JP4953357B2 - Cell invasion probe, cell invasion device having the cell invasion probe, and detailed invasion method - Google Patents

Description

  In the fields related to life science field, regenerative medicine, and the like, the present invention provides cells for introducing various solutions into cells, introducing substances such as sperm and eggs, and sucking contents in cells. The present invention relates to a cell invasion probe that invades inside, a cell invasion device having the cell invasion probe, and a cell invasion method.

In recent years, in the fields of life science, regenerative medicine, genomic drug discovery, etc., it has been practiced to introduce genes and various drugs into cells to modify the properties of the cells. This is because the gene function is analyzed by observing protein expression and cell dynamics (changes in cell size, shape, cell movement, etc.).
Also, in the field of cell biology, research has been conducted to examine the individuality of each cell, and in order to observe changes in gene expression in a single cell, Techniques for extracting and analyzing quantities of biomolecules are indispensable.
As described above, in recent years, various solutions have been introduced into individual cells, substances such as sperm and ovum have been introduced, and contents such as biomolecules have been aspirated and collected from within cells. It is done frequently.

  By the way, various methods are provided as introduction methods into cells. For example, an introduction method using electrical stimulation, an introduction method using a difference in electric charge, an introduction method using a liposome, and the like are known. However, these methods generally cannot target a single cell and statistically process the introduction of multiple cells. Specifically, the cell membrane (lipid bimolecular membrane) is temporarily destroyed by electrical stimulation, pH adjustment, or the like to promote introduction into the cell membrane. Therefore, it was not possible to meet the needs described above.

On the other hand, as an introduction method for a single cell, a method is known in which a microneedle such as a glass capillary is inserted into a desired cell to introduce substances such as various solutions and eggs (for example, Patent Document 1). reference). According to this method, since introduction can be performed for only one cell, it is a method that can meet the above-described needs.
JP 2006-34174 A

However, the following problems remain in the conventional method.
That is, in the method using a microneedle, the microneedle is invaded (stabbed) into a cell to introduce various solutions and substances, or to collect a substance such as a biomolecule from the inside of the cell. In this method, as shown in FIG. 13, when the microneedle 30 is inserted into the cell S, the cell S is recessed by its own softness (elasticity) before the hole is opened, and the microneedle 30 is surely connected to the cell. It was difficult to determine whether or not it entered the S.

  Even if the invasion into the cell S is possible, the microneedle 30 that is not sufficiently small with respect to the cell S physically penetrates the cell membrane S1 and invades inside as shown in FIG. There was a possibility of destroying S itself. In addition, various proteins present in the cell membrane S1 may be mixed into the cell S together with the microneedles 30. Further, when the microneedle 30 is pulled up from the cell S, there is a possibility that a part of the cell membrane S1 is taken away from the cell S as shown in FIG. This is because when the microneedle 30 is pulled out from the cell S, a part of the cell membrane S1 attached to the microneedle 30 is pulled out together with the microneedle 30.

  As described above, in the conventional method, various proteins (membrane proteins such as G protein) and sugar chains existing on the surface of the cell membrane S1 are physically destroyed or pulled out together with the microneedles 30. There was a fear. Therefore, there has been a problem that cell division expected after cell S is introduced, protein synthesis inside cell S, and the like are inhibited. This phenomenon was particularly remarkable in mammals and the like.

  On the other hand, the need to observe substances contained in the cells S has been increasing in recent years. In general high-resolution observation, a technique using an optical microscope is representative. However, although the resolution is high, the resolution is still limited, and it is difficult to accurately observe the inside of the cell S. Therefore, as an alternative to the optical microscope, after the cells S are dried, high-resolution observation is performed with a replica method or the like using a SEM (Scanning Electron Microscope), or the cells S are frozen and then observed with the SEM. The technique of performing etc. has been used.

However, since these methods are methods for observation after the cells S are once killed, accurate observation cannot be performed. In addition, it was not possible to observe over time due to the state of being alive.
Although single-molecule fluorescence observation in which a fluorescent substance is introduced into the cell S is also conceivable, this method is an observation method by tracking the fluorescence of a single molecule or a group thereof, and morphological knowledge is obtained. It wasn't something that could be done.

  Therefore, instead of the micro needle 30, a method of invading a probe of a scanning probe microscope having a probe at the tip into the cell S and observing using the probe can be considered. However, even in this case, the cell S may be killed when the probe is invaded. Even when the cell S does not die, as shown in FIG. 16, when the probe 31 at the tip of the probe is scanned, the probe 31 damages the cell membrane S1, and the probe from the cell membrane S1. Since the needle 31 receives minute resistance, accurate observation could not be performed.

  The present invention has been made in consideration of such circumstances, and its purpose is to be able to invade cells without killing them while suppressing the influence on the cell membrane as much as possible. It is an object to provide a cell invasion probe that can be accurately observed in a living state, a cell invasion device having the cell invasion probe, and a cell invasion method.

In order to achieve the above object, the present invention provides the following means.
The probe for cell invasion according to the present invention is disposed opposite to cells cultured on a substrate in a liquid and is relatively movable in an XY direction parallel to the substrate surface and a Z direction orthogonal to the XY direction. A probe for cell invasion, which is formed on the outer surface of the probe, the probe having a sharp tip, punctured into the cell, a lever portion that supports the probe in a cantilever state, and the probe A lipid bilayer membrane, and when the probe is punctured into the cell, the lipid bilayer membrane is fused and integrated with the cell membrane on the cell surface.

