JP5162738B2 - Methods for isolating and collecting living cells and structures in living cells - Google Patents

Description

  The present invention relates to a method for isolating and collecting living cells, structures in living cells, and the like.

  Currently, in the fields of pathology and molecular biology, research is being conducted to analyze the behavior of living cells (living cells) or various organelles (intracellular structures) existing in living cells. Interest is gathered. By directly analyzing living cells or intracellular structures, pathological mechanisms occurring in the cells can be analyzed more precisely.

  On the other hand, the collected pathological tissue pieces are actually very heterogeneous aggregates composed of many normal cells and cells not to be analyzed, and there are very few lesion cells (target cells) to be studied. Limited to. The laser microdissection method (LMD method) is effective as a method for collecting target cells distributed at a low density in the pathological tissue piece. The LMD method is a technique in which a target cell is separated from a tissue piece with a high-power laser while observing the tissue piece under a microscope using an apparatus in which a laser irradiation apparatus is connected to a microscope (Patent Documents 1 to 3). . The LMD method is an extremely powerful technique for analyzing the expression of pathogenic genes because only target cells can be isolated from a pathological tissue fragment in which normal cells and target cells coexist, and laser capture microdivision (LCM) It is also called.

Here, a method of collecting the separated target cells becomes important. As a method for collecting target cells, there are, for example, a method of pressing a transparent cap on which a thermoplastic film is attached (Patent Document 1), and a method of using a gelatin-coated slide glass (Patent Document 2). In addition, a method has been disclosed in which the peripheral region of the target cell is largely removed at the time of LMD and accurate collection is realized (Patent Document 3).
JP 2000-266649 A JP 2002-156316 A Japanese Patent No. 3820227

  However, when a high-power laser is irradiated onto living cells containing a large amount of water, the water is evaporated and scattered at a rapid rate due to the ablation effect, and accurate cutting at the laser irradiation site cannot be performed. The above-described LMD methods of Patent Documents 1 to 3 are all applicable only to a dried tissue specimen, and cannot be applied to isolation of living cells or intracellular structures containing a large amount of water.

  In addition, even if a sufficient distance from the outline of the target living cell is trimmed with a laser, the living cell containing a large amount of water generally tends to have a weak adhesive force, and is described in, for example, Patent Documents 1 and 2. It is difficult to collect by this method. In the first place, when trimming with a laser at a sufficient distance, a large amount of impurities are mixed. This problem becomes even more serious when targeting only minute intracellular structures. On the other hand, the method of Patent Document 3 is difficult to apply because the collection efficiency is poor when a large number of target living cells are scattered in the peripheral region.

  The purpose of the present invention is to accurately isolate target living cells or intracellular structures (target sites) distributed at low density in a sample containing a large amount of water from the sample, and to accurately isolate only this isolated target site. It is to provide the technology to collect.

  That is, the present invention comprises a step of preparing a specimen containing target living cells or intracellular structures, a step of imparting liquid permeability to the specimen, and controlling a water content in the specimen, Irradiating a laser to separate the target living cell or intracellular structure from the specimen, and a method for isolating the target living cell or intracellular structure. Here, the step of controlling the water content includes a step of obtaining a semi-dried specimen, whereby inconvenience at the time of ablation can be suitably avoided. The step of controlling the water content includes a step of semi-intacting such that a drug that forms a large number of micropores in a cell membrane is added to the specimen, and a liquid component in the cell is taken out, and each semi-intacted intracellular Including the step of causing the liquid component to flow out of the cell, the water content in the cell as well as outside the cell can be appropriately controlled, and more accurate isolation becomes possible. The step of detaching comprises generating light from a photomarker substance previously incorporated in the target living cell or intracellular structure, determining a dissection location based on light emission of the photomarker substance, Irradiating the specimen with a laser and separating the target living cells or intracellular structures from the specimen, thereby confirming a desired isolation target during observation with an optical marker. It can be easily isolated. The step of preparing the specimen includes a step of incorporating and visualizing a photomarker gene in the target living cell or intracellular structure, thereby allowing the genetic activity to be isolated and alive. Here, since the step of causing the light marker substance to emit light is an excitation light source (laser or mercury lamp spectrum light or the like) having a wavelength different from that of the cutout step, simultaneous operation can be performed without receiving optical interference with each other.

