JPH1128086A - Device for introducing molecule into cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a molecule-introducing device having an excellent operation performance and not giving a damage, etc., to cells. SOLUTION: A liquid supplied from a liquid-injecting device 4 is charged in a chamber 18. Pulse laser light hν introduced from a laser device 2 is guided with an optical fiber cable 12, gathered with a light-gathering lens 30, and focused at a breakdown position in the liquid. The energy of the laser hν induces impact waves due to a breakdown phenomenon in the liquid, and is propagated to outer cells and a molecule to be introduced through the liquid and the sound-propagating member 56. The molecule is introduced into the cell with the impact waves.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for introducing molecules into cells, and more particularly, to an apparatus for introducing molecules into cells by generating a shock wave in a liquid with laser energy and using the shock wave. It is.

[0002]

2. Description of the Related Art The introduction of molecules such as genes into cells has become an indispensable fundamental technology of biotechnology in the fields of biology and medicine. As a method for introducing a molecule such as a gene into a cell, a method using a vector such as a retrovirus, an adenovirus or a liposome, an electroporation method using electric energy,
A method using a gene gun is known as a physical method.

[0003] However, the method of introducing a viral vector or a liposome into cells is concerned about toxicity to cells. In addition, since the method for preparing a vector for introduction is complicated, special skills are required. The electroporation method requires the use of a solution with a low salt concentration, which is a severe condition for living cells. In addition, setting of optimal introduction conditions is complicated. In the method using a gene gun, the metal particles having the gene attached thereto are shot into the cells, so that the invasion of the cells is increased and the cells may be damaged.

In recent years, as a method of solving these problems, a method of introducing a molecule such as a gene into a cell using a shock wave or a stress wave has been studied.
Research report using an extracorporeal shock wave generator for lithotripsy as a source of shock wave generation (S. Gambihler,
et al., Permeabilization of the Plasma Membraneof
L1210 Mouse Leukemia Cells Using Lithotriper Shoc
k Waves, J MembraneBiol 141: 267-275 (1994)).

[0005]

However, in the prior art using an extracorporeal shock wave generator for crushing calculi, the entire apparatus was large and large. Furthermore, since this extracorporeal shock wave generator is originally a dedicated device used in the urology field, there is a problem that it lacks versatility.

[0006] The present invention has been made in view of such problems of the prior art, and has a small size, excellent operability, and can introduce molecules without damaging cells. The purpose is to provide.

[0007]

In order to achieve the above object, the present invention provides an apparatus for introducing a molecule into a cell.
Laser generating means, light guiding means for guiding pulsed laser light emitted from the laser generating means, optical means for condensing light guided by the light guiding means, and light condensing by the optical means And a chamber means for accommodating a liquid generating a shock wave by the pulsed laser light and transmitting the shock wave to the outside.

A laser generating means; a light guiding means for guiding pulsed laser light emitted from the laser generating means; an optical means for condensing light guided by the light guiding means; It is configured to contain a liquid that generates a shock wave by the pulsed laser light condensed by the optical means, and to have a stretchable balloon means for propagating the shock wave to the outside.

A laser generating means; a light guiding means for guiding pulsed laser light emitted from the laser generating means; an optical means for condensing light guided by the light guiding means; The apparatus includes a target member that generates a shock wave by the pulsed laser light condensed by the optical unit, and a balloon unit that accommodates the target member and the liquid and has elasticity that propagates the shock wave to the outside.

A laser generating means; a light guiding means for guiding pulsed laser light emitted from the laser generating means; an optical means for condensing light guided by the light guiding means; The apparatus includes a target member that generates a shock wave by the pulsed laser light condensed by the optical unit, and a chamber unit that accommodates the target member and the liquid and propagates the shock wave to the outside.

[0011]

The chamber means or the balloon means is used in such a manner that the chamber means or the balloon means is brought into contact with a solution containing a molecule to be introduced and cells, or is brought into contact with cells through the molecule to be introduced.