In the cell invasion probe according to the present invention, a lipid bilayer membrane having the same configuration as the cell membrane on the cell surface is formed on the outer surface of the probe supported by the lever portion. In other words, a single amphiphilic molecule having both hydrophilic and hydrophobic properties is arranged into two molecules with the hydrophobic portions inside, and a plurality of these two molecules are present on the outer surface of the probe. As a result, a lipid bilayer is formed.
Therefore, when the probe and the cell are relatively moved in the XY direction and the Z direction and the probe is brought close to the cell, the lipid bilayer film formed on the outer surface of the probe first comes into contact with the cell membrane. . Then, the cell membrane begins to fuse with the lipid bilayer membrane having the same configuration as the cell membrane, and the cell membrane and the lipid bilayer membrane gradually begin to integrate. That is, the cell membrane is pulled by the lipid bilayer membrane and starts moving to the outer surface of the probe, and the lipid bilayer membrane is pulled by the cell membrane and starts moving to the cell membrane. In this way, the cell membrane and the lipid bilayer membrane move and fuse with each other. Thereby, as the probe is pushed into the cell, the cell membrane and the lipid bilayer can be pushed away onto the outer surface of the probe.

  Therefore, only the probe portion can be punctured into the cell without physically breaking through the cell membrane. Therefore, unlike the conventional one, the influence on the cell membrane can be suppressed as much as possible, and the probe can be placed in the cell and invaded without killing the cell in culture. In particular, instead of physically puncturing the cells as in the past, the lipid bilayer membrane and the cell membrane can be fused and integrated, so when puncturing the probe, the cells will dent and escape from the probe There is nothing. Also from this point, it is possible to reliably invade without affecting the cells such as stimulation. In addition, the cell membrane is not mixed into the cell as in the prior art.

  As described above, according to this cell invasion probe, since it can invade into the cell without being killed while suppressing the influence on the cell membrane as much as possible, various probes can be used without affecting the various proteins present on the cell membrane. It is possible to make it possible to perform a measurement that has been difficult with the prior art.

Further, even when the probe is pulled out from the cell, there is no fear that the cell membrane is pulled out as in the conventional case. In other words, the lipid bilayer membrane formed on the outer surface of the probe is already integrated with the cell membrane, so that the lipid bilayer membrane is pulled by the cell membrane and hardly moves with the probe. It has become. Therefore, when the probe is pulled out, the lipid bilayer membrane is easily pulled by the cell membrane and remains on the cell side, and only the probe can be pulled out mainly. The control of the film residue on the probe side can be performed by an operation such as changing the pulling speed.
As a result, the probe can be extracted from the cell without extracting the cell membrane. Therefore, even after the probe is pulled out, the cells are not killed. Further, as the probe is pulled out, the cell membrane gradually begins to return to its original state, so that the cell surface can be covered again. A part of the lipid bilayer membrane may remain on the cell side, but in this case, it becomes a cell membrane as it is.

  The probe for cell invasion according to the present invention is characterized in that, in the probe for cell invasion according to the present invention, the outer surface of the probe is subjected to a hydrophilic treatment.

  In the probe for cell invasion according to the present invention, the outer surface of the probe is hydrophilized and the wettability is improved, so that the adhesion between the lipid bilayer membrane and the outer surface can be improved. Therefore, the lipid bilayer membrane is not peeled off in the middle, and the reliability of the probe can be improved.

  Moreover, the probe for cell invasion according to the present invention is the above probe for cell invasion according to the present invention, wherein the probe has a tube having an opening at the distal end extending from the distal end side to the proximal end side. It is characterized by.

  In the probe for cell invasion according to the present invention, after invading the probe into the cell, a predetermined substance, for example, a drug solution, a DNA solution, a sperm, an egg, or the like is introduced into the cell via the conduit, The contents, that is, various substances constituting the cell can be aspirated from inside the cell. As described above, by using the pipe line, various types of cell measurement (for example, electrical stimulation reaction measurement and ion measurement by introducing a conductive fluid) can be easily and reliably performed. Analysis can be performed. In particular, since the analysis can be performed without killing the cells, an accurate analysis can be performed.

  Further, the cell invasion probe according to the present invention is the specific substance to be introduced into the cell between the tip of the probe and the lipid bilayer in the cell invasion probe of the present invention. Is previously supported.

  In the probe for cell invasion according to the present invention, since the specific substance is previously supported between the tip of the probe and the lipid bilayer, the probe is reliably and promptly invaded after invading the cell into the cell. This specific substance can be introduced into cells. Therefore, it is possible to perform further multilateral analyzes such as observing cell reactions. In particular, since the analysis can be performed without killing the cells, an accurate analysis can be performed. In addition, it is easy to use because a specific substance can be introduced immediately by invading the probe into the cell.

  Further, the cell invasion probe according to the present invention is characterized in that, in the cell invasion probe of the present invention, the lipid bilayer membrane is a membrane composed of an amphipathic molecule of phospholipid or glycolipid. It is what.

  In the probe for cell invasion according to the present invention, the lipid bilayer membrane is a membrane composed of phospholipid (sphingophospholipid, glycerophospholipid) or an amphipathic molecule of glycolipid (sphingoglycolipid, glyceroglycolipid). ing. Specifically, it is a membrane composed of amphipathic molecules such as phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, sphingomyelin, cholesterol, galactocerebroside, GM1 ganglioside, sialic acid (NANA).

  Further, the cell invasive device according to the present invention is the probe according to any one of the cell invasive probes according to the present invention, the cell invasive probe and the substrate which are relatively moved in the XY direction and the Z direction. A moving means for puncturing the cell and an observation means for observing the cell.