  Further, the collection method of the present invention comprises a step of preparing a specimen containing target cells or intracellular structures, a step of controlling the water content of the specimen, a small-diameter collection rod having a flat tip, a laser Pressing the target living cells or intracellular structures separated by the step, releasing the target living cells or intracellular structures attached to the flat tip in a collection container containing a solution; It is characterized by including.

  Further, the collection method of the present invention comprises a step of preparing a specimen containing target cells or intracellular structures, a step of controlling the water content of the specimen, irradiating the specimen with a laser, Separating the intracellular structure from the specimen, and a method for isolating the target cell or the intracellular structure. Here, the specimen is pre-frozen or dried, and the step of controlling the water content is sufficiently increased once by the melting solution generated by freezing and the solution for restoring from the dry state. Then, the present invention can be applied to cells in various preserved states other than cultured cells by reducing the water content until a low dry state is achieved.

  By the method for isolating live target cells or intracellular structures of the present invention, live target cells or intracellular structures distributed at low density in a sample containing a large amount of water are accurately isolated from the sample, and the isolation is performed. Only the target site thus obtained can be accurately collected.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below are merely examples for explaining the configuration of the present invention in detail. Accordingly, the invention should not be construed as limited based on the description set forth in the following embodiments. The scope of the present invention includes all embodiments including various modifications and improvements of the following embodiments as long as they are within the scope of the invention described in the claims.

<First Embodiment>
FIG.
FIG. 1 is a flowchart showing a method in the first embodiment.

  First, a specimen containing target living cells or intracellular structures is prepared (11).

  “Target living cell” means a living cell (hereinafter, also simply referred to as “target cell”). Target cells include, but are not limited to, various abnormal cells that are available from any species or site and that are dispersed at low density in a pathological tissue piece. For example, in the case of a tissue piece, many normal cells are mixed in the entire tissue piece. For example, in order to perform gene expression analysis specific to a specific tumor cell, only the target tumor cell is collected from the tumor tissue piece. There is a need. The “specimen” means a cell dispersion system obtained by disassembling a pathological tissue piece or tissue piece.

  “Intracellular structures” means intracellular aggregates and various intracellular organelles. Intracellular organelles include any substance to which LMD can be applied, and include, for example, membrane-type structures such as the nucleus, mitochondria, lysosome, endoplasmic reticulum, and Golgi apparatus.

  In addition, the step of preparing the specimen may include a step of incorporating and visualizing a photomarker gene in the target living cell or an intracellular structure (hereinafter also referred to as “target site”). By visualizing the target site with the optical marker substance, only the target site can be reliably and accurately cut out from the specimen in the subsequent laser microdissection process.

  As the photomarker substance, a fluorescent protein that emits fluorescence by excitation light, such as GFP, is useful, as in Examples described later. The GFP used in the examples is pEGFP-N1, specifically a Clontech vector, and has an excitation wavelength of 488 nm. The photo marker gene includes a photoprotein gene that emits light by a chemical excitation agent such as an enzyme substrate solution. Moreover, the case where fluorescence by bioluminescence energy resonance transfer (BRET) is generated by a fluorescent protein gene and / or a photoprotein gene is also included. In terms of visualization, it is possible not only to incorporate and visualize a fluorescent marker gene, but also to react a fluorescently labeled secondary antibody with an antibody having specificity with the target protein (fluorescent antibody method) for visualization. is there. Then, LMD treatment can be performed on the target site labeled by the fluorescent antibody method.

  Second, liquid permeability is imparted to a specimen containing target living cells or intracellular structures (12).

  “Providing liquid permeability” means inducing the cell membrane of each cell constituting the specimen to a state in which the liquid component in each cell can flow out of the cell. The cell membrane imparted with liquid permeability allows permeation of liquid components in the cell but inhibits permeation of intracellular structures in the cell. Accordingly, the intracellular structure remains intact (intact) in the cell, and only liquid components containing various fluid microproteins (globulins and the like) can flow out. This technique is generally called “semi-intact”. Semi-intacted cells (semi-intact cells) can freely enter and exit liquid components including soluble components (such as ions and proteins), while the relative position between the cell membrane, cytoskeleton, and intracellular organelles, that is, intracellular It is a cell that retains the topology. By using semi-intact cells, protein functions and dynamics can be analyzed in the place and space where the protein originally functions. On the other hand, “cells are considered as a single test tube”, and cytoplasmic components, small molecule inhibitors, etc. Biochemical analysis such as removal or addition can be performed efficiently. In a first embodiment, this “semi-intact” technique is not used as an intracellular test tube, but as a technique for controlling the water content in a specimen containing living target cells or intracellular structures.

