JPH0662871A - Method for introducing and including foreign material in organism or cell and apparatus therefor - Google Patents

Abstract

PURPOSE:To prevent the contamination caused by the scattering of particles carrying foreign materials by closing a two-stage needle valve, charging fine particles carrying foreign material in a 1st inner hole and ejecting the fine particles through a nozzle by operating the two-stage needle valve. CONSTITUTION:A compressed gas having high pressure is ejected or stopped by opening or closing an air-operated valve 6 controlled by a controller 3 programmed to perform a prescribed on-off operation. A container 28 containing the specimen of cell or organism is placed on a thermostatic table to receive foreign material-supporting fine particles in optimum temperature state. In the preferable embodiment of the apparatus, the gas ejected from the tip of a nozzle 18a is not applied to the organism or the cell and the foreign material- supporting fine particles scattered to the surrounding are passed though a chamber 26 and transferred to a separator 30. The fine particles are captured by the liquid 32 in the separator 30 and removed from the gas. Exclusively the gas free from the fine particles carrying the foreign material is exhausted from an exhaust port 35.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention provides fine particles carrying foreign substances such as genes on fine particles of metals, polymers, etc., with minimal physical damage without chemical or biological damage. The present invention relates to an improvement in a method for introducing and incorporating a foreign substance into an organism or cell by implanting it in the organism or cell, and an apparatus used in the method.

[0002]

2. Description of the Related Art Japanese Unexamined Patent Publication No. 3-251187 discloses fine particles for carrying foreign matter, which carry foreign matter on fine particles, in a nozzle tip portion having a predetermined length and inner diameter, and in the nozzle tip hole. The foreign substance-carrying fine particle mounting hole of the nozzle, which is in communication with the shaft and has a predetermined length and a predetermined inner diameter sufficiently larger than the nozzle tip hole, into which the foreign substance-carrying fine particle mounting hole can be mounted. And ejecting the foreign matter-carrying fine particles from the nozzle tip while ejecting a compressed gas of a predetermined pressure to the nozzle for a predetermined time to strike the organism or cell maintained at a predetermined temperature, or the foreign matter-carrying fine particles are mounted as described above. A cylindrical stopper having a diameter smaller than the inner diameter of the mounting hole and larger than that of the nozzle tip hole at the compressed gas inlet of the foreign substance-carrying fine particle mounting hole after mounting in the hole. While supplying a compressed gas of a predetermined pressure to the nozzle for a predetermined time or a predetermined distance until the stopper stops at the communicating portion between the nozzle tip hole and the mounting hole, the foreign matter-carrying fine particles from the tip of the nozzle. Disclosed is a method for introducing and taking in a foreign substance into an organism or a cell, which comprises ejecting the gas into a living organism or a cell maintained at a predetermined temperature by injecting it with a gas filled in the system, and an apparatus used for the method. Has been done.

In the method and apparatus described in Japanese Unexamined Patent Publication No. 3-251187, the ejection direction of the foreign matter-carrying fine particles is limited to a substantially horizontal direction due to the structure of the nozzle, and especially the foreign matter-carrying fine particles due to the nozzle structure. When a cylindrical stopper is placed in the mounting hole, the operation of mounting the foreign particle-carrying particles in the foreign particle-carrying particle mounting hole is cumbersome, and when using a plug with a string that can be optionally mounted at the tip of the nozzle tip hole, its durability Is not sufficient, one part of the foreign matter-carrying fine particles ejected from the nozzle tip together with the high-pressure compressed gas scatters to the surroundings and causes contamination, and the high-pressure gas generated by the failure of the controller that sets the operating time of the on-off valve There were problems such as the inability to prevent spouting.

The present invention has been made to solve the above-mentioned problems in the prior art, and the ejection direction of the foreign matter-carrying fine particles ejected from the tip of the nozzle tip hole is not limited to a particular direction. The operation of mounting the foreign matter-carrying fine particles in the nozzle is easy, and it is possible to effectively and safely prevent the contamination by the foreign matter-carrying fine particles ejected from the tip of the nozzle and scattered around. (EN) A method for introducing and incorporating a foreign substance into an organism or a cell, which has an advantage that the supply of high-pressure compressed gas to a nozzle can be safely and effectively controlled, and an apparatus used for the method. It is intended.

