JP2006101718A - Cell disintegrator and disintegration method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell disintegrator for analyzing biological materials such as mRNAs, proteins and the like in an extremely small number of cells such as 1 cell and a new technical means for separating the cells in a state in which an extremely small number of molecular functions having activity to the cell can be traced. <P>SOLUTION: The apparatus is equipped with a pipet 11 for holding a solution containing two or more cells, holds droplets 15 containing the cells in a hydrophilic region 3 of a base plate comprising a hydrophobic periphery and the hydrophilic region 3 the size of which is smaller than that of the cells and disintegrate the cells by irradiating focused beams in a wavelength zone which is absorbed by the cells in the droplet. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cell destruction apparatus and a cell destruction method for analyzing biological substances such as mRNA and protein in a very small number of cells such as one cell.

  Regarding the separation of biological materials, for example, liquid chromatography is still the most important separation method for separation of amino acids and sugars, but variations thereof include paper chromatography, foil layer chromatography, column chromatography, etc. In addition to the variations in shape when performing a graph, there are various types of separation units and variations in separation solvents, so it is possible to separate many biochemical substances and chemical substances by optimizing the conditions. is there. Alternatively, a method in which the solution is transported by electroosmotic flow using a similar technique is also used.

  Electrophoresis is generally used to separate charged polymers such as proteins and polynucleotides (DNA and RNA). In electrophoresis, it is possible to identify high-separation, generally up to about 2% of the size, by selecting a single unit of separation and a solvent (in many cases, controlling pH and electrostatic force). Can be separated. Alternatively, in isoelectric focusing separated by charge difference, proteins can be separated by isoelectric point differences corresponding to 0.02 pH differences. In the field of polynucleotides, in the technique used for DNA sequencers, it is possible to separate 700 bp and 701 bp DNAs by the difference in length, as long as the DNAs have similar sequences.

  The field of biochemical research has been developed supported by such separation means. This is because it was thought that the whole life phenomenon could be reconstructed by separating the components of life into components and clarifying their characteristics. In recent omics research, including genome research, there are tens of thousands of components of living organisms alone, and besides that, there are a huge number of related chemical substances and interactions regardless of genomic information. It is becoming clear that For this reason, the classical interpretation that life phenomena are the result of complex interactions of matter has resurfaced.

  In order to proceed with such research, it is a general technique to first destroy cells, separate various substances contained therein, and analyze them by the above technique. Many cell destruction methods have already been implemented. For example, a method of mechanically grinding cells, a method of cutting with a cutter that rotates at high speed, a method of irradiating cells with ultrasonic waves, a method of boiling called the boiling water method, a method of using a strong alkali, an organic solvent The method used, yeast or plants to protoplast the cell surface using an enzyme, and then disrupt the cell membrane with a surfactant, for red blood cells etc., to burst the cells with a hypotonic solution, in addition to these Many cells that are suitable for each cell have been put into practical use.

  In any case, in general, in order to analyze a biological material in a cell, in many cases, the cell must be destroyed and solubilized for analysis. However, as described above, the fact that there is a cell destruction method suitable for each cell as a cell destruction method means that there are not many general-purpose cell destruction methods. A method of mechanically destroying and a method using ultrasonic waves are used for a relatively general purpose. However, when the number of cells is as small as one, for example, it is substantially difficult to use. This is because, in order to mechanically grind or irradiate ultrasonic waves, it is necessary to handle the solution as a solution of at least about 100 μl, which makes it difficult to handle a small amount of cells. In the method using an organic solvent, removal of the organic solvent after cell destruction becomes a problem. Genome and RNA can be recovered by alcohol precipitation, but the recovery rate decreases as the number of cells decreases.

  Usually, the cell suspension is put into a container for the operation, but if the amount of the cell suspension is reduced, the surface area of the container with respect to the volume of the solvent increases, and the phenomenon that the biological material as a solute is lost by adsorption occurs. . In general, when the cytoplasm is destroyed, various enzymes contained therein are activated, and for example, mRNAs can be rapidly degraded. For this reason, even if it is intended to destroy one cell and recover a biochemical substance contained therein, it is difficult to implement due to the limitation of the amount and damage to the substance itself.

  The present invention solves the problems of the prior art as described above, and not only from the viewpoint of simply separating biochemical substances, but also to clarify the function of the cell, so that a very small number of molecules active against the cell. It is an object of the present invention to provide a new technical means for separating functions in a form that allows function tracking.