  Generally, semi-intacting of a specimen is performed by adding an agent that imparts permeability to a cell membrane to the specimen. Agents that impart fluid permeability to cell membranes include thiol or sulfhydryl-activated toxins, oxygen-forming toxins that are capable of forming one or more lytic pores in cells. Bacterial toxins collectively referred to as oxygen labile, oxygen sensitive hemolysin. Particularly preferably, streptolysin O (storeptolysin O) SLO is used, but other usable agents include αHL, aerolysin, perfringolysin, pneumolysin, listeriolysin. (Listeriolysin), bacterial exotoxins such as Bacillus thuringensis toxin, or E. coli-derived lytic molecules such as hemolysin and colicin, or defensin, magainin, melittin And those derived from eukaryotic cells such as (mellitin), complement, perforin, yeast killer toxin, and histolysin. The method of the present invention is not particularly limited to chemical treatment with these drugs. For example, by adding streptricin O and semi-intacting each cell constituting the specimen, the liquid component in the cell is changed. It can be drained or created alive and semi-dry.

  Third, the semi-dried specimen is irradiated with a high-power pulse laser, and the target living cells or intracellular structures (target sites) are separated from the specimen (13).

  The technique used here is the laser microdissection (LMD) method. The LMD method is a method in which a target site on a tissue piece is specifically cut out and collected with a laser while observing the tissue piece under a microscope using an apparatus having a laser irradiation apparatus connected to a microscope. The LMD method is an extremely powerful technique for analyzing the expression of pathogenic genes because only pathological cells can be isolated from a pathological tissue fragment in which normal cells and pathological cells coexist, and laser capture microdigestion (LCM) It is also called. Since the wavelength and output of the laser used for LMD are known, detailed description thereof is omitted.

  When the LMD method is applied to living cells containing a large amount of water, the water is evaporated and scattered at an abrupt speed due to the ablation effect, and accurate cutting at the laser irradiation site cannot be performed. However, in the first embodiment, the semi-intacting process (12) keeps the specimen alive and is in a semi-dry state, so that evaporation of moisture due to the ablation effect is suppressed and the target site is accurately cut out by the LMD method. Is possible.

  In addition, the process of “semi-intact” generally referred to includes a step of containing a replacement liquid such as a buffer solution that does not contain a miscellaneous protein instead of the original intracellular solution. Also in the present invention, as in the examples described later, by washing away cytosol components and the like with an appropriate buffer solution for buffering, other liquids are accommodated in the cells while maintaining the intracellular structures. I have to. Thus, even when the liquid in the cell is exchanged with another liquid such as a buffer solution, the above-described effect by ablation is obtained by making the outside of the cell semi-dry.

  In addition, by removing the protein-containing solution with a diameter less than the micropore diameter through the many micropores generated in the cell membrane by the semi-intact treatment, it is possible to avoid inconveniences such as excess components remaining during isolation (or collection). The efficiency of will also increase. In addition, the formation of a large number of micropores by semi-intact treatment weakens the swellability of the cells before treatment, reduces the amount of fluid inside the cell, and the fluid pressure inside the cell approaches external pressure, so the cell membrane is perforated. Even if it is done, there is also an effect that there is almost no possibility that the liquid or the intracellular structure is blown out. Thus, there has been no technique that can specifically isolate (or collect) only desired intracellular structures from living cells without damage.

  Finally, the separated target site is collected (14).

  Living cells containing a large amount of water generally tend to have poor adhesion and are difficult to collect. However, in the first embodiment, after the semi-intacting treatment (12), the target site cut out by the laser microdissection (13) is alive and semi-dry, so that the target site is attached to the collection rod. Good and easy to collect.