[0005]

According to the present invention, firstly, foreign matter-carrying fine particles in which foreign matter is carried on fine particles are ejected from a nozzle tip together with a high-pressure compressed gas and are injected into an organism or a cell maintained at a predetermined temperature. In a method of introducing a foreign substance into an organism or a cell and incorporating the foreign substance, a hole of a nozzle tip having a predetermined length and an inner diameter, a first valve seat that is in continuous communication with the hole on the axis of the nozzle tip hole, a predetermined length and an inner diameter are set. An automatic airless spray coating having a first inner hole, a second valve seat, and a second inner hole having a predetermined length and inner diameter, and a foreign matter-carrying fine particle insertion hole with a cock valve communicating with the first inner hole. A nozzle mounted on the gun, and a valve element that operates back and forth on the axis of the nozzle tip hole in response to the operation of the automatic gun for airless spray coating to open and close at the first valve seat and the second valve seat. With the two-stage needle valve of the nozzle system configured as a two-stage needle valve, the first inner hole is closed and the foreign substance-carrying fine particles are introduced into the first inner hole from the foreign substance-carrying fine particle insertion port to operate the cock valve. After closing, the two-stage needle valve is operated to open the first inner hole, and the high-pressure compressed gas having a predetermined pressure is introduced into the first inner hole through the second inner hole for a predetermined time, and then introduced into the first inner hole. First with high pressure compressed gas
Ejecting foreign matter-carrying fine particles introduced into the inner hole from the nozzle tip through the nozzle tip hole at a predetermined speed,
Alternatively, a hole of a nozzle tip having a predetermined length and inner diameter, a first valve seat that is in axial communication with the nozzle tip hole, and an inner hole having a predetermined length and inner diameter and a cock valve that communicates with the inner hole. A foreign matter-carrying fine particle insertion port, which is attached to the automatic gun, and is preferably detachably connected in front of the insertion port, and the nozzle tip hole depending on the operation of the automatic gun for airless spray coating. A two-stage needle valve is formed by a valve element that is operated back and forth on the axis of the first valve seat and a second valve seat provided at the high pressure compressed gas outlet of the automatic gun for airless spray coating to open and close. The two-stage needle valve of the nozzle system closes the inner hole, introduces the foreign matter-carrying fine particles into the inner hole from the foreign matter-carrying fine particle insertion port, and closes the cock valve to close the two-stage needle. Is operated to open the inner hole to introduce a high-pressure compressed gas of a predetermined pressure into the inner hole for a predetermined time, and the foreign matter-carrying fine particles introduced into the inner hole together with the high-pressure compressed gas introduced into the inner hole are connected to the nozzle tip hole. Through a nozzle to eject at a predetermined speed from the nozzle tip, preferably, a high-pressure adjusting regulator is further provided at the high-pressure compressed gas source outlet to branch the adjusted high-pressure compressed gas, and a low-pressure adjusting regulator is provided on one side. The low-pressure compressed gas provided and decompressed is used as a compressed gas for control and a compressed gas for operation, and a high-pressure gas filter, an on-off valve, and a high-pressure accumulator are sequentially installed in the line leading to the other automatic gun, and high-pressure compression is performed in the line. After introducing gas, close the on-off valve,
The high-pressure compressed gas in the line from the on / off valve to the automatic gun is supplied to the automatic gun, and the portion of the ejected foreign matter-carrying fine particles scattered around is collected and introduced into the organism or cell. It provides a way to make it happen.

Secondly, the present invention relates to a controller for setting the operating time of the built-in on-off valve, and an air-operated valve for opening the on-off valve for a short time set when the controller is turned on and allowing compressed compressed gas to flow. , An automatic gun for airless spray coating that operates by compressed air for operation that has passed through an air operated valve and supplies high pressure compressed gas from a high pressure compressed gas source,
When the on-off valve is closed and the supply of the compressed control gas is stopped, the control compressed gas in the path is quickly discharged to NO.
A T-element and a quick exhaust valve, a nozzle system attached to the tip of the automatic gun, and a means for fixing organisms or cells, the nozzle system having a predetermined length and inner diameter, and a nozzle tip hole and the nozzle tip hole. A first valve seat which is in axial communication with the first valve seat, a first inner hole having a predetermined length and an inner diameter, a second valve seat, and a second inner hole having a predetermined length and an inner diameter and the first inner hole. A nozzle mounted on an automatic gun for airless spray coating, which has a foreign matter-carrying fine particle insertion port with a cock valve that communicates with the air nozzle, and operates forward and backward on the axis of the nozzle tip hole according to the operation of the automatic gun for airless spray coating. And a two-stage needle valve is constituted by a valve element that opens and closes at the first valve seat and the second valve seat, or the nozzle system has a nozzle having a predetermined length and inner diameter. Of the nozzle tip hole, a first valve seat which is in axial communication with the nozzle tip hole, and an inner hole having a predetermined length and inner diameter, and a foreign matter-carrying particle insertion port with a cock valve communicating with the inner hole. The nozzle attached to the automatic gun, preferably detachably connected in front of the insertion opening, and the nozzle operating in the axial direction of the nozzle tip hole in response to the operation of the automatic gun for airless spray coating. A two-stage needle valve is constituted by a valve body that opens and closes at a first valve seat and a second valve seat provided at the high-pressure compressed gas outlet of the automatic gun for airless spray coating, and preferably a high-pressure compressed gas source. From the line to the automatic gun for airless spray painting, high pressure regulator, high pressure compressed gas branch line, high pressure gas filter, on / off manual valve and high pressure accumulator. And a low-pressure adjusting regulator is provided in the high-pressure compressed gas branch line, and the low-pressure compressed gas decompressed by the low-pressure adjusting regulator is used as a control compressed gas and an operating compressed gas, and at a predetermined internal pressure. A chamber with a safety valve, which has a predetermined volume according to which a means for fixing organisms or cells is housed and which is detachably connected to the nozzle, expands in the chamber, and is agitated with the expanding gas exiting the chamber. A device for separating and collecting scattered foreign matter-carrying fine particles, and a device used in the above method, which is provided with a vacuum adjusting regulator and a vacuum pump for sucking the gas separated by the separator to prevent instantaneous pressure increase It is provided.