A shock wave is induced from the liquid or the target member by the energy of the pulsed laser light focused by the optical means. This shock wave propagates in the liquid and further propagates through the chamber means or the balloon means to the externally introduced molecules and cells. The impact force of this propagating shock wave (impact f
orce), the molecule is introduced into the cell.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
This will be described with reference to FIG. FIG. 1 is an explanatory diagram showing a main part structure of the molecule introduction device in a partially cutaway manner, and FIG. 2 is an explanatory diagram for explaining a method of using the molecule introduction device and the like.

In FIG. 1, this molecule introducing device includes a laser device 2 for generating pulsed laser light, a liquid injection device 4 having a built-in electric pump such as a so-called roller pump, and a laser injection device 4 including the laser device 2 and the liquid injection device 4. Control unit 6 for controlling operation
And a foot switch 8 for remote control, and a probe device 10.

The laser device 2 has a Q switch Nd: YA
A G laser and a coupling optical system such as a nonlinear optical crystal and an optical connector are provided. The pulse laser light emitted from the YAG laser is introduced into the nonlinear optical crystal, and the pulse laser light of the second harmonic component (wavelength 532 nm,
A pulse width of about 10 ns) hν is introduced into the optical fiber core 12F of the optical fiber cable 12 via the coupling optical system.
The optical fiber cable 12 has a flexible structure by coating an optical fiber core 12F for guiding the pulsed laser light hν with a synthetic resin tube.

The liquid injection device 4 has an output port OUT for outputting a liquid having fluidity such as water by the electric pump.
And an input port IN, an injection port 14 is connected to the output port OUT, and a discharge pipe 16 is connected to the input port IN. For both the injection pipe 14 and the discharge pipe 16, a flexible synthetic resin tube or the like is used.

The control section 6 is constituted by a microcomputer system or the like having an operation panel, and controls the operations of the laser device 2 and the liquid injection device 4 based on conditions specified by the operator on the operation panel. For example, the pulse laser beam intensity and the repetition frequency of the laser device 2, the number of pulses are adjusted, and the output flow rate and input flow rate of the liquid of the liquid injection device 4 are controlled. Further, remote control such as activation of the laser device 2 in synchronization with operation of the foot switch 8 is possible.

Next, the structure of the probe device 10 will be described. Optical fiber cable 12, injection pipe 14, and discharge pipe 1
A holding member 62 is provided for holding the bundle 6 in the vicinity of the probe device 10. An inlet port 20 and an outlet port 22 are provided at a side end of the substantially cup-shaped chamber 18 formed of a hard material. An injection pipe 14 is connected between the injection port 20 and the output port OUT of the liquid injection device 4, and a discharge pipe 16 is connected between the discharge port 22 and the input port IN of the liquid injection device 4, respectively. Thereby, the liquid from the liquid injection device 4 flows into the chamber 18 through the injection port 20, and the liquid in the chamber 18 flows out to the liquid injection device 4 through the discharge port 22.

A cylindrical connecting part 24 communicating with the inside of the chamber is integrally formed at the upper end of the chamber 18, and a funnel-shaped lens holder 26 is screwed to an inner peripheral wall of the connecting part 24. The interposition of the O-ring 28 hermetically seals the space between the connecting portion 24 and the lens holder 26.

In the lens holder 26, a condenser lens 30 is provided.
Are mounted by an annular spacer 32, an O-ring 34 and an annular fixing member 36. That is, the lens holder 2
6, an annular spacer 32 is mounted on a step formed on the inner peripheral wall of the lens holder 26. The annular spacer 32 and the O-ring 34 sandwich the peripheral portion of the condenser lens 30. The condenser lens 30 is mounted and fixed in the lens holder 26 by screwing it from the portion.

At the upper end of the lens holder 26, there is formed a vertical through-hole 38 whose optical axis is aligned with the condenser lens 30. The cylindrical through-hole 38 receives the distal end of the optical fiber cable 12 and the optical fiber core 12F. Connector shell 40 is inserted into the through hole 38.

Further, an O-ring 44 is mounted on an annular convex portion 42 integrally molded on the side surface of the connector shell 40, and an annular concave portion 4 integrally molded on the side surface of the upper end portion of the lens holder 26.
6 are provided with O-rings 48, and these O-rings 4
A cylindrical connector nut 50 is screwed to the upper end of the lens holder 26 with the intermediary of 4, 48. in this way,
The coupling mechanism including the O-rings 44 and 48, the lens holder 26, the connector shell 40, and the connector nut 50 achieves optical axis alignment between the optical fiber core 12F and the condenser lens 30 and provides excellent airtightness. Has been realized.