  In the present invention, a solution containing a small number of cells or cell clusters is placed on a substrate as droplets, and the droplets on the substrate are irradiated with convergent light.

  According to the present invention, it is possible to reliably destroy a very small amount of cells, ultimately one cell. Since the time required for destruction is instantaneous, cells can be destroyed in a shorter time than conventional cell destruction. This means that the biochemical reaction in the cell can be stopped instantaneously. Furthermore, while maintaining the shape of the droplet, the cells can be destroyed in the droplet, and the broken cells can be retained in the droplet. For this reason, there is an advantage that the state of the substance in the cell at the time when the cell is destroyed can be stably fixed.

  FIG. 1A is a plan view of a cell holding substrate 100 suitable for carrying out the present invention, and FIG. 1B is a cross-sectional view when viewed in the direction of the arrow at the AA position of the plan view. Reference numeral 1 denotes a silicon substrate, for example, having a thickness of 1 mm and a size of 20 mm × 20 mm. The surface of the substrate 1 is a hydrophobic region 2, in which hydrophilic regions 3 are arranged. The size of the hydrophilic region 3 is sufficiently smaller than the diameter of the droplet to be held in the hydrophilic region 3. Reference numeral 4 denotes a positioning marker, which is formed on one surface of the silicon substrate 1.

As a method of creating the hydrophilic region and the hydrophobic region, for example, the upper surface of the hydrophobic silicon substrate 1 is oxidized, and the entire region is once converted into a hydrophilic SiO ₂ thin film. Thereafter, the hydrophobic region may be created by dissolving and removing the SiO ₂ thin film in the region to be made hydrophobic with hydrofluoric acid. Alternatively, if the hydrophilic surface material of the substrate 1 is the surface is previously SiO ₂ film formation, fluorine-based resin, a hydrophobic material such as silicon-based resin, by disposing thereon, forming a hydrophobic region Just do it. In this case, the hydrophilic region present in the hydrophobic region is reduced by the thickness of the hydrophobic material. The example of FIG. 1 is an example in which the hydrophobic region 2 is formed by the latter method.

  FIG. 2A is a conceptual diagram illustrating an example of a system configuration that configures droplets containing cells in the hydrophilic region 3 of the cell holding substrate 100 suitable for the implementation of the present invention, and FIG. 10 is a cross-sectional view showing a result of forming droplets containing cells in the hydrophilic region 3 of the holding substrate 100. FIG.

  In FIG. 2A, a droplet containing the cell 12 is formed in the hydrophilic region 3 of the cell holding substrate 100 while optically monitoring the droplet at the tip of the pipette 11 for forming a droplet containing the cell 12. To do. Reference numeral 19 denotes a stage driven in the XY directions, and reference numeral 27 denotes a drive device for the stage 19. A cell holding substrate 100 is placed on the upper surface of the stage 19. On the upper part of the cell holding substrate 100, a pipette 11 in which a suspension 13 containing cells 12 to be included in droplets is sucked and held in advance is disposed. A syringe pump 31 is provided at the base of the pipette 11 via a tube 30, and a drive device 32 is attached to the syringe pump 31. When the syringe pump 31 is driven by the driving device 32, the suspension 13 in the pipette 11 is pushed out together with the cells 12. Note that the reason why the connection portion between the base portion of the pipette 11 and the tube 30 is separated is that the pipette 11 is enlarged and displayed.

  On the other hand, the tip of another pipette 20 for supplying a culture solution to the tip of the pipette 11 is disposed at the tip of the pipette 11. A syringe pump 35 is provided at the base of the pipette 20 via a tube 34, and a drive device 36 is attached to the syringe pump 35. When the syringe pump 35 is driven by the drive device 36, the culture solution in the syringe pump 35 is pushed out from the pipette 20.

  In addition, a pipette vertical movement drive device 37 for transferring a droplet formed on the tip of the pipette 11 to the hydrophilic region 3 of the cell holding substrate 100 is provided. Here, it is assumed that the vertical movement drive device 37 is linked to the pipette 11. When a signal for lowering the pipette 11 is given to the vertical movement drive device 37 by the user, the pipette 11 moves downward, and the droplet formed on the tip of the pipette 11 is transferred to the hydrophilic region 3 of the cell holding substrate 100. Transfer. When the user gives a signal for restoring the pipette 11 to the vertical movement drive device 37, the pipette 11 returns to the position shown in the figure. The recovery of the pipette 11 to the position shown in the drawing may be performed in a time sequential manner by the personal computer 26 from the lowering operation. A one-point difference line 39 means linkage between the vertical movement drive device 37 and the pipette 11.