In the present invention, examples of foreign substances that can be introduced into living organisms or cells include various chemicals and reagents such as genes, nucleic acids, enzymes, etc., as long as they are adsorbed on fine particles as carriers. There is no particular limitation. Also, other reagents may be introduced by adding various modifications to the surface of the fine particles as a carrier.

Examples of fine particles as a carrier for foreign substances that can be introduced in the present invention include metal fine particles such as iron and tungsten, inorganic fine particles such as quartz, and polymer fine particles having a high specific gravity. The size of these fine particles varies depending on the target organism or cell, but is usually about 0.1 μm to 10 μm in outer diameter.

Examples of organisms in the present invention include plants in general, animals, tissue culture cells, etc., whose cell walls are not extremely hard. In these organisms, the foreign matter-carrying fine particles are preferably implanted in a state in which the separated cells are fixed in a layer on the filter or in the state of a lump.

As an example of the cell in the present invention, a spatula (HeL
a) animal cells such as cells and cultured cells, green alga Chlorella,
Most of them include microalgae such as cyanobacteria and tissues such as callus.

Upon implanting the foreign matter-carrying fine particles, cells are preferably fixed by suction filtration on a filter. Any of these cells can be applied as long as it can be fixed on the filter. The thickness of the layer of cells fixed on these filters is about 2 times the diameter of the cells.
It is desirable that the amount be ˜3 times, but it is possible to introduce fine particles even if it is more than that. The cells on these filters were not completely dried and were half-wet.
t) It is desirable to use in such a state. This filter is fixed on a fixed platen connected to Coolnix.
This coolnix uses the one that can change the temperature from 0 ° C to 50 ° C, and the temperature change can be reproduced as it is on the fixed platen. Temperature change of fixed cells is 0 ℃
It is desirable that the cell viability is not extremely lowered in the range of -50 ° C. In the present invention, cellulase, lysozyme,
Known enzymes such as macerozyme and pectinase can be used. By using as a sample the cells or organisms that have been subjected to temperature control in the immobilization of the organisms or cells and treated with cell wall degrading enzymes and the like, it becomes possible to introduce and incorporate foreign particle-carrying fine particles at high frequency.

The controller used in the apparatus of the present invention incorporates an opening / closing valve, for example, a solenoid valve (hereinafter, an example of the solenoid valve will be described), and sets the operating time of the solenoid valve in units of 0.001 to 9.999 seconds. be able to. As the controller, for example, commercially available AD30 manufactured by Iwashita Engineering
A 00V controller can be used. When the controller is turned on, the solenoid valve opens only for the set short time, compressed gas for control flows, and the air operated valve opens. The working compressed gas that has passed through the compressed gas actuating valve operates the automatic gun for airless spray coating (made by Alloy Koki Co., Ltd.) to supply the high-pressure compressed gas from the cylinder or pump to the automatic gun. When the solenoid valve is closed and the supply of the control compressed gas is stopped, the NOT element works and the control compressed gas in the path from the quick exhaust valve to the air actuated valve is quickly discharged, so the supply time of the high pressure compressed gas is controlled in a short time. can do.

Examples of the high-pressure 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} 300 kg f / cm ² , preferably 10 to 200 kg f / cm ^2. Is. The pressure is 300
Exceeding kg f / cm ² is not preferable because it causes a problem in pressure resistance on the device, and is less than 2 kg f / cm ^{2 because} the probability of fine particles that can pass through the cell membrane and cell wall of an organism becomes extremely low, which is not practical. . In the method of the present invention, the high-pressure compressed gas is preferably supplied to the nozzle at a pressure of 10 to 200 kgf / cm ² , and 0.005 to 0.
It is jetted together with the foreign matter-carrying fine particles at an initial velocity of 100 to 400 m / sec for 01 second.