  The inventor has conducted intensive research on how to collect a target site without impairing the activity of the living target site. As a result, we have developed a sampling method that is completely different from existing sampling methods. The sampling method in the first embodiment is a method of pressing a thin glass rod having a flat tip portion against a target site cut by a laser and attaching the target site to the flat tip portion. This is a method of collecting a target site without impairing the activity. The target site and the tip of the glass tube can be easily confirmed on the screen. Therefore, it is possible to accurately and reliably collect only the target site.

FIG.
FIG. 2 is a diagram schematically showing an apparatus for performing the semi-interacting process in the first embodiment. The apparatus includes an injector 21 storing a drug that imparts liquid permeability to a cell membrane, and a micropipes that adds the drug ejected from the injector 21 onto a dish 22 on which target living cells A are seeded and cultured. It comprises a petter 23, collection containers 24 and 25 for collecting the culture solution aspirated by the micropipette 23, and a CPU 26 that can automatically control these series of operations.

  Agents that impart fluid permeability to cell membranes are not particularly limited, but include, for example, thiol or sulfhydryl-activated toxins, oxygen labile, oxygen sensitive hemolysin ) And bacterial toxins collectively referred to. In the examples described later, streptolysin O (SLO) was useful, but an optimal drug was selected for each target cell type, or a plurality of different drugs were used in combination. It is preferable to do this.

FIG.
FIG. 3 is a diagram schematically showing a semi-intacting process using the apparatus of FIG. First, a dish 22 in which target living cells A are seeded and cultured is prepared (FIG. 3a). Next, an appropriate amount of streptrin O stored in the injector 21 is added using the micropipette 23 (FIG. 3b). The addition amount of streptricin O varies depending on the type of specimen, but is, for example, 1 to 500 ng / ml, preferably 10 to 100 ng / ml, and most preferably about 40 to 70 ng / ml. The target living cell A exposed to streptricin O is imparted with fluid permeability and is in a state in which liquid components in the cell can flow out of the cell. Subsequently, the culture solution in the dish 22 is aspirated using the micropipette 23 (FIG. 3c). At this time, the liquid component in the cell flows out from the cell membrane of the target living cell A to which the liquid permeability is imparted, and is sucked together with the culture solution. Specifically, the cytosol removal buffer is added to the dish 22 several times, and the liquid component is gradually removed by incubation and shaking. The target living cell A is semi-intacted by the addition of streptolysin O and loses the liquid component by the subsequent suction operation, so that it becomes a semi-dried specimen while alive. The sucked liquid component and the culture solution are collected in the collection containers 24 and 25 through the four-way valve.

  In the first embodiment, the water content of the target living cell A is reduced to about 50 to 75% by weight with respect to the water content of the target living cell A in the natural environment such as an isotonic solution. A state can be generated. However, rather than measuring the moisture content as a reference, it is possible to determine whether or not the cell surface as a specimen is in a semi-dry state by checking with the naked eye the state in which the cell surface begins to dry. Preferably, when a plurality of cells are placed on a support such as a container in a dispersed state so as to be separated from each other, no moisture remains on the support surface at the peripheral edge of one or more cells of interest. As a guideline, it can be determined that the cell edge has been dried to the extent that it has not been dried, or the cell periphery has started to dry slightly. Such a semi-dry state may be executed by determination based on an image. In the first embodiment, by creating a semi-dry state showing such a low degree of dryness, the evaporation of water due to the ablation effect of the LMD method in the subsequent process is suppressed without impairing the cell activity. Can do. For example, semi-intacting with SLO provides living cells that are in a low dry state of about 60-70% by weight with respect to the water content.

  For convenience of explanation, the semi-intacting of the target living cell A has been described, but in actuality, the semi-intacting proceeds in the entire specimen and is not limited to the target living cell A alone.

FIG.
FIG. 4 is a diagram schematically showing an LMD apparatus for performing laser microdissection (LMD) processing in the first embodiment. The LMD apparatus includes a laser optical system 41 for performing LMD processing on a specimen and an observation optical system 42 for observing the specimen.

(Laser optical system 41)
Visible light output from the illumination light source 101 passes through the field stop 102 and the aperture stop 103 and is reflected by the dichroic mirror 104 below the optical axis p. The reflected light enters the laser objective lens 105, is condensed, and is applied to the specimen B.

  On the other hand, a pulsed laser beam having a wavelength in the ultraviolet region (hereinafter referred to as a laser beam) is output from a laser light source 123a in the UV laser head 123. The laser beam passes through the variable slit 125 and the UV imaging lens 124, enters the laser objective lens 105, and is irradiated onto the specimen B. The target living cell A or intracellular structure C is cut out by the laser beam applied to the specimen B.