  In the cell invasion device according to the present invention, the probe is securely invaded into the targeted cell by appropriately moving the cell invasion probe and the substrate in the XY direction and the Z direction by the moving means. can do. At this time, since the cells can be observed by the observation means, the invasion can be performed more accurately. In addition, it is possible to perform accurate analysis by observing the kinetics of cells after invasion in a living state.

  The cell invasive device according to the present invention is characterized in that in the cell invasive device of the present invention described above, there is provided an environment adjusting means for adjusting the surrounding environment of the cell to a predetermined environmental condition.

  In the cell invasive device according to the present invention, the environment surrounding the cells being cultured, for example, temperature, humidity, atmospheric pressure, carbon dioxide concentration, etc. can be adjusted to predetermined environmental conditions by the environment adjusting means. Therefore, the environment can be adjusted to an optimum state according to the type of cell. Therefore, the cells can be observed more accurately, and can be analyzed by long-term observation.

  The cell invasive device according to the present invention is characterized in that in the cell invasive device of the present invention described above, a displacement measuring means for measuring the deflection of the lever portion is provided.

  In the cell invasion device according to the present invention, since the deflection of the lever portion can be measured by the displacement measuring means, it is possible to more accurately determine whether or not the cell is invaded or pulled out. In addition, AFM observation can be performed with the probe invaded into the cell. Therefore, various cellular substances contained in the cells can be observed with high resolution and high accuracy in a living state. In particular, since the lipid bilayer membrane and the cell membrane are fused, even if the probe is scanned, the cell membrane is not damaged as in the prior art.

  The cell invasion method according to the present invention is a cell invasion method in which a probe that is supported in a cantilever state by a lever portion and has a sharpened tip is punctured into a cell cultured on a substrate in a liquid. A lipid film forming step of forming a lipid bilayer on the outer surface of the probe, and a moving step of moving the probe over the cell while observing the cell after the lipid film forming step, A puncturing step in which the probe and the cell are brought close to each other after the moving step to puncture the cell with the probe, and when the probe is punctured into the cell, the lipid bilayer membrane is It is characterized by being fused and integrated into the cell membrane.

  In the cell invasion method according to the present invention, first, a lipid bilayer membrane having the same configuration as the cell membrane on the cell surface is formed on the outer surface of the probe supported in a cantilever manner on the lever portion by a lipid membrane formation step. . As a formation method, for example, the tip is immersed in an aqueous solution in which a lipid membrane is spread in advance, and then slowly lifted to attach the lipid bilayer to the outer surface of the probe. In other words, a single amphiphilic molecule having both hydrophilic and hydrophobic properties is arranged in a state where the hydrophobic parts are inside each other to form two molecules, and a plurality of these two molecules are formed to form a lipid. A bilayer is formed.

  Next, a moving step of moving the probe onto the target cell is performed while observing the cultured cell. At this time, since the cell is observed, the probe can be accurately positioned on the target cell.

  Next, a puncturing step is performed in which the probe and the cell are brought close to each other from this state to puncture the cell with the probe. In carrying out this puncturing step, when the probe and the cell are brought close to each other, the lipid bilayer membrane formed on the outer surface of the probe first comes into contact with the cell membrane. Then, the cell membrane begins to fuse with the lipid bilayer membrane having the same configuration as the cell membrane, and the cell membrane and the lipid bilayer membrane gradually begin to integrate. That is, the cell membrane is pulled by the lipid bilayer membrane and starts moving to the outer surface of the probe, and the lipid bilayer membrane is pulled by the cell membrane and starts moving to the cell membrane. In this way, the cell membrane and the lipid bilayer membrane move and fuse with each other. Thereby, as the probe is pushed into the cell, the cell membrane and the lipid bilayer can be pushed away onto the outer surface of the probe.

  Therefore, only the probe portion can be punctured into the cell without physically breaking through the cell membrane. Therefore, unlike the conventional one, the influence on the cell membrane can be suppressed as much as possible, and the probe can be placed in the cell and invaded without killing the cell in culture. In particular, instead of physically puncturing the cells as in the past, the lipid bilayer membrane and the cell membrane can be fused and integrated, so when puncturing the probe, the cells will dent and escape from the probe There is nothing. Also from this point, it is possible to reliably invade without affecting the cells such as stimulation. In addition, the cell membrane is not mixed into the cell as in the prior art.

  As described above, according to this cell invasion method, it is possible to invade into the cell without killing it while suppressing the influence on the cell membrane as much as possible. Therefore, without affecting the various proteins existing on the cell membrane, Measurement can be performed, which was difficult in the prior art.

Further, even when the probe is pulled out from the cell, there is no fear that the cell membrane is pulled out as in the conventional case. In other words, the lipid bilayer membrane formed on the outer surface of the probe is already integrated with the cell membrane, so that the lipid bilayer membrane is pulled by the cell membrane and hardly moves with the probe. It has become. Therefore, when the probe is pulled out, the lipid bilayer membrane is easily pulled by the cell membrane and remains on the cell side, and only the probe can be pulled out mainly. The control of the film residue on the probe side can be performed by an operation such as changing the pulling speed.
As a result, the probe can be extracted from the cell without extracting the cell membrane. Therefore, even after the probe is pulled out, the cells are not killed. Further, as the probe is pulled out, the cell membrane gradually begins to return to its original state, so that the cell surface can be covered again. A part of the lipid bilayer membrane may remain on the cell side, but in this case, it becomes a cell membrane as it is.

