JP2008154528A - Cell injection apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell injection apparatus that readily carries out implantation operation of a micropipette with respect to cells. <P>SOLUTION: Cells CL moved in a feed route 1a are surely captured in a fixed implantation position, by driving a cylindrical part 2b to a first position by an actuator 2c, and further a micropipette MP inserted via an inlet route 1b is readily implanted in the captured cells CL. Sure implantation of the micropipette MP can be performed, especially so as to retain the cells CL by two cylindrical parts 2a. When the cells CL are adsorbed on the bottom of the feed route 1a, the cylindrical parts 2a are brought physically into contact with the cells CL, and the micropipette can be surely moved inside the feed route 1a. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell injection apparatus suitable for introducing into a living animal tissue or a cell an intracellularly introduced substance made of a biopolymer such as a gene or protein.

  Conventionally, in order to introduce intracellular introduction substances made of biopolymers such as genes and proteins into animal tissues or cells, a glass needle (micropipette) is directly inserted into animal cells or tissue cells. A microinjection method in which a substance introduced into the cell is injected from is used.

  However, when the microinjection method is executed while observing under an optical microscope, it is difficult to actually insert the micropipette appropriately because the cells can only be seen in a plane. In particular, it is difficult to trap microscopic cells and insert a micropipette. Unless you are an expert, efficient injection is not possible, and it is necessary to perform precise positioning while observing images with a light microscope. There was a problem that there was.

In order to increase the success rate of the micropipette insertion operation, a technique for reliably trapping cells on the substrate surface has been proposed. For example, using a method of fixing cells using suction or the like to a substrate having a through hole (see Patent Documents 1 and 2), or a microchannel array apparatus having an open end for particle trapping on the end surface (side surface) of the substrate There is disclosed a technique (see Patent Document 3) in which cells are inhaled and trapped, and the micropipette tip can be moved within the direction of microscopic observation and the depth of focus.
JP-A-1-112976 JP 2000-23657 A JP 2002-27969 A

  However, in the prior art, it is difficult to position the cell at a predetermined position, and therefore, the micropipette insertion operation is still difficult, and some countermeasure has been demanded.

  The present invention has been made in view of the problems of the prior art, and provides a cell injection device that can easily insert a micropipette into a cell, thereby enabling precise positioning depending on image observation. The purpose is to provide a technique that does not require the use of the substance, and to enable introduction of intracellular introduction substances into animal cells, fertilized eggs, etc., even for non-experts, and to obtain a high throughput of injection operation. .

The cell injection device of the present invention comprises:
A housing that forms a supply path through which cells move and an introduction path that intersects the supply path;
A stopper member movable between a first position entering the supply path and a second position retracted from the supply path;
Drive means for driving the stopper member between the first position and the second position;
The cells captured by the stopper member moved to the first position in the supply path are inserted by a micropipette inserted through the introduction path.

  The cell injection device of the present invention includes a housing in which a supply path through which cells move and an introduction path that intersects the supply path, a first position that enters the supply path, and a retreat from the supply path. A stopper member movable between the second position and drive means for driving the stopper member between the first position and the second position; When the driving means drives the stopper member to the first position, the driving means can be surely captured at a predetermined insertion position, and the captured cells are further passed through the introduction path. It can be easily inserted by the inserted micropipette.

  When the supply path and the introduction path are formed in a housing used for the cell injection apparatus according to the present invention by using MEMS (Micro Electro Mechanical Systems) technology, glass, silicon substrate, PDMS (poly-dimethylsiloxane), ceramic material As a material of the housing, it is possible to easily produce a fine structure by creating a minute groove on the surface, and it is possible to realize a miniaturization and a high pressure resistant structure at an extremely low cost, and various materials are used. Since it is possible to design, a wide range of material designs with excellent chemical resistance can be made.

  The housing is preferably formed by depositing platinum on a glass substrate, applying a resist, patterning the supply path and the introduction path, and then etching.

