed at the center of the carrying hole 2. The action time of an electromagnetic valve is set to about 0.001sec with a controller 13 built in the valve, the controller 13 is actuated to operate an automatic gun 21 for airless spray-coating and compressed air of about 100kgf/cm<2> pressure is supplied to eject the fine particles carrying the gene for about 0.005sec at a high speed from the tip end of a nozzle 23 controlled by the controller 13. The ejected fine particles are introduced into and taken by the cell 24 of a microalgae, etc., at about 40 deg.C.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is an object of the present invention to produce foreign substance-carrying fine particles, which are made by carrying foreign substances such as genes on fine particles of metal, polymer, etc., without any chemical or biological damage. The present invention relates to a method of introducing and ingesting a foreign substance into an organism or cell by impacting the organism or cell with only physical damage, and an apparatus used in the method.

(Prior Art) Methods for introducing foreign substances into living organisms or cells have been used for a long time, but these methods were mostly carried out after converting the cells into protoplasts. In addition, the foreign substances to be taken in are biopolymers that have a high affinity for the cells, and it is impossible to forcefully take in foreign substances that have no affinity for the living body except for special organisms or cells that have a high affinity for them. Ta. Therefore, foreign substances could not be taken up or introduced into cells that could not become protoplasts.

However, recently, a method of forcibly introducing foreign substances into organisms using the explosive power of gunpowder has been devised.

Methods for capturing foreign matter using the explosive power of gunpowder have the property of being able to obtain a sufficient rate of ejection of foreign matter, and have recently been used against several living organisms. This method is subject to the restrictions of the Firearms and Swords Act due to the requirement of using gunpowder, and the equipment itself is large and complex because the object must be placed under a certain degree of low pressure, making it difficult to use as a foreign material introduction method. It wasn't easy.

(Problems to be Solved by the Invention) In view of the current situation, it is possible to force cells or organisms that are difficult to take in foreign substances, such as cells or organisms that are difficult to convert into protoplasts, to take in foreign substances, and to use explosives. In order to obtain a method that is safe and easy to operate without the use of compressed air, the present inventors focused on a method that uses compressed air, which is regularly used in laboratories, and conducted various studies.

In other words, the key point of this method is that instead of using the explosive force of gunpowder to launch foreign objects, a pump is used to pump compressed air, and the pressure is used to release fine particles (carriers) to which the foreign objects to be introduced are attached. The goal is to fire it at a velocity sufficient for introduction and drive it into an organism or cell.

However, in this method, the use of gunpowder, which is restricted by the Firearms and Swords Act, is no longer used, but it is replaced by extremely high pressure (~2
A pump that produces compressed gas (20 atm) is required.
This results in a loss of simplicity.

(Means for Solving the Problems) The present inventors have developed a commonly used gas cylinder (~1
50 kgf/cm") is used as a pressure source for carrier ejection, and this compressed gas is ejected by instantaneous opening and closing of a solenoid valve, etc., making it possible to eject the carrier at the speed required for introduction. At the same time, we found that by attaching a stopper to the nozzle at the tip of the nozzle that spews compressed gas, the jetted compressed gas can effectively shoot out the foreign matter.Furthermore, we have added a part that fixes the organism or cell into which the foreign matter is introduced. The present invention was achieved by discovering that by controlling the temperature, the fluidity of the cell membrane can be changed and foreign substances can be introduced more easily than in conventional methods.

That is, in the present invention, foreign matter-carrying particles formed by carrying foreign matter are communicated with a hole in a nozzle tip portion having a predetermined length and inner diameter on the axis of the nozzle tip hole, and and a foreign matter-carrying fine particle mounting hole 7 having a predetermined inner diameter sufficiently larger than the nozzle tip hole, into which the foreign material-carrying fine particles can be mounted, and compressed gas at a predetermined pressure is applied to the nozzle. Either the foreign matter-carrying fine particles are ejected from the nozzle tip for a predetermined period of time and are shot into an organism or cell maintained at a predetermined temperature, or the foreign matter-carrying fine particles are installed in the mounting hole, and then the foreign substance-carrying fine particles are attached. A cylindrical stopper whose diameter is smaller than the inner diameter of the mounting hole and larger than that of the nozzle tip hole is arranged at the compressed gas inlet of the hole, and the stopper supplies compressed gas at a predetermined pressure to the nozzle. The foreign material-carrying fine particles are ejected from the nozzle tip along with the gas filled in the system while supplying the biological material for a predetermined time or a predetermined distance until it stops at the communication part with the mounting hole and maintained at a predetermined temperature. Alternatively, the present invention provides a method for introducing a foreign substance into an organism or a cell and causing the foreign substance to be incorporated into the organism or cell.