  The cell invasion method according to the present invention is characterized in that, in the cell invasion method of the present invention, a hydrophilization step of hydrophilizing the outer surface of the probe is performed before the lipid film formation step. Is.

  In the cell invasion method according to the present invention, the outer surface of the probe is hydrophilized by the hydrophilization step to improve the wettability, so that the adhesion between the lipid bilayer membrane and the outer surface can be improved. . Therefore, the lipid bilayer membrane is not peeled off in the middle, and the reliability of the probe can be improved.

  In the cell invasion method according to the present invention, in the cell invasion method according to the present invention, a tube having an opening at a distal end is formed on the probe from the distal end side to the proximal end side. After the step, an introduction suction step of introducing a predetermined substance into the cell using the pipe line or sucking the contents from the cell is provided.

  In the cell invasion method according to the present invention, after infiltrating the probe into the cell, an introduction and suction step is performed, so that a predetermined substance such as a drug solution, a DNA solution, sperm, An egg or the like can be introduced, or the contents, that is, various substances constituting the cell can be aspirated from inside the cell. As described above, by performing the introduction and suction step, it is possible to easily and surely perform a wide variety of cell measurements (for example, electrical stimulation reaction measurement or ion measurement by introducing a conductive fluid), and more versatile. Analysis can be performed. In particular, since the analysis can be performed without killing the cells, an accurate analysis can be performed.

  Further, the cell invasion method according to the present invention is the cell invasion method according to any one of the above-described present invention, wherein the specific substance to be introduced into the cell is supported in advance at the tip of the probe before the lipid film forming step. A carrying step is performed, and the specific substance is introduced into the cell during the puncturing step.

  In the cell invasion method according to the present invention, since the specific substance is supported in advance on the tip of the probe by the supporting process, the specific substance can be reliably and promptly introduced into the cell during the puncturing process. Therefore, it is possible to perform further multilateral analyzes such as observing cell reactions. In particular, since the analysis can be performed without killing the cells, an accurate analysis can be performed. In addition, it is easy to use because a specific substance can be introduced immediately by invading the probe into the cell.

  Further, the cell invasion method according to the present invention includes the observation step of performing AFM observation of the inside of the cell by scanning the probe inside the cell after the puncturing step in the cell invasion method according to any one of the present invention. It is characterized by that.

  In the cell invasion method according to the present invention, various types of cellular substances contained in the cells are made alive by performing an observation process of scanning the inside of the cells with a probe invaded into the cells and observing the inside of the cells with AFM. It can be observed with high resolution and high accuracy as it is. In particular, since the lipid bilayer membrane and the cell membrane are fused, the cell membrane escapes on the outer surface even when the probe is scanned. Therefore, the cell membrane is not damaged as in the prior art.

  The cell invasion method according to the present invention is characterized in that, in any of the above cell invasion methods of the present invention, an environment adjustment step for adjusting the surrounding environment of the cell to a predetermined environmental condition is provided. is there.

  In the cell invasion method according to the present invention, it is possible to adjust the surrounding environment of the cultured cells, for example, temperature, humidity, atmospheric pressure, carbon dioxide concentration, etc., to predetermined environmental conditions by the environmental adjustment step. Therefore, the environment can be adjusted to an optimum state according to the type of cell. Therefore, the cells can be observed more accurately, and can be analyzed by long-term observation.

According to the cell invasion probe and the cell invasion method according to the present invention, it is possible to invade into a cell without killing it while suppressing the influence on the cell membrane as much as possible.
Moreover, according to the cell invasive device according to the present invention, since the cell invasive probe is provided, it is possible to perform accurate analysis by observing the kinetics of the cell as it is.

Hereinafter, an embodiment of a cell invasion probe, a cell invasion device, and a cell invasion method according to the present invention will be described with reference to FIGS.
As shown in FIG. 1, the cell invasion device 1 of the present embodiment observes a cell (substrate) 2 for culturing the cell S in a liquid, a scanning probe microscope 4 having a cell invasion probe 3, and the cell S. This is an apparatus that combines an optical apparatus (observation means) 6 having an optical microscope 5.

  The scanning probe microscope 4 includes a cell invasion probe 3 disposed opposite to a cell S, and the cell invasion probe 3 and the cell 2 in an XY direction parallel to the bottom surface (substrate surface) of the cell 2 and these XY directions. A moving means 7 for puncturing the cell S with the probe 3a of the cell invasion probe 3 by relative movement in the Z direction perpendicular to the direction, and a displacement measuring means 8 for measuring the deflection of the lever portion 3b of the cell invasion probe 3. And a control unit 9 that comprehensively controls these components.

  The cell 2 is formed of an optically transparent material into a U-shaped cross section with an upper opening, and is held on the stage 10. Inside the cell 2, a medium (a culture solution containing elements for allowing the cells S to function normally) W is stored, and a plurality of cells S are cultured on the bottom surface. The cell 2 incorporates a heater, a carbon dioxide supply unit, and the like (not shown). For example, the temperature of the medium W is adjusted to 37 ± 0.5 ° C. and the carbon dioxide concentration is 5%. Yes. The cell 2 is housed in a sealed container 11. The container 11 has a vacuum pump 12 that adjusts the internal pressure to an arbitrary pressure (for example, a vacuum state), and a humidity adjusting unit that adjusts humidity by supplying air having a predetermined humidity into the container 11. 13 is connected.