  The stopper member is preferably formed by depositing a photo-curing resin or resist on a glass substrate, depositing a semitransparent metal thin film thereon, and etching while leaving a part.

  In the supply path and the introduction path, an etching mask and a resist are formed in this order on the material of the housing, and then a part of the resist is removed by exposure and development, and the resist and the etching are further etched. Preferably, it is formed by removing the mask.

  The supply path and the introduction path are formed by forming a film material, an etching mask, and a resist in this order on the material of the housing, and then removing a part of the resist by exposure and development, and further performing etching processing to remove the resist and the resist It is preferable that the film is formed by removing the etching mask and a part of the film material.

  The supply path and the introduction path are preferably formed by removing a part of the resist by exposure and development after forming a resist on the material of the housing.

  The supply path and the introduction path are preferably formed using a mold.

  Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view of a cell injection device according to the present embodiment, and FIG. 2 is a top view thereof. In FIG. 1, on the substrate 1 constituting the housing, a groove-shaped supply path 1a extends in the left-right direction, and the groove-shaped introduction path 1b extends from the front side so as to intersect the center of the supply path 1a. Exist. The width and height of the supply path 1a are such that one cell CL can be accommodated, and are generally about 10 μm to 100 μm. A scale Mk is preferably provided on the bottom surface of the supply path 1a (see FIG. 2). A plurality of cells CL move in one row in the cell culture solution filled in the supply path 1a.

  The driving device 2 includes two sets of a cylindrical portion 2a that can enter the supply path 1a, an arm portion 2b that is attached to the tip of the cylindrical portion 2a, and an actuator 2c that drives the arm portion 2b up and down, and an introduction path 1b. It is provided at a sandwiched position. The actuator 2c using a piezo element, an air cylinder, a hydraulic cylinder, a solenoid or the like as a drive source can independently drive the arm portion 2b up and down. Each arm portion 2b is made of glass, each cylindrical portion 2a is made of a photo-curing resin except for the bottom portion, and a semitransparent metal is deposited on the bottom portion. The cylindrical portion 2a constitutes a stopper member.

  A hollow glass micropipette MP with a sharp tip is inserted into the introduction path 1b so that the tip can enter the supply path 1a. A CCD camera 4 equipped with a microscope 3 is arranged above the intersection of the supply path 1a and the introduction path 1b. Although not shown, a moving stage placed under the substrate 1, a syringe pump for introducing a culture solution, an extraction device, and an introduction pipe connecting them are also provided. A personal computer or the like for automating the injection operation may be installed.

  Next, the operation of the cell injection device shown in FIG. 1 will be described. First, the driving device 2 drives each actuator 2c to place the cylindrical portion 2a in the second position retracted from the supply path 1a. The supply path 1a is filled with a cell culture solution, a syringe is connected to a pipette (not shown), the piston portion is pressurized, and cells (animal cells, fertilized eggs, etc.) CL are introduced from the supply side. At this time, the plurality of cells CL smaller in size than the supply path 1a move sequentially one by one along the supply path 1a (here, from right to left) by electrophoresis, pressure difference, liquid flow by a pipette, or the like. However, since the cylindrical portion 2a is retracted from the supply path 1a, the passage thereof is not hindered. The operator waits for the target cell CL to approach while observing with the CCD camera 4 depending on the scale MK on the supply path 1a.

  Here, when the target cell CL approaches, the operator turns on a switch (not shown), drives the actuator 2c on the discharge side (left side in FIG. 1), and moves the cylindrical portion 2a through the arm 2b. It moves to the 1st position which penetrates into supply way 1a (state shown in Drawing 1). Thereby, as shown to Fig.2 (a), the cell CL used as a target will be blocked | interrupted by the cylindrical part 2a, and will be captured. Further, the operator turns on a switch (not shown), and as shown in FIG. 2 (b), drives the actuator 2c on the supply side (right side in FIG. 1) to feed the cylindrical portion 2a to the supply path via the arm 2b. It moves to the 1st position which invades 1a. Thereby, the cells CL can be sandwiched and captured at the position intersecting the introduction path 1b by the two cylindrical portions 2a. In addition, since the bottom part of the cylindrical part 2a has a translucent metal film, positioning becomes easy while observing with the CCD camera 4 by the scale MK on the flow path substrate.