  Further, a light source 16 and a condensing lens 17 constituting an optical system for monitoring the size of droplets formed in and near the tip of the pipette 11 are provided, and at positions facing this. A collimating lens 18 and a monitor 25 are provided below the cell holding substrate 100. Therefore, the cell holding substrate 100 and the stage 19 need to be optically transparent. A so-called personal computer 26 is a control signal obtained from a predetermined program stored in advance according to an input signal from the monitor 25, and an operation input signal given by the user while viewing the display screen of the monitor 25. 28, the necessary signals are given to the driving devices 27, 32, 36 and 37. Although not shown here, it is convenient to display the same display on the monitor of the personal computer 26 as the screen detected by the monitor 25. Then, the monitor 25 can be a small CCD camera. The operation input signal 28 is given via an input device of the personal computer 26.

  In addition, when the cell holding substrate 100 and the stage 19 are not optically transparent, the monitor may be illuminated from the upper surface and monitored by reflected light. That is, when the substrate is opaque, the collimating lens and the monitor 25 may be installed on the same side as the light source 16 on the substrate, and the reflected image may be observed. For example, the light source is irradiated obliquely with respect to the substrate and observed from a right angle direction.

  Here, the size of the pipette 11 is considered as follows. The pipette 11 needs to be able to form an appropriately sized droplet having the required number of cells at its tip. On the other hand, in the pipette 11, the suspension 13 containing cells is used after being sucked up by the pipette 11, but when the droplet 21 is formed, the cells passing through the tip of the pipette 11 are detected without error by the monitor 25. It must be possible. Therefore, the diameter of the tip of the pipette 11 allows one cell or a lump of a predetermined number of cells to pass, but it is assumed that cells that cannot be counted cannot pass at once. In other words, it is not as thick as the culture pipette currently used for general purposes, it is transparent and the tip diameter is 20 to 100 μm for general animal cells, and for microorganisms such as bacteria. The thickness is preferably about 5 μm.

  An operation for forming the droplet 21 containing the cells 12 in the hydrophilic region 3 of the cell holding substrate 100 will be described below. First, when the system is activated, the user positions the cell holding substrate 100 so as to be at a predetermined activation position while paying attention to the marker 4 described with reference to FIG. Next, the stage 19 is operated by the drive device 27 in response to an operation input signal 28 for moving the placement position of the droplet 21 containing the cells 12 to a position corresponding to the tip of the pipettes 11 and 20. When the cell holding substrate 100 reaches a predetermined position, an operation of discharging the cell suspension 13 inside the pipette 11 together with the cells 12 is performed. At this time, the outside of the tip portion of the pipette 11 and the inside in the vicinity of the tip portion are monitored by an optical system including the light source 16 and the monitor 25. The output of the monitor 25 can be taken into the personal computer 26 and the drive device 32 can be operated based on the image calculation result of the personal computer 26 to control the liquid feeding of the syringe pump 31.

  While monitoring the tip of the pipette 11 with the monitor 25, the driving device 32 is operated, the syringe pump 31 is moved, the suspension 13 containing the cells 12 is discharged from the tip of the pipette 11, and the droplet 21 is applied to the tip of the pipette. Form. At this time, the personal computer 26 recognizes that a predetermined number of cells have been inserted into the droplet 21 through the monitor 25, issues a stop command to the drive device 32, and stops the syringe pump 31.

  Hereinafter, in order to simplify the description, the number of cells 12 inserted into the droplet 21 will be described as one, but the number of cells may be arbitrarily determined by the user according to the purpose. For example, 10 may be sufficient. The cell 12 may be recognized by directly detecting the cell 12 present in the droplet 21 at the tip of the pipette 11, but more efficiently, the cell 12 moving inside the pipette 11 is monitored by the monitor 25. Then, the position and moving speed of the cells in the pipette may be calculated by the personal computer 26, and the syringe pump 31 may be controlled by predicting the time when the cells are discharged from the tip of the pipette 11 into the droplet 21. If the latter recognition method is used, it is advantageous when only one cell is put in the droplet when, for example, a plurality of cells are moving in the pipette at short intervals.