The high-pressure regulating regulator used in the preferred embodiment of the present invention makes it possible to regulate the pressure of the compressed gas from the high-pressure compressed gas source to ² to 300 kgf / cm ² to obtain an arbitrary injection speed. And, for example, TESCOM 44-1115-24 manufactured by TESCOM CO., LTD.

The low pressure regulating regulator used in the preferred embodiment of the present invention regulates the pressure of the branched high pressure compressed gas to within the range of 1 to 6 kg / cm ² , and the low pressure regulated low pressure compressed gas is a controller, Air operated valve,
It can be used as an operating compressed gas for operating an automatic gun or the like, and specific examples thereof include TESCOM 44-1115-24, a trade name manufactured by TESCOM. Separately, the compressed gas from the compressor may be pressure-adjusted by a general-purpose regulator and used as the compressed gas for operation.

On used in the preferred embodiment of the present invention.
The off manual valve turns on / off the high-pressure compressed gas supplied to the automatic gun, and is safe because unnecessary high-pressure compressed gas does not enter the automatic gun, chamber, etc. when the controller etc. fails. A general-purpose high pressure valve can be used with the one provided above. The high-pressure gas filter used in the preferred embodiment of the present invention removes high-pressure compressed gas and dirt in piping by filtration,
The cleaning gas is supplied to the automatic gun, for example, a high pressure gas filter 0.2 manufactured by Japan Millipore Limited.
Examples include μ type ST00GCH00 and the like.

The high-pressure accumulator used in the preferred embodiment of the present invention sets the volume of the high-pressure compressed gas supplied to the automatic gun, and when the controller etc. fails together with the on / off manual valve. It is provided for safety so as to prevent unnecessary high pressure compressed gas from entering the chamber, etc.
2-13.8 liter Sunny Trading Co., Ltd.
Examples include manufactured products.

The size of the holes in the nozzle tip portion in the first embodiment of the nozzle system of the present invention varies depending on the fine particles, organisms or cells as the carrier to be introduced, but usually the inner diameter is 0.5 to 2 mm, preferably 1 .0-2mm,
The length is 1.0 to 20 mm, preferably 2.0 to 10 mm.
The size of the first inner hole of the nozzle in the first embodiment of the nozzle system of the present invention varies depending on the fine particles as carriers, organisms or cells, but usually the inner diameter is 1 to 10 mm, preferably 1 to 5 mm, and the length is 5 to 50 mm. , Preferably 10 to 3
The particle size is 0 mm, and the inner diameter of the space other than the cross-sectional area of the valve body of the two-stage needle valve in the first hole is about 2.0 to 5.0 times larger than the cross-sectional area of the nozzle tip hole. It is preferable in that it accelerates. First of the nozzle system of the present invention
The size of the second inner hole of the nozzle in the embodiment is not particularly limited, but for example, the inner diameter is 2 to 20 mm, preferably 2 to 10 mm, and the length is 5 to 50 mm, preferably 10 to 10.
It is 30 mm. The foreign matter-carrying fine particle insertion port with a cock valve, which constitutes the nozzle in the first aspect of the nozzle system of the present invention, communicates with the first inner hole, and the cock valve provided in the foreign matter-carrying fine particle insertion port is opened. The foreign matter-carrying fine particles are loaded into the closed first inner hole and then closed.
The cock valve needs to be pressure resistant in order to withstand high-pressure compressed gas. The size is not particularly limited, but usually has an inner diameter of 0.5 to 2 mm. The nozzle according to the first aspect of the nozzle system of the present invention includes a first valve seat and a first valve seat communicating with the nozzle tip hole and the first inner hole.
A second valve seat is provided that communicates with the inner hole and the second inner hole. The two-stage needle valve according to the first aspect of the nozzle system of the present invention operates forward and backward on the axis of the nozzle tip hole in response to the operation of the nozzle and the automatic gun, and simultaneously at the first valve seat and the second valve seat. It is composed of a valve body that opens and closes.

In a second aspect of the nozzle system of the present invention, the nozzle tip hole is the same as in the first aspect,
The inner hole has an inner diameter of 1 to 10 mm, preferably 1 to 5 mm, the total length is 20 to 100 mm, preferably 30 to 70 mm, and the foreign matter-carrying fine particle insertion port is the same as in the first embodiment, As a method of detachably connecting in front of the insertion port, for example, a joint such as a screw joint can be used. The first valve seat in the second aspect of the nozzle system of the present invention is the same as that in the first aspect, and the two-stage needle valve includes the first valve seat and the high pressure compressed gas outlet portion of the automatic gun. The second valve seat provided in the
And a valve body that opens and closes at the second valve seat.