  The variable slit 125 has, for example, a circular or quadrangular (for example, rectangular) opening, and adjusts the shape and size of the spot of the laser beam output from the UV laser head 123. The shape and size of the slit can be adjusted as appropriate.

  Further, the UV laser head 123 has an LED 126. The LED 126 outputs red LED light, and the output LED light is reflected on the optical axis p by the dichroic mirror 127. The reflected LED light is irradiated to the same position as the irradiation position of the laser beam on the specimen B. The red LED light irradiated on the sample B displays the irradiation position of the laser beam irradiated on the sample B.

  The dish 108 containing the specimen B is placed on a stage 106 that can be driven in the XYZ axial directions, and the target living cells A and intracellular structures C existing in the specimen B are moved to arbitrary locations. Can do. The target living cell A and the intracellular structure C are preferably labeled with a light marker substance in advance and the position in the specimen B is visualized. In addition, an air blower, an air suction machine, or the like may be installed in the vicinity of the specimen B, and the specimen debris after the LMD treatment may be treated.

(Observation optical system 42)
An observation objective lens 110, a dichroic mirror 111, a UV barrier filter 112, an imaging lens 113, and a half mirror 114 are provided on the optical axis p.

  Part of the reflected light from the sample B passes through the half mirror 114 and is reflected by the mirror 119. The light reflected by the mirror 119 enters the observer's field of view through the relay lens 120, the UV cut filter 121, and the eyepiece 122. On the other hand, part of the reflected light from the specimen B is reflected by the half mirror 114 and enters the CCD camera 115. The CCD camera 115 captures an image formed by the imaging lens 113 and outputs an image signal.

  The image processing and stage control device 116 acquires image data from the image signal output from the CCD camera 115 and displays the image data on the display 117. In addition, the image processing and stage control device 116 is connected to an operation unit 118 such as a keyboard and a mouse, receives an operation command for the movement direction and movement amount of the stage 106 by the observer, and moves the stage 106 according to the operation command. A control signal is transmitted to the stage drive unit 109.

  In addition, an excitation means for visualizing the target living cell A and the intracellular structure C labeled with the photomarker substance is provided. Specifically, a fluorescence excitation light source 128 and a UV bandpass filter 129 are provided on the reflected light path of the dichroic mirror 111, and the excitation light output from the fluorescence excitation light source 128 passes through the UV bandpass filter 129, The sample is reflected by the dichroic mirror 111, enters the observation objective lens 110, and irradiates the specimen B. The excitation light output from the fluorescence excitation light source 128 excites the fluorescent protein previously labeled on the target living cell A and the intracellular structure C. Here, when observing with the light intensity of the transmitted illumination light source 101 reduced, it is possible to simultaneously observe that cells incorporating a fluorescent protein gene as a light marker are shining in a cell group visualized as a bright field image. .

  In the figure, an inverted observation method in which the observation optical system is arranged below the specimen is adopted, but an upright observation method in which the laser optical system 41 and the observation optical system 42 are vertically inverted is adopted. May be.

FIG.
FIG. 5 is a diagram schematically showing a state of LMD performed using the LMD apparatus of FIG. The observer operates the operation unit 118 to perform LMD processing while confirming the image of the sample B displayed on the display 117 and the coloring display unit indicating the irradiation position of the laser beam. For example, when the intracellular structure C inside the target living cell A is separated from the specimen B as a target site (FIG. 5a), one shot is made in each of the top, bottom, left and right around the intracellular structure C emitting fluorescence with excitation light. A laser beam is irradiated with a rectangular laser spot 51 one by one (FIG. 5b). Since the sample site irradiated with the laser disappears and only the central region surrounding the target site remains, the intracellular structure C can be separated from the sample B.

  The laser spot 51 does not need to be rectangular, and can have an arbitrary shape according to the shape and size of the target site to be separated. For example, by irradiating a laser beam with a circular laser spot having various diameters around the target site in the same manner, accurate cutting along the contour of the target site becomes possible.