  Here, when the cell concentration of the cell suspension 13 is low, the droplet 21 starts to form immediately before it exits from the tip of the pipette 11, and if the droplet formation is stopped after a predetermined time, the size of the droplet 21 is increased. The thickness can be made predetermined. When it is not desired to form droplets, for example, the liquid coming out from the tip of the pipette 11 may be blown off with a blower. Alternatively, a drain may be provided outside the substrate 1 and discharged there.

  On the other hand, when the cell concentration of the cell suspension 13 is high, the amount of liquid discharged from the pipette 11 varies. That is, since the frequency of discharging the cells 12 discharged from the pipette 11 increases, if the time for discharging the liquid is fixed at a predetermined time, the next cell enters the liquid 21 within that time. There is a possibility. In such a case, the pipette 20 is used. In the pipette 20 and the syringe pump 35 connected thereto, only the culture solution or the cell dilution solution is placed. That is, when the personal computer 26 confirms that the cell 12 enters the droplet 21 through the monitor 25, a stop command is issued to the driving device 32 to stop the syringe pump 31, and the droplet 21 is formed by this time. The volume of the droplet 21 at that time is determined from the feed amount of the syringe pump 31 that is driven. The personal computer 26 calculates the difference between this volume and the desired volume of the droplet 21. In accordance with the calculation result, an operation signal is sent from the personal computer 26 to the driving device 36 to drive the syringe pump 35 so that the culture solution or the cell dilution solution is added to the droplet 21 formed at that time with the pipette 20. The liquid is added using the pipette 20 until the volume of the droplet 21 reaches a predetermined value.

  At this time, it is preferable that the tip of the pipette 20 has a size that prevents passage of cells, for example, 0.2 μmφ so that the cells in the droplets do not flow back into the pipette 20. Alternatively, it is preferable to have a filter structure with a tip of 0.2 μm.

  The droplet 21 containing one cell prepared in this way is brought into contact with the hydrophilic region 3 on the substrate 1 placed on the stage 19 by the vertical movement drive device 37 of the pipette 11, and the liquid The droplet 21 moves to the hydrophilic region 3 of the substrate 1. The droplet 21 is repelled by the hydrophobic region 2 and fixed in a self-forming manner at the position of the energetically stable hydrophilic region 3. When it is confirmed that the droplet 21 containing the cells 12 has moved to the hydrophilic region 3 of the substrate 1, that is, the hydrophilic region 3 of the cell holding substrate 100, the user stops the operation.

  FIG. 2B shows a droplet containing cells in the hydrophilic region 3 of the cell holding substrate 100 by the system for forming droplets containing cells on the cell holding substrate 100 described with reference to FIG. It is sectional drawing which shows the result by which was mounted. Cells 12 and droplets 15 that wrap around the cells 12 are disposed in the hydrophilic region 3 on the silicon substrate 1.

  FIG. 3 is a perspective view showing an outline of an embodiment of an apparatus for destroying cells in a droplet with the droplet 15 formed on the substrate as a target, as described with reference to FIGS. 1 and 2. is there. The apparatus described in FIG. 3 is an independent form, but is configured to be integrated and adjacent to the system that forms the droplet described in FIG. 2, and the movement of the cell holding substrate 100 by the control of the stage 19, the laser It is convenient that the personal computer 26 can control beam irradiation and the like.

  In FIG. 3, the droplet 15 on the cell holding substrate 100 can be irradiated with light from two directions.

  The light irradiated from the upper light source 41 is adjusted to a specific wavelength by the filter 42, condensed by the condenser lens 43, and irradiated to the droplet 15. The irradiated light is guided as transmitted light to the camera 52 through the objective lens 47, the dichroic mirror 48, the mirror 49, and the filter 51, and the transmitted light image inside the droplet 15 is formed on the light receiving surface of the camera 52. . Therefore, it is desirable that the cell holding substrate 100 and the stage 19 are optically transparent materials as in the case of droplet formation. Specifically, a glass such as borosilicate glass or quartz glass, a resin or plastic such as polystyrene, or a solid substrate such as a silicon substrate is preferable. When a silicon substrate is used as the substrate 1 of the cell holding substrate 100, the upper light source 41 is preferably light having a wavelength of 900 nm or more.