  In this state, if the operator inserts the micropipette MP from the introduction path 1b, the introduction path 1b serves as a guide, guides the sharp tip of the micropipette MP, and is appropriate for the cell CL sandwiched between the cylindrical portions 2a. The substance in the micropipette MP can be injected into the cell CL. The operator extracts the injected cells CL using an apparatus (not shown) for extracting the cells CL after the injection.

  FIG. 3 is a diagram illustrating a manufacturing process of the arm portion 2b and the cylindrical portion 2a. With reference to FIG. 3, the manufacturing method of the arm part 2b and the cylindrical part 2a is shown. First, a photosensitive resin OR such as a photo-curing resin or a resist is formed on the glass substrate GS constituting the arm portion 2b, and a metal thin film MM having a semi-transparent reflectance adjusted is further deposited thereon (see FIG. 3 (a)). Next, the semi-transparent metal thin film MM is etched to leave only a part to be the bottom of the cylindrical part 2a (FIG. 3B), and the photo-curing resin OR is further etched to leave only a part of the cylindrical part 2a. Other than the bottom portion (FIG. 3C), the arm portion 2b and the cylindrical portion 2a can be formed as described above (FIG. 3D).

  FIG. 4 is a diagram illustrating a manufacturing process of the substrate 1. With reference to FIG. 4, the manufacturing method of the board | substrate 1 is demonstrated. First, a plurality of scales MK are formed by vapor deposition on the glass substrate GS using platinum or the like (FIG. 4A). Then, resist RS is apply | coated on glass GS (FIG.4 (b)). Furthermore, after patterning the supply path and the introduction path, etching is performed to form the supply path and the introduction path (FIG. 4C). If necessary, the introduction path 1b of the micropipette MP may be hydrophobized using a water repellent or the like. Thus, the substrate 1 having the supply path 1a and the introduction path 1b can be formed (FIG. 4D).

Another method for manufacturing the substrate 1 will be described. In the following example, the scale of platinum is not formed on the bottom surface of the supply path, but it can be provided in the same manner as described above.
(1) Etching channel processing method:
This is a method of processing the supply path 1a and the introduction path 1b for the circulation path on the substrate S using wet etching or dry etching. FIG. 5 shows a process flow of the etching channel processing method. First, as an etching mask layer EM for protecting other than the etched portion, chromium or the like is formed on the upper surface of the substrate S by vacuum deposition or sputtering (see FIG. 5A). Next, a resist RS is coated on the upper surface of the etching mask layer EM with a uniform thickness by spin coating (see FIG. 5B). Then, after performing RS patterning (pattern transfer) of the resist on the shape of the supply path 1a and the introduction path 1b by photolithography and other exposure techniques (see FIG. 5C), the etching mask layer EM is etched ( 5D), the substrate S is etched (see FIG. 5E), and finally the unnecessary etching mask layer EM and the resist RS are removed to form the supply path 1a in the substrate S. I do. The introduction path 1b can be formed in the same manner.

Examples of the material structure used for the etching channel processing method are shown below.
(Example 1) Substrate: quartz or glass / etching mask: Cr or the like / resist: photoresist / mask etching solution: Cr etchant / substrate etching: hydrofluoric acid solution (wet etching)
(Example 2) Substrate: silicon / etching mask: SiO ₂ + Cr, etc./resist: photoresist / mask etching solution: Cr etchant (diammonium cerium nitrate + perchloric acid), SiO ₂ etchant (hydrofluoric acid buffer solution) / Substrate etching: Wet etching using KOH or TMAH, or dry etching using SF ₆ , plasma etching, RIE etching)