2a and 2b are sectional views for explaining the operation of the two-stage needle valve in the first aspect of the nozzle system of the present invention, in which 17a is a valve body, 18a is a nozzle, and 19a is a nozzle tip hole. , 20a is the first valve seat, 21
a is a first inner hole, 22a is a second valve seat, 23 is a second inner hole,
Reference numeral 4a is a foreign matter carrying fine particle insertion port, and FIG. 2a shows that the first inner hole 21a has the first valve seat 2 when the automatic gun 16 is stopped.
0a and the second valve seat 22a are in the closed state, and FIG. 2b shows the case where the first inner hole 21a is in the open state due to the operation of the automatic gun 16.

FIG. 2c shows a second nozzle system of the invention.
FIG. 2C is a cross-sectional view for explaining the operation of the two-stage needle valve in the embodiment of FIG.
Is a nozzle, 19b is a nozzle tip hole, 20b is a first valve seat, 21b is an inner hole, 22b is a second valve seat, 24b is a foreign particle carrying fine particle insertion port, and 25 is a joint.
Of the first valve seat 2
The inner hole 21b is closed or opened at 0b and the second valve seat 22b. After use, it is possible to remove the nozzle 18b at the joint 25 portion and easily clean the valve seat and the like contaminated with foreign matter-carrying fine particles to maintain good performance, and easily replace it with a desired nozzle. There is an advantage that can be.

In the method of the present invention, when the foreign matter-carrying fine particles are charged into the first inner hole of the nozzle, they can be charged as they are, but as a suspension such as a buffer solution containing a foreign matter stabilizer. It is preferable to use a microsyringe, a pipetter, or the like to charge the foreign matter in terms of stability of the foreign matter in the nozzle hole and prevention of deterioration of the foreign matter.

The means for immobilizing organisms or cells in the device of the present invention comprises a filter by which the organisms or cells are suction-filtered and immobilized, and this filter is immobilized on the immobilizing plate connected to the Coolnix. . This coolnix uses the one that can change the temperature from 0 ° C to 50 ° C, and the temperature change can be reproduced as it is on the fixed platen. Temperature control can be performed with a thermostat. It is possible to set the container in which the organisms or cells fixed on the above-mentioned fixed plate are placed with a certain distance from the nozzle tip on the nozzle axis, for example, 10 to 100 mm, and adjusted by, for example, a lifter. Preferably,
These organisms or cells-containing containers, lifters, etc. are mounted in a chamber that is detachably attached to the nozzle tip. As the container, for example, a Petri dish can be used and can be set with a Petri dish guide.

The chamber used in a preferred embodiment of the present invention is contaminated by foreign matter-carrying fine particles that accept and scatter a portion of the high pressure compressed gas jetted from the tip of the nozzle and the foreign matter-carrying fine particles jetted out. This is for preventing the above-mentioned phenomenon and for expanding and decompressing the high-pressure compressed gas that is ejected. A safety valve is provided in the chamber as a safety measure when the high-pressure compressed gas supply control device malfunctions. The volume of the safety valve-equipped chamber is not particularly limited, but in consideration of pressure resistance and downsizing, for example, the pressure of the high-pressure compressed gas to be ejected 2
2-5 in case of 00 kg / cm ² and ejection time of 0.05 seconds
L is preferred. The above-mentioned chamber is equipped with a container containing organisms or cells set with a lifter or the like as described above. The chamber is detachably connected to the nozzle tip portion on the nozzle shaft.

The separator used in the preferred embodiment of the present invention may be a conventionally known gas-liquid separator. For example, the separator is inserted into the separator from the top of the separator,
A pipe that introduces a gas that stirs the dispersed foreign matter-carrying fine particles in the separator, the liquid such as water that was put in the bottom of the separator to capture the introduced foreign matter-carrying fine particles, and the scattered foreign matter-carrying fine particles were removed. It is possible to use a discharge port provided at the top of the separator for discharging gas. The distance between the tip of the pipe and the liquid surface is usually 1 to 20 mm, but the separation effect is small. In this case, the pipe may be placed in the liquid.

The vacuum pump used in a preferred embodiment of the present invention sucks the gas separated by the separator to prevent instantaneous pressure increase, and thus prevents contamination by scattered foreign particles carrying fine particles. As the vacuum pump, known ones can be used, and examples thereof include VP0660 (vacuum degree −600 mmHg) manufactured by Shimada Scientific Instruments Co., Ltd. When operating the vacuum pump, a vacuum degree adjusting regulator may be provided to set the degree of vacuum to, for example, about -600 mmHg.