The inner wall of the chamber 18 has an elliptical concave shape centered on the focus position (hereinafter, referred to as a breakdown position) TG of the condenser lens 30.

An acoustic transmission member 56 formed by attaching a thin film 54 to an annular cap member 52 is screwed into an opening at the lower end of the chamber 18. That is, by sandwiching the peripheral portion of the thin film 54 between the annular packing members 58 and 60 and screwing the cap member 52 into the lower opening of the chamber 18, the thin film 54 is tightly attached to the lower opening of the chamber 18. Is being worn. The thin film 54 is made of a material having high mechanical strength and having good acoustic impedance matching with a sample solution or gel described later and a liquid filled in the chamber 18. For example, a silicon thin film or the like is used. Further, by removing the cap member 52, the thin film 54 can be replaced.

Next, the operation and use of the molecule introducing apparatus will be described with reference to FIG. The case where a molecule is introduced into cells taken out of a living body (in vitro) will be described.
The case where a sample solution containing cells and molecules to be introduced into the cells is used will be described.

First, a sample solution containing cells and molecules to be introduced into the cells is prepared. This solution 64 is placed in a sample container 66. As a specific experimental example, NIH / 3T3 cells (mouse-derived fibroblasts) were used as cells.
And FITC dextran (molecular weight 71200) can be used as the introduced molecule.

The chamber 18 is placed in the sample container 66, and the thin film 54 is immersed in the sample solution 64. Next, by operating the injection device 4, a liquid such as water is supplied into the chamber 18 through the injection port 20. At the same time, by supplying the liquid while exhausting the inside of the chamber 18 through a discharge path constituted by the discharge port 22, the discharge pipe 12 and the input port IN, automatic control is performed so that the pressure in the chamber 18 does not excessively increase. Is performed.

When the chamber 18 is full of liquid, the liquid injection device 4 automatically stops, and the liquid pressure in the chamber 18 is kept constant. Alternatively, the liquid pressure in the chamber 18 is automatically kept constant by circulating the liquid so that the supply flow rate to the chamber 18 and the discharge flow rate from the chamber 18 are made equal. The automatic stop or circulation operation of the liquid injection device 4 can be selected by the operator instructing the control unit 6. In addition, if this circulation operation is continued,
Air bubbles that may be generated in the chamber 18 due to a breakdown phenomenon described later can be discharged, and the filling density of the liquid can be increased.

Next, the foot switch 8 is operated. Thereby, the laser device 2 is started and the pulse laser light hν is introduced into the optical fiber cable 12. Pulsed laser light h
ν is emitted from the tip of the optical fiber core wire 12F, is collected by the condenser lens 30 having a predetermined focal length, and
It converges to the breakdown position (focal position) TG in Q. At this breakdown position TG, the laser energy is concentrated at a high density, so that a breakdown phenomenon occurs, and a stress wave or a shock wave (shock wave) is generated.
ve) is induced.

Here, the wave SW is a general term for waves such as a compression wave, a pressure wave, and a sound wave propagating in a medium. However, since the present invention utilizes an impact force, , And the wave SW is hereinafter referred to as a shock wave.

This shock wave SW isotropically radiates and propagates in the liquid LQ, and is propagated directly or reflected on the inner wall of the chamber 18 to the thin film 54 side efficiently. And the shock wave S
W propagates to the thin film 54 and propagates through the thin film 54 to the sample solution 64.

In the sample solution 64, molecules are introduced into cells through the cell membrane by the energy of the shock wave SW. By appropriately shifting the position of the thin film 54, molecules can be introduced into all the cells in the sample container 66.

The mechanism by which molecules are introduced into cells is as follows:
It is considered that some opening is transiently formed in the lipid bilayer membrane of the cell by receiving the energy of the shock wave SW, and the molecule permeates into the cell through this opening. Therefore, the molecule is introduced without damaging or damaging the cells.