  The light emitted from the lower light source 47 is wavelength-selected by the filter 46, guided to the objective lens 47 by the dichroic mirror 48, and used as excitation light for fluorescence observation inside the droplet 15. The fluorescence emitted from the inside of the droplet 15 is observed again by the objective lens 47, and the fluorescence after the excitation light is cut by the filter 51 can be observed by the camera 52.

  At this time, by adjusting the combination of the filters 42, 46 and 51, only the transmitted light is observed with the camera 52, only the fluorescence is observed, or the transmitted light image and the fluorescence image are simultaneously observed with the camera 52. You can also.

  The image data obtained by the camera is analyzed by the personal computer 26, and the stage 19 can be controlled so that the laser beam 63 is aimed at the droplet 15. When the laser beam 63 is an ultraviolet laser, it is dangerous, and observation is performed with the CCD camera 52. Again, although not shown, it is convenient to display the image signal detected by the CCD camera 52 on the monitor of the personal computer 26.

  Reference numeral 61 denotes a laser beam source, and 62 denotes a filter for wavelength selection. In the first embodiment, 355 nm, which is the third harmonic of the YAG laser, can be irradiated. The intensity of the laser beam 63 is about 200 μJ or more, and the laser beam 63 is focused to irradiate the cells. Here, the laser beam 63 is directly irradiated from the laser beam source 61 to the droplet 15. However, when there is a structural limitation and the droplet 15 cannot be directly irradiated, the laser beam 63 is used. An appropriate mirror can be arranged and guided in the optical path. Irradiation of the laser beam source 61 can be controlled by the personal computer 26. The operator can control the irradiation of the laser beam source 61 by giving an operation signal 28 to the personal computer.

  When the laser beam 63 is aimed at the cell 12 in the droplet 15 and the cell is irradiated with a laser pulse of 200 μJ under a microscope, it is observed that the cell membrane is instantaneously broken and scattered. Since this laser is an ultraviolet laser, all laser irradiation optical systems are compatible with ultraviolet rays. When the droplet is large or when the number of internal cells is large, the intensity of the laser beam 63 may be increased. It is easy to produce a power of about 5 mJ.

  What is important here is the wavelength of light and the manner of irradiation. When light having a wavelength that is absorbed by water as a solvent is used, the water as the solvent itself is vaporized. Therefore, although the cells have absorption, the solvent is heated in a wavelength band where absorption of water is negligible. Specifically, one method is to use an ultraviolet region, which is a band in which intracellular biochemicals, proteins, and nucleic acids absorb light and convert it into heat. Alternatively, cells can be destroyed even with visible light for unknown reasons. It has also been found that by irradiating the cells as focused light, the cells can be killed and destroyed instantaneously.

  The present invention aims to efficiently destroy a very small amount of cells such as one cell and efficiently use it for subsequent analysis of the contents and collection of the contents. Dilution diffusion is prevented by holding the contents of the cells at the time of disruption in the droplets. In general, light absorption is slightly different between cells and water, which is a solvent, so that water is hardly absorbed, and the cells are irradiated with light of a wavelength that is absorbed by various cellular organs, and only the cells are heated. , Destroy and keep the contents of the broken cells in the droplets. If the light absorption and the temperature increase are slow, the temperature of water, which is a component of the solvent, also increases. For this reason, it is important to increase the speed of light increase in the portion where the cells are absorbed rather than the temperature increase of the solvent by applying strong light for a moment, and as a result, solubilize the cells.

  FIG. 4 is a conceptual diagram illustrating a specific example in which a biological material is directly recovered from a suspension of cell fragments in a droplet 15 in which cells have been destroyed as described in the embodiment. A predetermined amount (0.1 μl) of RNA intercalator Cyber Green II for RNA is added to the suspension of cell pieces in the droplet 15 in which the cells are broken. For this, the droplet 15 is directly injected by a capillary. A capillary 72 having an inner diameter of 50 μm filled with a platinum electrode 71 and an electrophoretic separation medium mainly composed of a linear dimethylpolyacrylamide polymer is brought into contact with the droplet 15. The other end of the capillary 72 is immersed in a buffer solution stored in the container 73. Further, one end of the platinum electrode 74 is immersed in the buffer solution. A voltage is applied for 10 seconds so that an electric field of 50 v / cm is applied to the capillary 72 between the two electrodes with the platinum electrode 71 being negative and the platinum electrode 74 being positive. Thereafter, 50 μl of electrophoresis buffer (Tris-HCl) is added to the droplet 15, and electrophoresis is continued so that an electric field of 200 V / cm is applied. Argon laser is irradiated from a 488 nm argon laser source 75 to a position 10 cm from the droplet 15, and the resulting fluorescence is monitored by a detector 76.