The present invention also provides a built-in controller that sets the operating time of the on-off valve, a compressed gas operated valve that opens when the controller is turned on, the on-off valve opens only for a set short time, and compressed gas flows through the compressed gas operated valve. An automatic gun for airless spray painting that operates with compressed gas and supplies compressed gas from a compressed gas source, and operates when the on-off valve closes and the supply of compressed gas is stopped, and the compressed gas in the path from there to the automatic gun. comprising a NOT element and a quick exhaust valve that operate to rapidly discharge the gun, a nozzle attached to the tip of the automatic gun, and means for fixing the organism or cells, or further comprising a stopper in the nozzle.
The present invention provides an apparatus for use in the method of the present invention.

In the present invention, examples of foreign substances that can be introduced into living organisms or cells include genes, nucleic acids, enzymes, and various other chemicals and reagents; There isn't. Furthermore, by adding various modifications to the surface of the microparticles used as carriers, it may be possible to introduce other reagents.

Examples of fine particles that can be introduced as carriers of foreign substances in the present invention include metal fine particles such as iron and tungsten, inorganic fine particles such as quartz, and high specific gravity polymer fine particles.

The size of these fine particles varies depending on the target organism or cell, but usually has an outer diameter of about 0.1 μm to 10 μm.

The size of the hole in the nozzle tip of the present invention varies depending on the microparticles, organisms, or cells introduced as a carrier, but usually has an inner diameter of 0.5 to 2 mm and a length of 1.0 to 20 mm.
It is.

The size of the foreign matter-carrying fine particle mounting hole in the nozzle of the present invention is as follows:
Although it varies depending on the microparticle, organism, or cell used as a carrier, the inner diameter is usually 160 to 10 mm and the length is 10 to 100 m.
This inner diameter is 2.0 m compared to the inner diameter of the nozzle tip hole.
It is preferable that the particle size is about 5.0 times larger in terms of accelerating the carrier fine particles.

In the method of the present invention, when the foreign matter-carrying fine particles are attached to the mounting hole of the nozzle, it is possible to attach them as they are, but it is better to attach them as a suspension of a buffer solution or the like containing the foreign matter stable side. This is preferable in terms of stability within the pore and prevention of deterioration of foreign substances.

Examples of the compressed gas in the method of the present invention include nitrogen, air, argon, and other inert gases, and the pressure thereof is usually 2 to 200 kgf/clI+2, preferably 10
~100 kgf/cva".The pressure is 200 kgf/cva"
If it exceeds "kgf/cta", the amount of outflow gas per unit time will increase and the sample will start to scatter, which is undesirable.
If it is less than kgf/cm2, the probability of fine particles passing through the cell membrane and cell wall of living things becomes extremely low, which is not preferable.

In the method of the present invention, the compressed gas is usually 10 to 120 kg.
It is supplied to the nozzle at a pressure of f/cmt, and is ejected from the nozzle tip for 0.005 to 0.01 seconds together with the foreign matter-carrying fine particles.

In the method of the present invention, when the stopper is used, the stopper is moved while supplying compressed gas having a pressure of usually 10 to 120 kgf/cm2, and the foreign matter-carrying fine particles are filled into the mounting hole from the nozzle tip. Approximately 50m with gas at a pressure of 2 to 200 kgf/cm2
Spray a distance of m. This method requires the inside of the hole and the stopper to be made more precisely than when no stopper is used, but the amount of outflowing gas can be adjusted by adjusting the nozzle inner diameter and length, and the outflowing gas can also be turned on and off. It has the advantage that it does not have to be done exactly.