As described above, according to the present embodiment, the following effects can be obtained. Compared with the prior art, it is possible to realize a molecule introduction device having a simple and small device configuration and excellent in operation performance. Molecules can be introduced without damaging cells. By using the second harmonic component as the pulsed laser light, the absorption coefficient of the laser light in the water in the chamber 18 is reduced. For this reason, the attenuation of the laser light hν from the condenser lens 30 to the breakdown position TG can be ignored,
The energy of the laser light hν can be used with high efficiency.

Further, since the inner wall surface of the chamber 18 is made concave so that the shock wave SW is reflected toward the thin film 54, the energy of the shock wave SW can be transmitted to the sample solution 64 with high efficiency. The laser device 2 with low power consumption can be used. When the inner wall of the chamber 18 has an elliptical shape, the propagation efficiency of the shock wave SW can be optimized by adjusting the center position of the elliptical shape to the breakdown position TG.

The thin film 54 can be reused by washing it. In addition, replacement of the contaminated thin film 54 and collection lens 3
Since the replacement of 0 is easy, the maintenance performance is excellent. The convergence position (breakdown position) TG of the pulse laser beam hv can be finely adjusted by adjusting the amount of screwing of the lens holder 26 to the connecting portion 24 shown in FIG. Therefore, it is possible to easily adjust the propagation efficiency of the shock wave SW optimally.

Although the case of using water has been described, another liquid may be supplied into the chamber 18. Although the case where the sample solution 64 is used has been described, a gel containing cells and introduced molecules is used as a sample, and the thin film 54 is brought into contact with the gel to give a shock wave SW, whereby molecules can be introduced into the cells. .

Although the method for using the molecule introduction device in vitro has been described, the invention can be applied in vivo.
For example, the thin film 54 of the probe device 10 is brought into contact with a living tissue via an agent containing at least a molecule to be introduced,
By applying the shock wave SW to the living tissue, molecules can be introduced into the tissue cells. Thus, the molecule introduction device can be applied irrespective of in vitro and in vivo, and has excellent versatility.

The laser device 2 for generating the pulsed laser light hν is not limited to the Q switch Nd: YAG laser, but may use another laser such as an excimer laser or a semiconductor laser. . Further, the case where the second harmonic component is used for the pulsed laser light hν has been described, but the present invention is not limited to this, and a fundamental wave or another harmonic component may be used.

Although the case where the pulse laser light hν is guided by the optical fiber cable has been described, the present invention is not limited to this, and light guide means such as a hollow waveguide or a multi-joint mirror type manipulator is used. Is also good.

Various molecules can be applied as the introduced molecule. For example, genes (such as DNA fragments and RNA), proteins such as enzymes, drugs, and the like can be introduced into cells.

The solution containing cells and introduced molecules may be added with a chemical such as a thickener or a pH adjuster. Thereby, the processing efficiency of the apparatus, the handling of the solution, and the like can be improved.

(Second Embodiment) A second embodiment of the present invention will be described with reference to FIGS. FIG. 3 is an explanatory view showing a main part structure of the molecule introduction device, partially cut away, and FIG.
FIG. 3 is an explanatory diagram for explaining a method of using the molecule introduction device and the like. In FIGS. 3 and 4, FIGS.
The same or corresponding parts as those shown in FIG.

In FIG. 3, a holding member 6 for holding the optical fiber cable 12, the injection pipe 14 and the discharge pipe 16 in a bundle.
Balloon-shaped balloon member 7 having elasticity
0 is closely attached. Balloon member 70
Is made of a material having a high mechanical strength and good acoustic impedance matching with a sample solution or gel to be described later and a liquid filled in the balloon member 70. For example, it is formed of silicon rubber.

In the balloon member 70, the condenser lens 30 is provided.
And the optical fiber cable 1
2, a connector shell 40 and a connector nut 50 for receiving the optical fiber core 12F, the O-ring 34,
A highly airtight coupling mechanism composed of 44, 48, etc. is accommodated. Further, the respective distal end portions of the injection pipe 14 and the discharge pipe 16 are also accommodated in the balloon member 70.

To attach the balloon member 70 to the holding member 68, the connecting mechanism is housed in the balloon member 70, and the open end of the balloon member 70 is
At the same time, a structure is adopted in which the airtightness is maintained by attaching these to the holding member 68 and tightening and fixing them with an annular holding member 74 screwed to the holding member 68.