These heater, carbon dioxide supply unit, vacuum pump 12 and humidity adjustment unit 13 can adjust the surrounding environment of the cells S in the container 11 to predetermined environmental conditions suitable for culture. That is, the container 11, the heater, the carbon dioxide supply unit, the vacuum pump 12, and the humidity adjustment unit 13 constitute an environment adjustment unit 14.
In addition, the culture medium W circulates in the cell 2 via a pipe line (not shown), and a new culture medium W is always supplied.

  The optical microscope 5 is arranged below the stage 10 so that the culture state of the plurality of cells S can be observed from the lower side of the cell 2 through the opening 10 a formed at the approximate center of the stage 10. Further, the optical microscope 5 outputs the observed image to a monitor 15 connected to the control unit 9. As a result, the operator can specify the position of the cell S with the operation unit while viewing the image displayed on the monitor 15. That is, the optical microscope 5 and the monitor 15 constitute the optical device 6. The monitor 15 is arranged outside the container 11.

The cell invasion probe 3 has a tip 3 which is sharpened and punctured into the cell S, and a lever portion 3b which supports the probe 3a in a cantilever state with the base end supported by the main body. And a lipid bilayer membrane 3c formed on the outer surface of the probe 3a.
As shown in FIG. 2, the lipid bilayer membrane 3c is formed on the outer surface of the probe 3a so as to enclose the probe 3a. This lipid bilayer membrane 3c is a bilayer in which a single amphiphilic molecule having both hydrophilic and hydrophobic properties is arranged with the hydrophobic portion P1 on the inside and the hydrophilic portion P2 on the outside. A plurality of these two molecules are formed on the outer surface of the probe 3a to form a film.

  The lipid bilayer membrane 3c of the present embodiment is a lipid membrane made of amphiphilic molecules of phospholipid (sphingophospholipid, glycerophospholipid) or glycolipid (sphingoglycolipid, glyceroglycolipid). Specifically, it is a membrane composed of amphipathic molecules such as phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, sphingomyelin, cholesterol, galactocerebroside, GM1 ganglioside, sialic acid (NANA).

  Here, the phospholipid will be described in more detail. This phospholipid is a lipid having phosphoric acid in the hydrophilic part, and phosphoric acid is ester-bonded at one of the inner ends of the three hydroxyl groups of glycerin, and the remaining fatty acid is an ester bond. It is a thing. A fatty acid is a hydrophobic molecule having a carboxylic acid at the end of a saturated or unsaturated carbon chain having 12 to 18 carbon atoms. Examples of the fatty acid include lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, arachidonic acid, linoleic acid, linolenic acid, and the like.

  The lipid bilayer membrane 3c described above is a lipid membrane having the same configuration as the cell membrane S1 covering the surface of the cell S as shown in FIG. Therefore, when the probe 3a is punctured into the cell S, the lipid bilayer membrane 3c is fused and integrated with the cell membrane S1. This will be described in detail later.

  As shown in FIG. 1, the cell invasion probe 3 is detachably fixed to a slope block 17 fixed to the lower surface of the XYZ scanner 16 through a main body 3d by a wire or the like. At this time, the cell invasion probe 3 is fixed by the inclined block 17 so that the lever portion 3b is inclined at a predetermined angle with respect to the bottom surface of the cell 2. The position of the probe 3 for cell invasion is adjusted so that at least the probe 3a is immersed in the medium W.

  The XYZ scanner 16 is a piezoelectric element made of PZT (lead zirconate titanate) or the like, and is provided in the container 11. Then, when a voltage is applied to the XYZ scanner 16 from the drive circuit 18 disposed outside the container 11, the XYZ scanner 16 is in the XY direction (direction parallel to the bottom surface of the cell 2) according to the voltage application amount and polarity. ) And the Z direction (direction perpendicular to the bottom surface of the cell 2). Thereby, the cell invasion probe 3 can be moved in the XY direction and the Z direction. That is, the XYZ scanner 16 and the drive circuit 18 constitute the moving means 7.

  In this embodiment, the cell invasion probe 3 is moved in the XYZ directions. However, the present invention is not limited to this, and the stage 10 side may be moved in the XYZ directions. Even in this case, the same operational effects as those described below can be obtained only by different scanning methods. The cell invasion probe 3 side and the stage 10 side may be configured to move in the XYZ directions.

Further, above the cell invasion probe 3 and outside the container 11, a semiconductor laser light source 20 for irradiating the laser light L and a laser for receiving the laser light L reflected by a reflection surface (not shown) of the lever portion 3b. The light receiving unit 21 is attached to a gantry (not shown). The laser light receiving unit 21 is, for example, a four-divided photodetector, and detects a change in the bending of the lever unit 3b based on the incident position of the laser light L. The laser light receiving unit 21 converts the detected change in bending of the lever portion 3b into a bending signal, and then outputs it to the control unit 9. That is, the semiconductor laser light source 20 and the laser light receiving unit 21 constitute the displacement measuring means 8.
The laser light L enters the container 11 or exits from the container 11 through a window 22 attached to the upper part of the container 11.

  The control unit 9 operates the drive circuit 18 in accordance with the deflection signal sent from the laser light receiving unit 21. The control unit 9 is connected to an operation unit 9a for an operator to input various conditions and operate the drive circuit 18 and the like. Thus, the operator can appropriately move the XYZ scanner 16 in the three-dimensional direction via the operation unit 9a.