  The size of the central region surrounded by the laser spots described above is within the range of 1 μm to 100 μm, preferably 5 μm to 50 μm, and most preferably 7.5 μm to 30 μm. By arbitrarily changing the area of the non-irradiated portion, the spotting according to the size of the target site can be appropriately selected.

FIG.
FIG. 6 is a diagram schematically showing a sampling device for performing the sampling process in the first embodiment.

  The collection device includes an operation arm 64, a collection unit 63 attached to the tip of the operation arm, and a drive unit that can move the operation arm 64 to an arbitrary spatial position.

  The sampling means 63 is preferably fixed in the vertical direction at the tip of the operation arm 64. The collecting means 63 is preferably a glass rod having a flat tip. The flat tip of the glass rod has a diameter of 1 to 500 μm, preferably 5 to 100 μm, and most preferably 10 to 30 μm.

  The driving means preferably has an imaging means (camera) for imaging the collection site, an image display means (display) for displaying an electrical signal output from the camera as image data, and the collection means 63 at an arbitrary position. It comprises an operation means (controller) for inputting an operation command to be moved, and a drive control unit for driving an operation arm to which the sampling means is fixed based on the operation command.

  The collection device may also include a collection container 62 for collecting the target site and a fixing means 65 for the collection container 62. The collection container 62 is a transparent container, and the target site collected in the collection container 62 can be confirmed with a microscope. The collection container 62 is fixed by the fixing means 65.

  The shape and size of the collection container 62 are not particularly limited, but as an example, the PCR tube cap can be removed from the tube body and used as a collection container. That is, by reversing and fixing the cap portion of the PCR tube upward, the target site is recovered in a recess formed on the inner wall of the cap. In this case, sampling is completed by rejoining the cap portion and the PCR tube main body portion after collection.

  In addition, as a background of using the cap portion of the PCR tube in the present invention, (1) Since the actual work of the LDM is performed on the stage of the microscope, a tall tube body is formed because the distance between the objective lens and the stage is narrow. (2) Since the bottom of the tube body is generally an elongated conical shape and has a small radius of curvature (from 1R to 3R), cells attached to the tip surface of the glass rod can be placed on the objective on the lower side of the stage. In the case of observation through the bottom of the tube, aberration occurs due to the shape (curvature radius) of the tube bottom, and it is impossible to observe whether it is attached or not. On the other hand, the cap portion of the PCR tube is usually made of plastic with sufficient light transmission, and is easy to install on the stage because it is dish-shaped and short, and the movement of the glass rod is small. Since the radius of curvature of the inner wall of the cap is relatively large, there are advantages that cells attached to the end surface of the glass rod can be observed without the above-mentioned aberration. Further, as a characteristic of the PCR tube as a whole, there are advantages such as being a chemical-resistant material and being subjected to a surface treatment for preventing the collected cells and the like from being peeled off from the inner wall of the tube. Furthermore, since the PCR tube main body has a shape to be set in a centrifuge, etc., the cap portion and the tube main body are rejoined after the recovery, so that the post-processing after the recovery can be quickly continued. There is also an advantage of being able to.

FIG.
FIG. 7 is a cross-sectional view along the line AA of the fixing means 65 of FIG. The fixing means 65 includes a cavity 72 and a fixing plate 73 on the fixing base 71. The collection container 62 fitted in the cavity 72 is fixed by a fixed plate 73 that can move in the direction of the arrow in the figure. In the hollow portion 72, the transparent bottom of the collection container is exposed, so that the target site in the collection container can be confirmed by the objective lens installed below. In addition, when using the cap part of a PCR tube as the container 62 for collection | recovery, sampling can be completed easily by rejoining a tube main-body part to a cap part after collection | recovery of a target site | part.

  The collection device can be attached to the LMD device shown in FIG. 4 to perform isolation and collection of target living cells or intracellular structures as a series of operations. When the sampling device is attached to the LMD device, the imaging means (camera), image display means (display) and operation means (controller) of the LMD device can be used, and only the manipulator (sampling means and driving means) is newly added. It only has to be installed, which is advantageous in terms of cost. The collection container 62 and its fixing means 65 may be installed on the stage 106.