Next, the operation and use of the molecule introducing device will be described with reference to FIG. The case where a molecule is introduced into cells taken out of a living body (in vitro) will be described.

A sample solution containing cells and molecules to be introduced into the cells is prepared.
Put in 6. Balloon member 7 in sample container 66
0 is immersed in the sample solution 64 and the injection device 4 is operated to supply a liquid such as water into the balloon member 70 via the injection tube 14. When the balloon member 70 expands to a predetermined size, the liquid injection device 4 automatically stops,
The fluid pressure in the balloon member 70 is kept constant. Alternatively, by circulating the liquid supply amount by the injection pipe 14 and the liquid discharge amount by the discharge pipe 16 so as to automatically keep the liquid pressure in the balloon member 70 constant,
Eliminate any air bubbles that may occur due to the breakdown phenomenon.

Next, the laser switch 2 is started by operating the foot switch 8, and the pulse laser light hν is introduced into the optical fiber cable 12. The pulsed laser light hν is emitted from the tip of the optical fiber core wire 12F, is condensed by a condenser lens 30 having a predetermined focal length, and converges at a breakdown position (focal position) TG in the liquid LQ. At this breakdown position TG, a breakdown phenomenon occurs, and a shock wave SW is induced.

The shock wave SW isotropically radiates and propagates in the liquid LQ, and passes through the balloon member 70 to the sample solution 64.
Propagate to

In the sample solution 64, molecules are introduced into the cells through the cell membrane by the energy of the shock wave SW.

According to the present embodiment, since the shock wave SW generated at the breakdown position TG in the liquid LQ propagates in the sample solution 64 through almost the entire balloon member 70, a large amount of molecules can be introduced into cells at one time. It can be carried out.

The balloon member 70 can be reused by cleaning it. Further, since the replacement of the dirty balloon member 70 and the replacement of the condenser lens 30 are easy, the maintenance performance is excellent.

Although the case of using water has been described, another liquid may be supplied into the chamber 18. Although the case where the sample solution 64 is used has been described, it is possible to introduce a molecule into cells by using a gel containing cells and introduced molecules as a sample and bringing the balloon member 70 into contact with the gel to give a shock wave SW. it can.

Further, the method of using the molecule introduction device in vitro has been described, but application in vivo is possible.
For example, as shown in FIG. 5A, when targeting a tissue 78 such as a liver, a stomach wall, or skin, a drug (eg, solution or gel) 80 containing a molecule to be introduced is applied to the tissue 78, The molecules can be introduced by bringing the balloon member 70 into contact with the tissue 78 via the medicine 80 and applying a shock wave SW. Further, as shown in FIG.
In the case of targeting the inner wall of the luminal tissue 84 such as the second, the target site of the tissue 84 is perfused with a drug containing a molecule to be introduced,
Molecules can be introduced by inserting the balloon member 70 transvascularly and applying a shock wave SW. When these processes are performed, the use of a conventionally used endoscope is effective in confirming a target portion and the like.

As described above, the molecule introducing apparatus is in vit
It can be applied regardless of ro and in vivo, and has excellent versatility.

(Third Embodiment) A third embodiment of the present invention will be described with reference to FIG. FIG. 6 is an explanatory view showing a main part structure of the molecule introduction device, partially cut away.
In FIG. 6, the same or corresponding parts as those in FIG. 3 are denoted by the same reference numerals.

In FIG. 6, a holding member 6 for holding the optical fiber cable 12, the injection pipe 14 and the discharge pipe 16 in a bundle.
A balloon member 70 is closely attached to and fixed to the distal end of 8 by a holding member 74 via an O-ring 72.

In the balloon member 70, the lens holder 26 having the condenser lens 30 and the like, and the optical fiber cable 12
The connector shell 40 and the connector nut 50 for receiving the tip of the optical fiber and the optical fiber core 12F, and the O-rings 34 and 4.
A highly airtight connecting mechanism composed of 4, 48, etc. is accommodated. Further, the respective distal end portions of the injection pipe 14 and the discharge pipe 16 are also accommodated in the balloon member 70. Further, a target holder 88 is attached to the lower end of the lens holder 26.