Next, a cell invasion method in which the probe 3a is invaded into the cell S and the inside of the cell S is observed using the cell invasion apparatus 1 configured as described above will be described.
The cell invasion method of the present embodiment includes a lipid film forming step of forming a lipid bilayer 3c on the outer surface of the probe 3a, and a probe on the cell S while observing the cell S after the lipid film forming step. A moving step of moving 3a, a puncturing step in which the probe 3a and the cell S are brought close to each other after the moving step, and the probe 3a is inserted into the cell S after the puncturing step. And an observation step of scanning and AFM observation of the inside of the cell S. Moreover, in this embodiment, the environmental adjustment process of adjusting the circumference | surroundings of the cell S to the predetermined environmental conditions suitable for culture | cultivation is also performed simultaneously. Hereinafter, each of these steps will be described in detail.

First, a lipid bilayer membrane 3c having the same configuration as the cell membrane S1 is formed on the outer surface of the probe 3a by a lipid membrane formation step. As a formation method at this time, for example, the lipid bimolecular film 3c can be formed on the outer surface of the probe 3a by immersing the probe 3a in an aqueous solution in which a lipid membrane is spread in advance and then slowly pulling it up. .
Then, after forming the lipid bilayer membrane 3 c, the cell invasion probe 3 is set on the slope block 17 and the cell 2 is set on the stage 10. Subsequently, an environmental adjustment process is performed in which the heater, the carbon dioxide supply unit, the vacuum pump 12 and the humidity adjustment unit 13 are appropriately operated to adjust the environmental conditions to the optimum conditions for culture. Accordingly, long-term cell S culture can also be performed.

  Next, the culture state of the plurality of cells S cultured in the cell 2 is observed with the optical microscope 5 from the lower surface side of the cell 2. Then, the operator identifies the cell S that invades the probe 3 a while confirming the observed image on the monitor 15. Subsequently, after the position of the cell S is specified, a moving step of moving the probe 3a onto the cell S is performed. That is, the probe 3a is positioned on the specified cell S by appropriately moving the XYZ scanner 16 in the XYZ directions. At this time, since the image captured by the optical microscope 5 is confirmed on the monitor 15, the probe 3 a can be accurately positioned on the specified cell S.

  Next, from this state, the XYZ scanner 16 is slightly moved in the Z direction to bring the probe 3a and the cell S closer to each other, and a puncturing step is performed to puncture the cell S with the probe 3a. In performing this puncturing step, when the probe 3a and the cell S are brought closer to each other, as shown in FIG. 4, first, the lipid bilayer membrane 3c formed on the outer surface of the probe 3a contacts the cell membrane S1. . Then, the cell membrane S1 begins to fuse with the lipid bilayer membrane 3c having the same configuration as the cell membrane S1, and the cell membrane S1 and the lipid bilayer membrane 3c begin to gradually integrate. That is, the cell membrane S1 is pulled by the lipid bilayer membrane 3c and starts moving to the outer surface of the probe 3a, and the lipid bilayer membrane 3c is pulled by the cell membrane S1 and starts moving to the cell membrane S1. In this way, the cell membrane S1 and the lipid bilayer membrane 3c move and fuse with each other. Thereby, as shown in FIGS. 5 and 6, as the probe 3a is pushed into the cell S, the cell membrane S1 and the lipid bilayer membrane 3c can be pushed out onto the outer surface of the probe 3a.

As a result, as shown in FIG. 7, only the probe 3a portion can be punctured into the cell S without physically breaking through the cell membrane S1. Therefore, unlike the conventional one, the influence on the cell membrane S1 can be suppressed as much as possible, and the probe 3a can be placed in the cell S and invaded without killing the cell S in culture.
In particular, instead of physically puncturing the cell S as in the prior art, the lipid bilayer membrane 3c and the cell membrane S1 can be fused and integrated. Therefore, when the probe 3a is punctured, the cell S is recessed and probed. There is no escape from the needle 3a. Also from this point, the cell S can be reliably invaded without affecting the cell S. Moreover, the cell membrane S1 is not mixed into the cell S as in the prior art.

Thus, according to the cell invasion device 1 and the cell invasion method of the present embodiment, since it can enter the cell S without being killed while suppressing the influence on the cell membrane S1 as much as possible, it affects various proteins present in the cell membrane S1. It is possible to make it possible to perform various types of measurement with the probe 3a without providing the above, which was difficult in the prior art.
Moreover, since the cell S can be observed with the optical microscope 5, the dynamics of the cell S after the invasion can be observed as it is, and an accurate analysis can be performed.
In particular, in this embodiment, since the surrounding environment of the cell S being cultured can be adjusted to the optimum environmental conditions suitable for the culture, the cell S can be observed more accurately and a long-term observation can be performed. Can be analyzed.

Further, whether or not the probe 3a has surely invaded the cell S can be observed with the optical microscope 5, but in this embodiment, the scanning probe microscope 4 having the displacement measuring means 8 is provided. Therefore, it is possible to determine whether or not the invasion is possible with higher accuracy.
That is, when the lipid bilayer 3c and the cell membrane S1 are brought into contact with each other by bringing the probe 3a and the cell S close to each other, the lever portion 3b starts to bend due to fusion of the lipid bilayer 3c and the cell membrane S1. Therefore, the position of the laser beam L (laser beam L reflected by the reflecting surface) incident on the laser receiving unit 21 changes. The laser receiving unit 21 outputs a deflection signal corresponding to this change to the control unit 9. In this way, the control unit 9 can immediately know the displacement of the lever portion 3b that occurs in the process of puncturing the probe 3a. And the control part 9 can judge with high precision whether it invaded in the cell S as mentioned above by comparing this deflection | deviation signal and the predetermined value defined beforehand.