The method and apparatus of the present invention are illustrated in FIG.
A description will be given below with reference to FIGS. FIG. 1 is a schematic diagram showing an example of a preferred embodiment of the method and apparatus of the present invention. 2a to 2c are as described above, and in FIG. 1, 1 is a power supply, 2 is a switch, 3 is a controller,
4 is a pressure reducing valve, 5 is a NOT element, 6 is an air operated valve, 7a and 7b are rapid exhaust valves, 8 is a gas for automatic gun on / off operation, 9 is a high pressure compressed gas source, 10 is a high pressure adjusting regulator, 11 is a high pressure Compressed gas branch line, 12 low pressure regulator, 13 on / off valve, 14 high pressure accumulator, 15 high pressure compressed gas line, 16 airless spray coating automatic gun, 18a nozzle, 24a foreign matter carrying fine particle insertion port, 26 is a chamber, 27 is a safety valve, 28 is a container for living things or cells, 29 is a lifter,
30 is a separator, 31 is a pipe, 32 is a liquid, 33 is a vacuum adjusting regulator, 34 is a vacuum pump, 35 is an exhaust port, and 36 is a high-pressure gas filter. 1 and 2a
2c, for example, the controller 3 causes 0.
When the operation time of the solenoid valve in the controller 3 is set in units of 001 seconds and the controller 3 is turned on, the solenoid valve opens only for the set short time and the low pressure regulator 1
The control compressed gas regulated by 2 flows, the air actuated valve 6 controlled by the controller 3 opens, and the actuated compressed gas 8 that has passed through this actuates the automatic gun 16 for airless spray painting to operate a cylinder or the like. High-pressure compressed gas source 9
From the high pressure adjusting regulator 10 directly into the automatic gun 16, preferably through the high pressure adjusting regulator 10 and further through the high pressure gas filter 36.
And after passing through the on / off valve 13, the on / off valve 1
3 is turned off, and the high pressure compressed gas in the line from the on / off valve 13 through the high pressure accumulator 14 to the automatic gun 16 is supplied to the automatic gun 16. In this way, the high-pressure compressed gas supplied to the second inner hole 23 of the nozzle in the first aspect of the nozzle system through the automatic gun 16 is in a state where the two-stage needle valve is opened by the operation of the automatic gun 16. In this case, the particles are supplied to the first inner hole 21a and are ejected from the tip of the nozzle 18a together with the foreign matter-carrying fine particles previously charged in the first inner hole 21a through the foreign matter-carrying fine particle insertion port 24a. It is driven into the organism or cells in the cell-containing container 28. When the solenoid valve is closed and the supply of the control compressed gas is stopped, the NOT element 5 operates to quickly discharge the working compressed gas in the path to the quick exhaust valve 7a, so that the high-pressure compressed gas supply time is 0.0
It is possible to control in a short time of about 01 seconds. In this way, by opening and closing the air-operated valve 6 controlled by the controller 3, which can be turned on and off for up to 0.001 seconds, a high pressure compressed gas of up to 300 kg f / cm ² can be ejected and stopped. . The container 28 containing a sample of cells or organisms is placed on a temperature control table (not shown) so that the foreign matter-carrying fine particles are introduced in an optimum temperature state. According to a preferred embodiment of the present invention, the nozzle 18
The gas ejected from the tip of a and the foreign-particle-carrying fine particles that are not hit by the organisms or cells and are scattered around are sent to the separator 30 through the chamber 26, and the scattered foreign-particle-carrying fine particles are in the liquid 32 in the separator 30. Captured and removed by
Only the gas from which the scattered foreign matter-carrying fine particles have been removed passes through the vacuum degree adjusting regulator 33 and the vacuum pump 34 and is discharged from the exhaust port 35.

[0028]

According to the present invention, the ejection direction of foreign matter-carrying fine particles ejected from the tip of the nozzle is not limited to the horizontal direction, and the foreign matter-carrying fine particles can be easily mounted in the nozzle. Even if an abnormality such as a failure occurs in the control system of the high-pressure compressed gas supplied to the nozzle, it is possible to prevent the adverse effects on the automatic gun and subsequent ones, and to easily remove the contamination of the nozzle system by foreign particles carrying foreign particles. A method of introducing and incorporating a foreign substance into an organism or cell, which has advantages such as being possible, and being capable of eliminating contamination by foreign substance-carrying fine particles scattered around without being driven into the organism or cell, and the method A device for use in is provided.

[0029]

The present invention will be described in detail with reference to the following examples.

Example 1 Nozzle tip inner hole having an inner diameter of 1.5 mm and a length of 5 mm, a first inner hole having an inner diameter of 5 mm and a length of 10 mm, a second inner hole having an inner diameter of 8 mm and a length of 10 mm, and a valve having an outer diameter of 2 mm. A two-stage needle valve composed of a body and a shape as shown in FIGS. 2a and 2b is attached to an automatic gun for airless spray coating (made by Alloy Koki), and chlorella gene-1 μg has a particle size of 0.6.
10 mg of tungsten fine particles having a diameter of 10 μm were suspended and suspended in a buffer solution to obtain a suspension, and the suspension was inserted into the first inner hole through a foreign matter-supporting fine particle insertion port using a microcylinder. On the other hand, a solenoid valve built-in controller (AD3000V, manufactured by Iwashita Engineering Co., Ltd.) reduces the solenoid valve operating time to 0.
Set 005 seconds, high pressure adjust the pressure of the N ₂ gas from the N ₂ gas cylinder regulator (Telecom Co., 44-1
115-24) to 200 kg / cm ² to obtain high-pressure compressed gas, and a part of it is branched and a regulator for low-pressure adjustment (Telecom Co., 44
-1115-24) is set to 5 kg / cm ² as the compressed gas for operation on the controller side, the controller is turned on to operate the automatic gun for airless spray painting and the needle valve, and it is installed in the first inner hole. The fine tungsten particles carrying the chlorella gene were ejected from the tip of the nozzle together with the high-pressure N ₂ compressed gas, and were injected into the cells attached to the fixing means. As a result, the operating time of the compressed working gas was extremely short, so that the degree of scattering of cells by the air flow was extremely small, and the chlorella gene-supported tungsten fine particles were satisfactorily introduced into the cells.