Examples of living organisms in the present invention include plants in general whose cell walls are not extremely hard, animals, tissue culture cells, and the like. For these organisms, foreign substance-carrying microparticles are implanted, preferably in a state in which separated cells are fixed in a layer on a filter or in a state in which they form a mass.

Examples of cells in the present invention include animal cells such as HeLa cells, cultured cells, microalgae such as the green alga Chlorella and all cyanobacteria, and tissues such as callus.

When implanting the foreign material-carrying microparticles, cells are preferably suction-filtered and fixed onto a filter.

Any type of cells that can be fixed on a filter can be used as these cells. The thickness of the cell layer fixed on these filters is approximately 2-3 cell diameters.
Although it is desirable to double the amount, it is possible to introduce fine particles even if the amount is more than that. The cells on these filters should not be completely dried, but should be kept half-wet (half-wet).
It is desirable to use it in a state of ivet).

This filter is fixed on a fixed plate connected to Coolnics. This Coolnics uses a device that can change the temperature from 0°C to 50°C, and the temperature change can be reproduced directly on a fixed plate.

The temperature change of the fixed cells is preferably within the range of 0°C to 50°C, within which the survival rate of the cells does not drop significantly.

In the present invention, known enzymes such as cellulase, lysozyme, macerozyme, and pectinase can be used to increase the ability of cells to take up foreign substance-carrying microparticles.

By controlling the temperature in the immobilization of organisms or cells as described above and using cells or organisms treated with cell wall degrading enzymes as samples, it becomes possible to introduce and incorporate foreign substance-carrying microparticles at high frequency.

The controller used in the device of the present invention has a built-in on-off valve, such as the tM1 valve (an example of a solenoid valve will be described below), and can set the operating time of the solenoid valve in units of 0.001 to 9.999 seconds. . As the controller,
For example, a commercially available AD3000V controller manufactured by Lower Right Engineering Co., Ltd. can be used.

When the controller is turned on, the solenoid valve opens for a set short time, compressed gas flows, and the compressed gas operated valve opens.

The compressed gas that has passed through the compressed gas operating valve operates an automatic gun for airless spray painting (manufactured by Aloeki), and high-pressure compressed gas is supplied from the cylinder or pump to the automatic gun.

When the solenoid valve closes and compressed gas supply stops, the NOT element activates and quickly exhausts the compressed gas in the path from the quick exhaust valve to the automatic gun, making it possible to control the supply time of high-pressure compressed gas to a short time. .

The shape and size of the nozzle in the device of the present invention are as described above, and it is fixed to the tip of the airless automatic gun with a cap nut or the like. This will be explained in more detail with reference to FIG. 1, FIG. 2a, and FIG. 2b.

FIG. 1 is a cross-sectional view of the nozzle when no stopper is provided in the nozzle, in which 1 is the nozzle body, 2 is the foreign matter-carrying particulate attachment hole, 3 is the communication part, and 4 is the nozzle tip hole. 5 is a foreign matter-supporting fine particle as a sample.

Figures 2a and 2b are cross-sectional views of the nozzle when the stopper 6 is provided, Figure 2a shows the state before compressed gas is supplied to the nozzle, and Figure 2b shows the state before compressed gas is supplied to the nozzle. This shows a state in which the stopper 6 moves to the communication part 3 and the foreign matter-carrying fine particles 5 are ejected from the nozzle tip.
5 is as described above, 6 is a stopper, and 7 is an optionally attached stopper with a string.

The means for immobilizing organisms or cells in the device of the present invention consists of a filter through which the organisms or cells are suction-filtered and immobilized, and this filter is immobilized on a fixing plate connected to Coolnics. This Coolnics uses a device that can change the temperature from 0°C to 50°C, and the temperature change can be reproduced directly on a fixed plate.

The above fixing means will be explained with reference to FIG. 3. In FIG. 3, 1 is a nozzle, 8 is the filter, and 9 is a nozzle.
is a filter fixing plate such as an iron plate, and 10 is a heating wire for heating or a Coolnics terminal for cooling attached in a spiral shape to the bottom of the fixing plate 9, and the temperature of these is controlled by a thermostat. It is possible.