(2) Channel processing method using membrane material:
This is a method of processing a fine groove for a channel in a film material formed on a substrate. 6 and 7 show the process flow of the flow path processing method using a membrane material. In this method, a fine groove can be formed by exposing a photosensitive film material, i) when using a photosensitive material, and ii) when using a non-photosensitive material that processes the film material on the substrate by etching. and so on.

i) When a photosensitive material is used, a photoresist PR is applied on the substrate S (see FIG. 6A), patterned by photolithography or other exposure technique (see FIG. 6B), and further developed. Thus, a part of the photoresist PR can be removed to form the supply path 1a and the introduction path 1b (see FIG. 6C).

ii) When a non-photosensitive material is used, a manufacturing method similar to the etching channel processing method described with reference to FIG. 5 is used. More specifically, the film material PR, the etching mask EM, and the resist RS are formed on the upper surface of the substrate S in this order (see FIG. 7A). Next, after performing RS patterning (pattern transfer) of the resist on the shape of the supply path 1a and the introduction path 1b by photolithography and other exposure techniques (see FIG. 7B), the etching mask layer EM is etched. (See FIG. 7 (c)), the film material PR is etched (see FIG. 7 (d)), and finally the unnecessary etching mask layer EM and the resist RS are removed to supply the film material PR with a supply path. 1a and the introduction path 1b are formed. The accommodating portion can be formed in the same manner.

  Examples of the material configuration used in the flow path processing method using a membrane material are shown below. Note that glass or silicon can be used as the substrate, but various materials can be used as long as the material is not specified and the material is not specified. In particular, when silicon is used as the film material, an SOI substrate can be used.

(Example 1 using photosensitive material) Film material: photo-curing resin, thick film resist or photosensitive polyimide (Example 2 using non-photosensitive material) Film material: SiO ₂ glass, etc./resist: photoresist / etching mask : Cr, etc./film etching: hydrofluoric acid solution (wet etching)
(Example 3 using non-photosensitive material) Film material: Polyimide etc./Resist: Photoresist / Etching mask: Cr, SiO ₂ etc./Etching of film: Wet etching using KOH or TMAH)
(Example 4 using non-photosensitive material) Film material: Resist etc./Resist: Photoresist / Etching mask: SiO ₂ etc./Etching film: Dry etching using O ₂ gas (Example using non-photosensitive material) 5) Film material: silicon etc./resist: photoresist / etching mask: Cr, SiO ₂ etc./film etching: wet etching using KOH or TMAH or dry etching using SF ₆

Note that the SiO ₂ etchant is a hydrofluoric acid buffer solution, and the etching of the silicon substrate is preferably wet etching using KOH or TMAH or dry etching using SF ₆ . In the flow path processing method using the film material, the depth of the flow path may be processed to be shallower than the film thickness of the film material.

(3) Channel processing method by molding using a mold:
This uses a mold prepared for transferring a fine groove pattern for a flow path, and forms a fine groove for the flow path by transferring a shape that is opposite to the surface shape of the mold. Although it is a technique, it is suitable for mass production. A molding method in which PDMS (polydimethylsiloxane) or silicon rubber or the like is molded on a mold and a molding method in which the plastic is pressed against the mold while being pressed may be used.

  FIG. 8 is a diagram showing a process flow for forming fine grooves from a mold to PDMS. In FIG. 8A, a mold M having convex portions corresponding to fine grooves is manufactured. Subsequently, as shown in FIG. 8B, the supply path 1 a (and the introduction path 1 b) is transferred and formed on the PDMS by the convex portion of the mold M. Further, as shown in FIG. 8C, by releasing the PDMS from the mold M, a housing or the like having the supply path 1a can be formed.

  FIG. 9 is a diagram showing a process flow for forming fine grooves in the resin from the mold. In FIG. 9A, a mold M having protrusions corresponding to fine grooves is manufactured. Subsequently, as shown in FIG. 9B, the supply path 1 a is transferred and formed by covering the convex portion of the mold M with the molten resin PL. Further, after the resin PL is solidified, as shown in FIG. 9C, by releasing the resin PL from the mold M, a housing or the like having the supply path 1a can be formed.