  In addition, after the puncturing process is completed, the XYZ scanner 16 scans the probe 3a invaded into the cell S in the XY direction, and performs an observation process of AFM observation in the cell S. As a result, various substances that are the contents in the cell S, such as pores of the cell nucleus, microtubules, actin filaments, and the like can be observed with high resolution and high accuracy while they are alive. In particular, since the lipid bilayer membrane 3c and the cell membrane S1 are fused, even when the probe 3a is scanned, the cell membrane S1 escapes onto the outer surface of the probe 3a as the probe 3a moves. Therefore, the cell membrane S1 is not damaged unlike the conventional case.

Even when the probe 3a is pulled out from the cell S, there is no possibility that the cell membrane S1 is pulled out as in the prior art. That is, since the lipid bilayer membrane 3c formed on the outer surface of the probe 3a is already united with the cell membrane S1, the lipid bilayer membrane 3c is pulled by the cell membrane S1 and thus the probe 3a. It is difficult to move with. Therefore, when the probe 3a is pulled out, the lipid bilayer membrane 3c is easily pulled by the cell membrane S1 and remains on the cell S side, and only the probe 3a can be pulled out mainly. The control of the remaining film on the probe 3a side can be performed by an operation such as changing the pulling speed of the probe 3a.
As a result, the probe 3a can be extracted from the cell S without extracting the cell membrane S1, and therefore the cell S is not killed even after the probe 3a is extracted. Further, as the probe 3a is pulled out, the cell membrane S1 begins to gradually return to the original state, so that the surface of the cell S can be covered again. A part of the lipid bilayer membrane 3c may remain on the cell S side, but in this case, it becomes the cell membrane S1 as it is.

In the above embodiment, a hydrophilization step of hydrophilizing the outer surface of the probe 3a may be performed before the lipid film formation step.
Specifically, it is made hydrophilic by irradiating plasma on the outer surface of the probe 3a, or made hydrophilic by surface modification by UV ozone environment, or an extremely thin SiO ₂ film on the outer surface of the probe 3a. And is hydrophilized by utilizing the moisturizing effect due to the fine cracks present in the SiO ₂ film. Thus, by performing various hydrophilic treatments, the wettability of the outer surface can be improved, and the adhesion between the lipid bilayer 3c and the outer surface of the probe 3a can be improved. Therefore, the lipid bilayer membrane 3c is not peeled off in the middle, and the reliability of the cell invasion probe 3 can be improved.

In the above-described embodiment, a supporting step of supporting the specific substance N to be introduced into the cell S at the tip of the probe 3a in advance may be performed before the lipid film forming step. By performing this step, as shown in FIG. 8, some specific substance N (eg, egg) can be supported between the tip of the probe 3a and the lipid bilayer 3c.
In this way, when the probe 3a is invaded into the cell S by the puncturing step, the supported specific substance N can be reliably and promptly introduced into the cell S as shown in FIG. Therefore, further multilateral analysis can be performed such as observing the reaction of the cells S. In particular, since analysis can be performed without killing the cells S, accurate analysis can be performed. Further, since the specific substance N can be immediately put in by simply invading the probe 3a into the cell S, it is easy to use.

Moreover, in the said embodiment, as shown in FIG. 10, you may form the pipe line 3e which has opening at the front-end | tip of the probe 3a from a front end side to a base end side. By using such a cell invasion probe 3, it is possible to easily and reliably measure a wider variety of cells S. That is, after the puncturing step, an introduction suction step of introducing a predetermined substance into the cell S using the conduit 3e or sucking the contents from the cell S can be performed.
Specifically, as shown in FIG. 11, various substances such as a chemical solution, a DNA solution, sperm, and an egg can be easily and reliably introduced into the cell S through the conduit 3e. In addition, as shown in FIG. 12, various substances as contents can be sucked from the cell S through the conduit 3e. By performing the introduction and suction step in this manner, various types of cell S measurements (for example, electrical stimulation reaction measurement by introducing a conductive fluid, ion measurement, etc.) can be easily and reliably performed. Analysis can be performed. In particular, since analysis can be performed without killing the cells S, accurate analysis can be performed.

  The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

  For example, in the above-described embodiment, the displacement measuring unit 8 measures the deflection of the lever portion 3b by the optical lever method using the laser light L, but is not limited to the optical lever method. For example, you may comprise so that the bending of the lever part 3b may be measured by the self-detection system which provided the displacement detection mechanism (for example, piezoresistive element etc.) in the lever part 3b itself.

Moreover, although the case where the optical microscope 5 was used was demonstrated in the said embodiment, as long as the confirmation of the cell S and the alignment of the probe 3a can be performed, what kind of observation means may be used. For example, a CCD or the like may be used. However, an inverted biological optical device is preferably used.
Furthermore, in the above embodiment, the case where the scanning probe microscope 4 and the optical device 6 are combined has been described as an example, but the scanning probe microscope 4 is not essential. For example, a combination of a micromanipulator having the cell invasion probe 3 and an optical device may be used. As described above, the cell invasion device can be configured as long as it includes at least the moving means for operating the cell invasion probe 3 to puncture the cell 3 with the probe 3a and the observation means for observing the cell S. .