The operation of the device of the present invention will be explained below with reference to FIG. FIG. 4 is a schematic diagram for explaining the device of the present invention, in which 11 is a power source, 12 is a switch, 13 is a controller, 14 is a high-pressure compressed gas source, 15 is a pressure reducing valve, 16 is a NOT element, and 17 is a compressed gas source. Gas-operated valve, 18 is an air source for operation, 19 is a rapid exhaust valve, 20 is a gas for automatic gun on-off operation, 21 is an automatic gun for airless spray painting, 22 is a high-pressure air source for operation, 23 is a nozzle, 24 is a biological or a cell-containing container; 25 is a temperature control table;

In FIG. 4, for example, the controller 13
．． Set the operation time of the 'Wi magnetic valve in the controller 13 in units of 001 seconds, and turn on the controller I3.The solenoid valve opens for the set short time, allowing compressed gas to flow, and the compression controlled by the controller 13 is activated. The gas-operated valve 17 opens, and the compressed gas 20 that has passed through it operates the automatic gun 21 for airless spray painting, and high-pressure compressed gas is supplied from the high-pressure compressed gas source 14 of a cylinder or pump to the automatic gun 21, and accordingly the nozzle The foreign matter-carrying fine particles installed inside are ejected from the nozzle tip and driven into cells or organisms. When the solenoid valve closes and the supply of compressed gas stops, the NOT element 16 operates to quickly exhaust the compressed gas in the path from the rapid exhaust valve 19 to the automatic gun 21, so the supply time of high-pressure compressed gas is o, ooi. It is possible to control in a short time of about seconds. 0.00 like this
Controller 13 that can be set on-off for up to 1 second
By opening and closing the compressed gas operating valve 17 controlled by the compressed gas valve 17, compressed gas of up to 100 kgf/cm2 can be spouted and stopped. The container 24 containing the cell or biological sample is placed on a temperature control table 25 so that the foreign substance-carrying particles are introduced at an optimum temperature. When a stopper is placed inside the nozzle, it is possible to introduce foreign matter-carrying fine particles without being exposed to excess compressed gas, and when a stopper is not installed inside the nozzle, the compressed gas is ejected at the same time as the foreign body-carrying fine particles which are ejected at high speed. Through this interaction, the foreign substance-carrying fine particles are effectively introduced into cells or organisms. Furthermore, since the nozzle and stopper are made of metal, they can be sterilized using an autoclave or the like.

(Example) A device of the present invention in which no stopper is provided in the nozzle has a figurine-carrying particulate attachment hole with an inner diameter of 5 mm and a length of 18III+, and a nozzle tip hole with an inner diameter of 1.0 mm and a length of 5a+a+, as shown in FIG. In the center of the mounting hole of the nozzle having the shape shown in , an amount of 1 μg of Chlorella gene as a foreign substance supported on 10 mg of tungsten fine particles with a particle size of 0.6 μm is suspended in a buffer solution. Set the solenoid valve operating time to 0.001 seconds using the controller with a built-in solenoid valve (manufactured by Engineering, below right, ^D3000V), turn on the controller, and turn on the automatic gun for airless spray painting (manufactured by Aloeki). When activated, compressed air was supplied from the 100 kgf/cm2 compressed air source in the cylinder to the automatic gun for 0.001 seconds, and at the same time gene-carrying microparticles were delivered from the nozzle tip at high speed for 0.001 seconds.
It was ejected for 5 seconds and then shot into the above cells whose temperature was controlled at 40°C. As a result, since the operating time of the high-pressure compressed air was extremely short, the degree of scattering of the cells by the airflow was extremely small, and fine particles were successfully introduced into the cells.