8 and 9
FIGS. 8 and 9 are diagrams schematically showing a target site collecting step using the collection device shown in FIGS. 6 and 7. First, the intracellular structure C in the target living cell A is separated from the specimen B on the dish 108 by microdissection (LMD) processing by the laser irradiation means 81 (FIG. 8a). The glass rod 63 having a flat tip is moved above the intracellular structure C, and the intracellular structure C is pressed from above (FIG. 8b). When the glass rod 63 is moved upward after the pressing, the intracellular structure C adheres to the flat portion at the tip of the glass rod (FIG. 8c). Next, the glass rod 63 is moved above the collection container 62 (FIG. 9d), and the glass rod 63 is lowered into the collection container 62 (FIG. 9e). The collection container 62 contains a small amount (for example, 5 to 20 μl) of a solution (for example, a buffer solution) for facilitating the collection. The glass rod 63 is lowered in the buffer solution, and the collection container 62 is further lowered. The intracellular structure C is released from the tip of the glass rod by appropriately performing an operation of bringing the attached intracellular structure C lightly into contact with the wall surface (for example, the bottom surface) of the glass rod one or more times. After the intracellular structure C is released, the glass rod 63 is moved upward to complete sampling (FIG. 9f). The collecting means is not limited to a glass rod, and other materials can be used. The sampling means can be attached to various microscope apparatuses equipped with an LMD device via a manipulator fixture and an operation knob that can be driven three-dimensionally (xyz direction). As shown in the figure, the LMD apparatus is inclined to extend to the vicinity of the specimen B so as to be out of the optical path by an optical system such as an objective lens of the LMD apparatus, and is about 0.5 to 3 cm from the tip of the sampling rod. By providing a straight portion whose length is along an axis parallel to the optical path (vertical direction in the figure), only the distal end surface of the sampling rod can be observed and access to the specimen B is facilitated. It becomes possible.

10 and 11
FIG. 10 and FIG. 11 are diagrams showing how the target site is collected on the display.

  On the display, the specimen B is projected in a fluorescent state. Each part of the target living cell A excluding the intracellular structure C is removed by the LMD method, and the intracellular structure C is cut out from the specimen B (FIG. 10a). In order to collect the intracellular structure C, the glass rod 63 having a flat tip is moved to the vicinity of the upper part of the intracellular structure C. At this time, since the tip 91 of the glass rod is projected on the display, it can be easily aligned (FIG. 10b).

  Next, by slowly lowering the tip 91 of the glass rod from above the intracellular structure C, the tip 91 is pressed against the bottom of the container to the extent that the intracellular structure is not destroyed (FIG. 10c). At this time, the intracellular structure C adheres to the vicinity of the center of the tip 91 of the glass rod in a semi-dry state as described above.

  Then, when the glass rod 63 is pulled up and moved, when the tip 91 of the glass rod moves, if the fluorescent image of the intracellular structure C disappears from the screen as shown (FIG. 11d), It can be confirmed that the intracellular structure C adheres to the tip of the glass rod and is taken out of the cell. After removing the intracellular structure C from the cell in this way, the glass rod 63 is moved toward a predetermined transfer destination while the intracellular structure C is attached to the glass rod tip 91 (FIG. 11e). Collect in another container.

  In this way, the operator can easily and reliably perform sampling by driving (automatically or manually) the sampling means while looking at the display.

(Second Embodiment)
As a sampling method in the second embodiment, there is a sampling method using a manipulator type suction machine after performing the above-mentioned LMD processing. Specifically, a manipulator equipped with an operation arm that can be driven and controlled at an arbitrary position in a three-dimensional space is attached with a suction machine (cuvette). While dropping pure water on the biological specimen, while observing the semi-intact target cell or intracellular structure (target part) separated from the specimen with a microscope, the suction port of the cuvette is brought close to the target part, Aspirate this. Thereafter, the aspirated target site is discharged into a PCR container containing a buffer solution and collected.