It is a lineblock diagram showing one embodiment of a cell invasion device concerning the present invention. It is the figure which expanded the front-end | tip of the probe for cell invasion of the cell invasion apparatus shown in FIG. It is an enlarged view of the cell currently cultured with the cell of the cell invasion apparatus shown in FIG. It is process drawing in the case of performing the cell invasion method based on this invention using the cell invasion apparatus shown in FIG. 1, Comprising: A probe and a cell are made to adjoin and the state which made the lipid bilayer membrane and the cell membrane contact is shown. FIG. It is a figure which shows the state which brought the probe and the cell closer from the state shown in FIG. It is a figure which shows the state which brought the probe and the cell closer from the state shown in FIG. It is a figure which shows the state which made the probe approach the cell further from the state shown in FIG. 6, and invaded the probe in the cell. It is a figure which shows the modification of the probe for cell invasion which concerns on this invention, Comprising: It is the front-end | tip enlarged view of the probe for cell invasion with which the specific substance is carry | supported beforehand at the front-end | tip of a probe. It is a figure which shows the state which has made the probe shown in FIG. 8 invade into a cell, and has injected the specific substance in the cell. It is a figure which shows the modification of the probe for cell invasion concerning this invention, Comprising: It is the front-end | tip enlarged view of the probe for cell invasion in which the pipe line which has opening is formed in the front-end | tip of a probe. It is a figure which shows the state which has made the probe shown in FIG. 10 invade into a cell, and has introduce | transduced various substances into the cell through the pipe line. It is a figure which shows the state which is making the probe shown in FIG. 10 invade into a cell, and attracting the contents from the inside of a cell through a pipe line. It is a figure for demonstrating the conventional cell invasion method, Comprising: It is a figure which shows the state which has pressed the microneedle against the cell membrane. It is a figure which shows the state which penetrated the cell membrane from the state shown in FIG. 13, and punctured the microneedle in the cell. It is a figure which shows the state which extracted the microneedle from the inside of a cell from the state shown in FIG. It is a figure for demonstrating the conventional cell invasion method, Comprising: It is a figure which shows the state which is making the probe which penetrated the cell membrane and invaded in the cell scanned.

Explanation of symbols

N specific substance S cell S1 cell membrane 1 cell invasion device 2 cell (substrate)
3 Probe for Cell Invasion 3a Probe 3b Lever Part 3c Lipid Bilayer 3e Pipe 6 Optical Device (Observation Means)
7 Moving means 8 Displacement measuring means 14 Environmental adjusting means

Claims (14)

A cell invasion probe that is disposed opposite to cells cultured on a substrate in a liquid and is relatively movable in an XY direction parallel to the substrate surface and a Z direction orthogonal to the XY direction,
A probe whose tip is sharpened and punctured into the cell;
A lever portion for supporting the probe in a cantilever state;
A lipid bilayer formed on the outer surface of the probe,
A probe for cell invasion, characterized in that, when the probe is punctured into the cell, the lipid bilayer membrane is fused and integrated with the cell membrane on the cell surface. The probe for cell invasion according to claim 1,
A probe for cell invasion, characterized in that the outer surface of the probe is hydrophilized. The probe for cell invasion according to claim 1 or 2,
A probe for cell invasion, characterized in that a pipe line having an opening at the tip is formed in the probe from the tip side to the base end side. The probe for cell invasion according to any one of claims 1 to 3,
A cell-invasive probe characterized in that a specific substance to be introduced into the cell is supported in advance between the tip of the probe and the lipid bilayer. The probe for cell invasion according to any one of claims 1 to 4,
The probe for cell invasion, wherein the lipid bilayer membrane is a membrane composed of amphiphilic molecules of phospholipid or glycolipid. The cell invasion probe according to any one of claims 1 to 5,
Moving means for relatively moving the probe for cell invasion and the substrate in the XY direction and the Z direction to puncture the cell with the probe;
A cell invasion device comprising observation means for observing the cells. The cell invasive device according to claim 6,
A cell invasive device comprising an environment adjusting means for adjusting an environment surrounding the cell to a predetermined environmental condition. The cell invasive device according to claim 6 or 7,
A cell invasion device comprising displacement measuring means for measuring the deflection of the lever portion. A cell invasion method in which a probe that is supported in a cantilevered state by a lever part and has a sharpened tip is punctured into a cell cultured on a substrate in a liquid,
A lipid film forming step of forming a lipid bilayer on the outer surface of the probe;
After the lipid film forming step, a moving step of moving the probe onto the cell while observing the cell;
After the moving step, the probe and the cell are brought close to each other, and a puncturing step for puncturing the cell with the probe is provided.
A cell invasion method characterized in that when the probe is punctured into the cell, the lipid bilayer membrane is fused and integrated with the cell membrane on the cell surface. The cell invasion method according to claim 9,
A cell invasion method comprising performing a hydrophilization step of hydrophilizing the outer surface of the probe before the lipid film formation step. The cell invasion method according to claim 9 or 10,
In the probe, a pipe having an opening at the tip is formed from the tip side to the base side,
A cell invasion method comprising an introduction and aspiration step of introducing a predetermined substance into the cell using the duct or aspirating contents from the cell after the puncturing step. The cell invasion method according to any one of claims 9 to 11,
Prior to the lipid film formation step, a loading step of loading a specific substance to be introduced into the cell at the tip of the probe in advance,
A cell invasion method, wherein the specific substance is introduced into the cell during the puncturing step. The cell invasion method according to any one of claims 9 to 12,
A cell invasion method comprising: an observing step of scanning the inside of the cell with the probe after the puncturing step and observing the inside of the cell with AFM. The cell invasion method according to any one of claims 9 to 13,
A cell invasion method comprising an environment adjustment step of adjusting an ambient environment of the cell to a predetermined environmental condition.

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