  As described above, according to the present embodiment, the actuator 2c drives the cylindrical portion 2b to the first position so that the cell CL that has moved in the supply path 1a is reliably placed at a predetermined insertion position. The captured cells CL can be easily inserted by the micropipette MP inserted through the introduction path 1b. In particular, since the cells CL are held by the two cylindrical portions 2a, the micropipette MP can be surely inserted. Further, when the cell CL is adsorbed to the bottom of the supply path 1a, the cylindrical portion 2a can be moved in the supply path 1a reliably by physically contacting the cell CL.

  Further, the scale MK provided in the supply path 1a can accurately perform the positioning of the cylindrical portion 2a for physically operating the cell CL in the supply path 1a regardless of the depth of focus of the microscope 3. Since the supply path 1a is thin, a plurality of cells CL can be introduced in a line, so that the micropipette MP can be inserted one by one in order. Since the micropipette MP is guided to the introduction path 1b, the micropipette MP can be accurately positioned without depending on the microscope image of the CCD camera 4.

  The present invention has been described above with reference to the embodiments. However, the present invention should not be construed as being limited to the above-described embodiments, and can be modified or improved as appropriate.

It is a perspective view of the cell injection device concerning this embodiment. It is a top view of the cell injection device concerning this embodiment. It is a figure which shows the manufacturing process of the arm part 2b and the cylindrical part 2a. It is a figure which shows the manufacturing process of the board | substrate 1. FIG. It is a figure which shows the process flow of an etching flow-path processing method. It is a figure which shows the process flow of the flow-path processing method using the photosensitive film | membrane material. It is a figure which shows the process flow of the flow-path processing method using a non-photosensitive film | membrane material. It is a figure which shows the process flow which forms a fine groove | channel from a metal mold | die to PDMS. It is a figure which shows the process flow which forms a supply path and an introductory path from a metal mold | die to resin.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Board | substrate 1a Supply path 1b Introduction path 2 Drive apparatus 2a Cylinder part 2b Arm part 2c Actuator 3 Microscope 4 CCD camera CL Cell EM Etching mask GS Glass GS Glass substrate M Mold MK Scale MM Metal thin film MP Micro pipette OR Photosensitive resin OR Photocurable resin PL Resin PR Photoresist PR Film material RS Resist S Substrate

Claims (7)

A housing that forms a supply path through which cells move and an introduction path that intersects the supply path;
A stopper member movable between a first position entering the supply path and a second position retracted from the supply path;
Drive means for driving the stopper member between the first position and the second position;
The cell injection device, wherein the cells captured by the stopper member moved to the first position in the supply path are inserted by a micropipette inserted through the introduction path.   2. The housing according to claim 1, wherein the housing is formed by depositing platinum on a glass substrate, applying a resist, patterning the supply path and the introduction path, and then etching. Cell injection device.   2. The stopper member according to claim 1, wherein the stopper member is formed by depositing a photo-curing resin or resist on a glass substrate, depositing a semitransparent metal thin film thereon, and etching while leaving a part. The cell injection device described.   In the supply path and the introduction path, an etching mask and a resist are formed in this order on the material of the housing, and then a part of the resist is removed by exposure and development, and the resist and the etching are further etched. The cell injection device according to claim 1, wherein the cell injection device is formed by removing the mask.   The supply path and the introduction path are formed by forming a film material, an etching mask, and a resist in this order on the material of the housing, and then removing a part of the resist by exposure and development, and further performing etching processing to remove the resist and the resist The cell injection device according to claim 1, wherein the cell injection device is formed by removing the etching mask and a part of the film material.   2. The cell according to claim 1, wherein the supply path and the introduction path are formed by removing a part of the resist by exposure and development after forming a resist on the material of the housing. Injection device.   The cell injection apparatus according to claim 1, wherein the supply path and the introduction path are formed using a mold.

Images