Example 2 Chamber with a capacity of 5 liters, capacity 6 with water in the bottom
Liter separator, vacuum regulator (manufactured by Shimada Scientific Instruments Co., Ltd.) and vacuum pump (manufactured by Shimada Scientific Instruments Co., Ltd., trade name VPO 660) with the degree of vacuum set to -600 mmHg
As a result of carrying out the same experiment as in Example 1 except that the chlorella gene-carrying tungsten fine particles that were not taken up by the cells and were scattered were separated and collected from the jet gas using
The scattered chlorella gene-carrying tungsten fine particles were completely separated and collected by the above separator.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of a preferred embodiment of the method and apparatus of the present invention.

FIG. 2a is a sectional view for explaining the operation of the two-stage needle valve in the first aspect of the nozzle system of the present invention.

FIG. 2b is a sectional view for explaining the operation of the two-stage needle valve in the first aspect of the nozzle system of the present invention.

FIG. 3a is a sectional view for explaining the operation of the two-stage needle valve in the second aspect of the nozzle system of the present invention.

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[Procedure amendment]

[Submission date] December 3, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of a preferred embodiment of the method and apparatus of the present invention.

FIG. 2a is a sectional view for explaining the operation of the two-stage needle valve in the first aspect of the nozzle system of the present invention.

FIG. 2b is a sectional view for explaining the operation of the two-stage needle valve in the first aspect of the nozzle system of the present invention.

FIG. 2c is a sectional view for explaining the operation of the two-stage needle valve in the second aspect of the nozzle system of the present invention.

Claims (8)