The target holder 88 has an annular base 90 screwed to the lower end of the lens holder 26 and a base 9.
A plurality of thin support rods 9 integrally molded to extend downward
2 and 94, and an annular target holding portion 96 integrally formed at the lower ends of the support rods 92 and 94, between the target holding portion 96 and an annular target holding member 100 screwed to the lower end thereof. Then, a target member 98 is attached. Further, by making the optical axis of the condenser lens 30 coincide with the center position of the target member 98, the pulse laser light hν emitted from the optical fiber core 12F is condensed by the condenser lens 30 and
Is to be irradiated.

The target member 98 is formed of a polymer material or the like that absorbs laser energy with high efficiency and generates a shock wave. As a specific example, a polyimide film (thickness: about 75 μm) having good shock wave generation efficiency and easy handling was used.

Next, the operation and use of the molecule introducing apparatus will be described with reference to FIG. The case of in vitro will be described. As shown in FIG. 4, a sample solution containing cells and molecules to be introduced into the cells is prepared.
Is placed in the sample container 66. The balloon member 70 is put in the sample container 66 and immersed in the sample solution 64,
By operating the injection device 4, a liquid such as water is supplied into the balloon member 70 via the injection tube 14. At this time, the liquid is supplied while being exhausted through the discharge pipe 16.

When the balloon member 70 is filled with the liquid and swells to a predetermined size, the liquid injection device 4 automatically stops, and the liquid pressure in the balloon member 70 is kept constant. Alternatively, by circulating the liquid so that the liquid supply amount by the injection pipe 14 and the liquid discharge amount by the discharge pipe 16 are equalized, the liquid pressure in the balloon member 70 is automatically kept constant, and laser irradiation on the target member 98 is performed. Eliminates ablation products and bubbles that may occur due to

Next, the laser device 2 is started by operating the foot switch 8, and the pulse laser light (wavelength 355 nm) hν of the third harmonic component generated here is introduced into the optical fiber cable 12. The pulsed laser light hν is emitted from the tip of the optical fiber core wire 12F, collected by the condenser lens 30 and irradiated on the target member 98. The target member 98 absorbs laser energy and induces a shock wave. The shock wave isotropically propagates through the liquid, and the balloon member 70
Through the sample solution 64, and the molecules of the shock wave energy penetrate the cell membrane and are introduced into the cells.

As specific examples of the laser incidence conditions, the repetition frequency of the pulse laser light hν is 1 Hz, the number of irradiation pulses is 10, and the laser energy of the pulse laser light hν is 1
5 to 85 mJ, the irradiation beam diameter at the target member 98 is 3 mmφ, and the fluence is 0.2 to 1.2 J / cm.
^{And 2} . As a result, the peak value of the impact (stress) generated in the target member 98 made of the polyimide film becomes
For a fluence of 0.2 to 1.2 J / cm ² , 0.1 to 1
It is on the order of 0 MPa. At this time, a shock wave was induced without penetrating the target member 98 by laser ablation, and molecules could be introduced into cells.

As described above, the present embodiment is characterized in that a shock wave is generated using laser energy absorption or ablation of the target member 98, and molecules can be introduced by the shock wave. As in the second embodiment, molecules can be introduced without damaging cells or the like. In addition, the molecule introduction device,
It can be applied both in vitro and in vivo, and has excellent versatility.

Further, since the third harmonic component is used as the pulse laser beam hν, the efficiency of the shock wave generation in the target member 98 is improved, and the balloon member 7
The absorption coefficient of laser light in a liquid such as water within 0 becomes small. Therefore, the energy of the laser light hν can be used with high efficiency. The lens holder 2 shown in FIG.
By adjusting the screwing amount of 6 to the base 90, the irradiation beam diameter of the pulse laser beam hv on the target member 98 can be finely adjusted. For this reason, it is possible to easily adjust the generation efficiency of the shock wave SW optimally.

In FIG. 6, the entirety of the target holder 88 including the target member 98 is immersed in the liquid. However, instead of the support rods 92 and 94, a truncated cone-shaped cylindrical portion is used for the base 90 and the target. The structure may be such that the liquid does not enter the space between the light emitting end of the optical fiber core wire 12F and the inside of the target member 98 by integrally molding between the holding parts 96 and sealing the inside.