  Although not shown in FIG. 4, the electrode 71 and the capillary 72 are held by a personal computer 26 having an operating arm whose tip can be moved to an arbitrary position, such as the operating device 37 described in FIG. 2. It is good to operate by.

  FIG. 5 is a waveform diagram showing an example of an electrophoresis pattern obtained by electrophoresis. The horizontal axis represents the migration time, and the vertical axis represents the fluorescence intensity. Two sharp peaks 81 and 82 are derived from two types of rRNA, a broad band 83 is derived from mRNA, and 84 is a high molecular genome. As can be seen from FIG. 5, after a time corresponding to the migration time, these biological materials are discharged from the other end of the capillary 72 to the container 73. That is, according to the present invention, the biological materials can be recovered from a very small amount of cells. . Moreover, since this is obtained by destroying the cells in the droplets, it is clear that the substance state in the cells at the time of the destruction of the cells can be stably fixed.

(A) is a top view of the cell holding substrate 100 suitable for implementation of this invention, (B) is sectional drawing when it sees in the arrow direction in the AA position of a top view. (A) is a conceptual diagram illustrating an example of a system configuration that configures droplets containing cells in the hydrophilic region 3 of the cell holding substrate 100 suitable for the implementation of the present invention, and (B) is a diagram of the cell holding substrate 100. It is sectional drawing which shows the result of having formed the droplet containing a cell in the hydrophilic region. It is a perspective view which shows the outline | summary of the Example of the apparatus which destroys a cell within a droplet using the droplet 15 formed on the board | substrate as a target as demonstrated with reference to FIG. 1, FIG. It is a conceptual diagram explaining the specific example which collect | recovers a biological material directly from the suspension of the cell piece in the droplet 15 in which the cell was destroyed. It is a wave form diagram which shows the example of the electrophoresis pattern obtained by electrophoresis.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Silicon substrate, 2 ... Hydrophobic area | region, 3 ... Hydrophilic area | region, 4 ... Marker for positioning, 11 ... Pipette, 12 ... Cell, 13 ... Suspension, 15 ... Droplet, 16 ... Light source, 17 ... Collection Optical lens, 18 ... Collimating lens, 19 ... Stage, 25 ... Monitor, 26 ... Personal computer, 27, 32, 36, 37 ... Drive device, 28 ... Operation input signal, 30, 34 ... Tube, 31, 35 ... Syringe pump, DESCRIPTION OF SYMBOLS 41 ... Light source, 42, 46, 51 ... Filter, 43 ... Condenser lens, 47 ... Objective lens, 48 ... Dichroic mirror, 49 ... Mirror, 52 ... Camera, 61 ... Laser beam source, 62 ... Filter, 63 ... Laser beam, 71, 74 ... platinum electrode, 72 ... capillary, 73 ... container, 75 ... argon laser source, 76 ... detector, 100 ... cell holding substrate.

Claims (4)

  A cell disruption apparatus comprising a substrate for holding a droplet containing a predetermined cell on a surface, and an optical system for irradiating the droplet with focused light having an absorption band on the cell.   2. The cell destruction according to claim 1, wherein the substrate holding the droplets containing cells in the hydrophilic region has a structure in which the periphery is water-repellent and the range narrower than the size of the droplet is composed of the hydrophilic region. apparatus. A pipette capable of holding a solution containing a plurality of cells and having a tip opening diameter suitable for passing a cell or cell mass of a predetermined size;
Means for observing cells at the tip of the pipette;
Means for extruding a solution containing cells at the tip of the pipette from inside the pipette to form droplets;
Means for determining that a predetermined cell is contained in the droplet and the droplet has a predetermined size;
An apparatus for dropping the droplet formed on the pipette tip at a predetermined position on the substrate;
A cell disruption apparatus comprising: an optical system that irradiates the droplet with focused light having an absorption band on a cell. A cell that contains droplets containing cells in the hydrophilic region of the substrate that is water-repellent and has a region narrower than the cell size, and that emits focused light in a wavelength band that the cells in the droplet absorb light Crushing method.

Images