[Claims] 1. A method for introducing foreign substances into a living organism or cells, in which the foreign substances are supported by fine particles, and the foreign substances-carrying fine particles are ejected from a tip together with a high-pressure compressed gas into a living organism or cells maintained at a predetermined temperature. At
A hole of a nozzle tip having a predetermined length and inner diameter, a first valve seat which is in axial communication with the hole of the nozzle tip hole, a first inner hole having a predetermined length and inner diameter, and a second valve seat and a predetermined A second inner hole having a length and inner diameter and the first
A nozzle mounted on an automatic gun for airless spray coating, which has a foreign matter-carrying particle insertion port with a cock valve communicating with the inner hole, and the front and rear on the axis of the nozzle tip hole depending on the operation of the automatic gun for airless spray coating. Act on the first
The first inner hole is closed by the two-stage needle valve of the nozzle system which comprises the two-stage needle valve, which is constituted by the valve body that opens and closes at the valve seat and the second valve seat, and the first inside from the foreign substance-carrying particulate insertion port. The foreign matter-carrying fine particles are inserted into the hole, the cock valve is closed, the two-stage needle valve is operated to open the first inner hole, and the high-pressure compressed gas having a predetermined pressure is kept for the second time for the second time.
The foreign matter-carrying fine particles introduced into the first inner hole together with the high-pressure compressed gas introduced into the first inner hole through the inner hole are passed through the nozzle tip hole to a predetermined speed from the nozzle tip. A method for introducing and incorporating a foreign substance into the organism or cell, which comprises ejecting the foreign substance into the organism or cell. 2. A method for introducing foreign substances into a living organism or cells, wherein foreign substances-carrying fine particles comprising foreign substances carried on fine particles are ejected from a nozzle tip together with a high-pressure compressed gas to strike an organism or cell maintained at a predetermined temperature. A hole of a nozzle tip having a predetermined length and inner diameter, a first valve seat that is in axial communication with the nozzle tip hole, and an inner hole having a predetermined length and inner diameter and a cock valve that communicates with the inner hole A nozzle mounted on the automatic gun, wherein the nozzle part from the insertion port to the tip of the nozzle is detachably connected, and the automatic airless spray coating The nozzle tip hole is axially actuated according to the operation of the gun, and is installed at the high pressure compressed gas outlet of the first valve seat and the airless spray coating automatic gun. The two-stage needle valve of the nozzle system, which is composed of a valve body that opens and closes at the second valve seat, closes the inner hole, and carries the foreign matter from the foreign matter-carrying fine particle insertion port to the inner bore. The fine particles are introduced, the cock valve is closed, the two-stage needle valve is operated to open the inner hole, and the high-pressure compressed gas of a predetermined pressure is introduced into the inner hole for a predetermined time, and the high-pressure compressed gas introduced into the inner hole. At the same time, the foreign matter-carrying fine particles introduced into the inner hole are ejected from the tip of the nozzle through the nozzle tip hole at a predetermined speed to introduce and incorporate the foreign matter into the organism or cell. 3. A high pressure adjusting regulator is provided at the outlet of the high pressure compressed gas source to branch the high pressure compressed gas whose pressure is regulated, and a low pressure adjusting regulator is provided on one side to control the low pressure compressed gas for controlling compressed gas and operation. It is used as compressed gas for the automatic gun, and a high-pressure gas filter, an on-off valve and a high-pressure accumulator are sequentially installed on the other side of the automatic gun. 3. The method according to claim 1, wherein the high-pressure compressed gas in the entire line is supplied to the automatic gun. 4. The method according to claim 1, wherein a portion of the ejected foreign matter-carrying fine particles scattered around is collected. 5. A controller for setting an operating time of a built-in on-off valve, when the controller is turned on, the on-off valve opens only for a set short time, and a compressed compressed gas for control opens to pass through the air-operated valve and the air-operated valve. The automatic gun for airless spray painting that operates from the high-pressure compressed gas source to supply high-pressure compressed gas from the high-pressure compressed gas source to the nozzle tip, and the control compressed gas in the path when the on-off valve closes and the supply of control compressed gas stops A nozzle having a predetermined length and inner diameter, which includes a NOT element and a rapid exhaust valve that operate to expel water rapidly, a nozzle system attached to the tip of the automatic gun, and a means for fixing organisms or cells. A first valve seat that sequentially communicates with the hole of the tip and the axial direction of the nozzle tip hole, a first inner hole having a predetermined length and inner diameter, a second valve seat, And a nozzle having a second inner hole having a predetermined length and inner diameter and a foreign particle carrying fine particle insertion hole with a cock valve communicating with the first inner hole, the nozzle being mounted on an automatic gun for airless spray coating, and the airless spray. A two-stage needle valve is configured by a valve element that is operated back and forth on the axis of the nozzle tip hole according to the operation of the automatic coating gun to open and close at the first valve seat and the second valve seat. An apparatus for use in the method according to claim 1. 6. A controller for setting an operating time of a built-in on-off valve, an air-operated valve for opening the on-off valve for a set short time when the controller is turned on and a control compressed gas flows, and an air-operated valve. The automatic gun for airless spray painting that operates from the high-pressure compressed gas source to supply high-pressure compressed gas from the high-pressure compressed gas source to the nozzle tip, and the control compressed gas in the path when the on-off valve closes and the supply of control compressed gas stops A nozzle having a predetermined length and inner diameter, which includes a NOT element and a rapid exhaust valve that operate to expel water rapidly, a nozzle system attached to the tip of the automatic gun, and a means for fixing organisms or cells. The hole of the tip portion, the first valve seat which is in axial communication with the hole of the nozzle tip hole, and the inner hole having a predetermined length and inner diameter A nozzle having a foreign particle-carrying fine particle insertion hole with a cock valve communicating with the hole, which is mounted on the automatic gun, and is preferably removably connected in front of the insertion port, and which corresponds to the operation of the automatic gun for airless spray coating. And a two-stage needle that operates back and forth on the axis of the nozzle tip hole to open and close at the first valve seat and the second valve seat provided at the high-pressure compressed gas outlet of the automatic gun for airless spray coating. An apparatus for use in a method according to claim 2, characterized in that it comprises a valve. 7. A high pressure adjusting regulator, a high pressure compressed gas branch line, a high pressure gas filter, a line from a high pressure compressed gas source to an automatic gun for airless spray coating.
An on-off manual valve and a high pressure accumulator are sequentially provided, and a low pressure adjusting regulator is provided in the high pressure compressed gas branch line, and the low pressure compressed gas decompressed by the low pressure adjusting regulator is used as a control compressed gas and an operating compressed gas. The device according to claim 5 or 6, which is provided. 8. Having a predetermined volume according to a predetermined internal pressure,
A chamber equipped with a means for fixing organisms or cells and equipped with a safety valve that is detachably connected to the nozzle, expands in the chamber, and separates and collects scattered foreign matter-carrying fine particles that are agitated by the expanded gas that exits the chamber. 7. The apparatus according to claim 5, further comprising: a separator, a vacuum degree adjusting regulator and a vacuum pump for sucking the gas separated by the separator to prevent instantaneous pressure increase.