Further, the case where the target member 98 is housed in the balloon member 70 has been described. However, as in the first embodiment, the target member 98 may be housed in a chamber sealed with a sound propagation member. . That is, the target holder 88 including the target member 98, the lens holder 26 including the condenser lens 30, and the like, the optical fiber cable 12
The connector shell 40 and the connector nut 50 for receiving the tip of the optical fiber and the optical fiber core 12F, and the O-rings 34 and 4.
4, 48 and the like may be accommodated in the chamber 18 which is sealed by the sound propagation member 56 shown in FIG.

The case where a polymer film is used for the target member 98 has been described, but the present invention is not limited to this. For example, a metal film, a polymer piece, a metal piece, a rubber material, or the like can be used.

[0071]

As described above, according to the present invention, a shock wave is induced in a liquid or a target member by the energy of a pulsed laser beam, and this shock wave is propagated to the outside via the liquid to cause the introduced molecules and cells to be introduced. The molecule can be introduced without damaging the cells. It is possible to realize a molecule introduction device that has a simple and small device configuration and is excellent in maintenance performance and operation performance. Further, the molecule introduction device of the present invention can be applied irrespective of in vitro and in vivo, and exhibits excellent versatility.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram illustrating a structure of a molecule introduction device according to a first embodiment.

FIG. 2 is an explanatory diagram illustrating an operation and a method of using the molecule introduction device according to the first embodiment.

FIG. 3 is an explanatory diagram illustrating a structure of a molecule introduction device according to a second embodiment.

FIG. 4 is an explanatory diagram for explaining an operation and a usage method of a molecule introduction device according to a second embodiment.

FIG. 5 is an explanatory diagram for explaining another method of using the molecule introduction device according to the second embodiment.

FIG. 6 is an explanatory diagram illustrating a structure of a molecule introduction device according to a third embodiment.

[Explanation of symbols]

2 laser device, 4 liquid injection device, 6 control unit, 10, 7
6,86 ... probe device, 12 ... optical fiber cable,
12F: optical fiber core wire, 14: injection tube, 16: discharge tube, 18: chamber, 20: injection port, 22: discharge port, 24: connecting portion, 26: lens holder, 30: condenser lens, 40: connector Shell: 54: Thin film, 56: Sound propagation member, 70: Balloon member, 88: Target holder, 96: Target holding part, 98: Target member, 100: Target holding member.

Claims (5)

[Claims] 1. a laser generating means; a light guiding means for guiding pulsed laser light emitted from the laser generating means; an optical means for condensing light guided by the light guiding means; An apparatus for introducing molecules into cells, comprising: chamber means for containing a liquid generating a shock wave by pulsed laser light condensed by an optical means and for propagating the shock wave to the outside. 2. A laser generating means, a light guiding means for guiding pulsed laser light emitted from the laser generating means, an optical means for condensing light guided by the light guiding means, An apparatus for introducing molecules into cells, comprising: a balloon means for containing a liquid that generates a shock wave by a pulse laser beam condensed by an optical means and having elasticity for propagating the shock wave to the outside. 3. A laser generating means, a light guiding means for guiding pulsed laser light emitted from the laser generating means, an optical means for condensing light guided by the light guiding means, A target member for generating a shock wave by the pulsed laser light condensed by the optical means; and a balloon means for containing the target member and the liquid and having elasticity for propagating the shock wave to the outside. A device for introducing molecules into cells. 4. A laser generating means, a light guiding means for guiding pulsed laser light emitted from the laser generating means, an optical means for condensing light guided by the light guiding means, A target member for generating a shock wave by pulsed laser light condensed by optical means, and a chamber means for containing the target member and a liquid and for propagating the shock wave to the outside, comprising: Molecular introduction device. 5. The apparatus according to claim 1, wherein the chamber means includes a sound propagation member for propagating a shock wave generated in the liquid, and a hard cup member holding the sound propagation member and accommodating the liquid together with the sound propagation member. The apparatus for introducing a molecule into cells according to any one of claims 1 and 4.