Example 2 A device of the present invention in which a stopper is provided in the nozzle has a foreign matter-carrying particulate attachment hole with an inner diameter of 5 mm and a length of 54 mm, and a nozzle tip hole with an inner diameter of 1.0 mm and a length of 6 mm, as shown in FIGS. 2a and 2a. Using a nozzle having a shape as shown in Figure 2b, an outer diameter of 1.9 m was applied to the compressed gas port of the mounting hole.
m Arrange a cylindrical stopper with a length of 5 mm, and 100 k
By supplying compressed gas at a pressure of 150 kgf/cm'' and moving the stopper, the gene-carrying microparticles were ejected from the tip of the nozzle along with the gas filled in the system over a distance of approximately 45 mm at a pressure of 150 kgf/cm''. The same experiment as in Example 1 was conducted except for the above.

As a result, since the amount of air ejected was small and almost equal to the amount pushed out by the stopper from inside the nozzle, the cells were hardly scattered by the airflow, and fine particles were successfully introduced into the cells.

(Effects of the Invention) According to the present invention, first, foreign substances such as genes can be stably transferred to living organisms or cells without chemical or biological damage and with minimal physical damage. Methods and apparatus are provided that can be introduced and incorporated into.

According to the present invention, secondly, there is provided a method and device for introducing and incorporating foreign substances into living organisms or cells, using extremely simple devices, with extremely little scattering of living organisms or cells as samples by air jets.

According to the present invention, thirdly, a foreign substance can be effectively introduced into an organism or a cell, and the introduction can be facilitated by controlling the temperature of the organism or cell as a sample. Methods and devices are provided for introducing and incorporating foreign substances into living organisms.

[Brief explanation of drawings]

FIG. 1 is a sectional view of the nozzle in the case where no stopper is provided in the nozzle in the present invention, and FIG.
Figure b is a sectional view of the nozzle for explaining the operation of the stopper and bar when a stopper is provided in the nozzle in the present invention, and Figure 3 is a cross-sectional view for explaining the means for fixing cells or living organisms in the present invention. FIG. 4 is a schematic diagram for explaining the apparatus of the present invention. Applicant Kansai Paint Co., Ltd. Figure 3 Figure 2a Figure 2b

Claims (1)

[Claims] 1. Foreign matter-supporting fine particles made by supporting foreign matter on fine particles,
a hole in the nozzle tip having a predetermined length and inner diameter;
The foreign matter of the nozzle is comprised of a foreign matter-supporting fine particle mounting hole that is on the axis of the nozzle tip hole, communicates with the nozzle tip hole, has a predetermined length and a predetermined inner diameter sufficiently larger than the nozzle tip hole, and is capable of mounting the foreign matter-supporting fine particles. It is characterized in that it is attached to a supported particle mounting hole, and while jetting compressed gas at a predetermined pressure through the nozzle for a predetermined period of time, the foreign substance-supporting particles are ejected from the nozzle tip and driven into an organism or cell maintained at a predetermined temperature. A method of introducing and ingesting foreign substances into living organisms or cells. 2. A cylindrical stopper whose diameter is smaller than the inner diameter of the mounting hole and larger than that of the nozzle tip hole is arranged at the compressed gas inlet of the foreign matter-supporting fine particle mounting hole, and compressed gas at a predetermined pressure is supplied to the nozzle. , the foreign matter-carrying fine particles are ejected from the nozzle tip together with the gas filled in the system while supplying the particles for a predetermined time or a predetermined distance until the stopper stops at the communication portion between the nozzle tip hole and the mounting hole. 2. The method of claim 1, wherein the method is applied to an organism or cell maintained at a temperature. 3. A controller that sets the operating time of the built-in on-off valve; when the controller is turned on, the on-off valve opens for a set period of time, allowing compressed gas to flow through and open; the compressed gas that has passed through the compressed gas operating valve; An automatic gun for airless spray painting that is activated to supply compressed gas from a compressed gas source, and when the on-off valve closes and the supply of compressed gas is stopped, it activates to immediately remove the compressed gas from the path to the automatic gun. Apparatus for use in the method according to claim 1, characterized in that it comprises a NOT element and a quick evacuation valve operative to eject, a nozzle attached to the tip of the automatic gun and means for immobilizing organisms or cells. 4. An apparatus for use in the method according to claim 2, characterized in that a stopper is provided in the nozzle in the apparatus according to claim 3.