(Modification)
In the first embodiment, the above-mentioned semi-intacting is achieved by expanding the “step of imparting liquid permeability to the specimen and controlling the water content in the specimen” to the “step of controlling the moisture content of the specimen”. In addition, the method of the present invention can be applied to dead cells that create a semi-dry state and contain a large amount of water. For example, cryopreserved dead cells contain a large amount of water. By controlling the water content of dead cells upon thawing, dead cells to which the LMD method can be applied can be produced. In the present invention, as a method for obtaining a semi-dry state, an air dry method in which a gas in a dry state is blown onto a specimen, or heating that heats various supports for specimens such as containers (however, a high temperature that does not alter cells). Laws can be used. When similarly applied to living cells (or biological tissues) stored at a cryogenic temperature of minus 60 degrees or less, it is contained in a gas (eg, carbon dioxide) environment that keeps the ph of living cells good. It is preferable to control the amount of water. Therefore, the LMD method can also be applied to the analysis of pathological tissue pieces that have been cryopreserved, and a large number of pathological tissue pieces can be frozen and stored, and the analysis of each pathological tissue piece can be performed comprehensively at a later date. Become. On the other hand, when controlling the water content of freeze-dried cells (or biological tissues), the cells (or biological tissues) were once revived by restoring them with a sufficient amount of lysis solution. It is preferable to perform the process after the process of providing liquid permeability as described above.

Example 1
1. Transfection of plasmid into target cells <Devices used>
・ CO ₂ INCUBATOR (ESPEC)
・ 50mm diameter, 14mm measurement hold dish (Mat Tek # P50G-O-14-F)
(Dish is coated with 0.5mg / ml POLY-L-LYSINE beforehand)
<Cells, reagents, etc.>
- cells; mouse neuroblastoma Neuro-2a (ATCC # CCL- 131)
・ Plasmids: GFP-PrP ^c (Δ140-254) /pcDNA3.1
Opti-MEM I (GIBCO # 31985)
・ LipofectAMINE2000 (Invitrogen # 11668-019)
Antibiotic-free MEM medium (SIGMA # M5650)
・ POLY-L-LYSINE (SIGMA # P6282)
<Procedure>
(1) Spread cultured cells in a dish.

(I) the day before Transfection, sow the mouse neuroblastoma Neuro-2a (N2a) cells so as to be 1500 cells / dish.

(Ii) Add 3 ml / dish of antibiotic-free MEM medium and incubate at 37 ° C. for 24 hours in a CO ₂ incubator. (At this point, the cells are spread so as to be 30-50% confluent)
(2) Perform transfection (* according to the protocol attached to Lipofect AMINE2000)
(I) Add 300 μl of Opti-MEM I to 6 μl of Lipofect AMINE2000 and incubate at room temperature for 10 minutes.

  (Ii) Add 300 μl of Opti-MEM I to 3 μg of plasmid.

  (Iii) Gently mix both reagents (i) and (ii) and incubate at room temperature for 20 minutes.

  (Iv) During the 20-minute incubation, replace the cultured cell dish medium with 3 ml of fresh antibiotic-free MEM.

(V) Incubate for 4 hours at 37 ° C in a CO ₂ incubator.

(Vi) After 4 hours, replace with 3 ml of fresh antibiotic-free MEM and incubate at 37 ° C for 24 hours in a CO ₂ incubator.

2. Semi-intacting with streptolysin-O <Devices used>
・ Bouble Shaker NR-3 (TAITEC)
・ Water Bath (TAITEC)
・ LOW TEMP. INCUBATOR LTI-601SD (EYELA)
<Cells, reagents, etc.>
・ Cells in glass bottom dish ・ streptolysin-O (SIGMA # S5265)
· 5 × KOAc Buffer (575mM KOAc , 125mM Hepes-KOH (ph7.4), 12.5mM MgCl 2)
・ 1M DTT
・ 200mM EGTA
Cytosol removal buffer (1 × KOAc Buffer, 1mM DTT 2mM EGTA)
・ PBS (-)
<Procedure>
(1) Susplicin O is suspended at a concentration of 60 ng / ml in 1 × KOAc Buffer containing 10 mM DTT and incubated in a 37 ° C. water bath for 30 minutes (activation of streptricin O).

(Make it immediately before use. If there is a gap, place it at 4 ° C and use it within 1 hour. Store it in small portions at -80 ° C.)
(2) Activated 60 ng / ml streptricin O is added at 1.4 ml / dish and incubated at 4 ° C. for 10 minutes.

(3) Wash twice with ice-cold PBS (-).

(4) Add cytosol-removed buffer that has been brought to 37 ° C. at 3 ml / dish, and incubate at 37 ° C. for 5 minutes.

(5) Wash twice with cytosol removal buffer at room temperature.

(6) Add cytosol removal buffer at room temperature at 3 ml / dish and shake at room temperature for 10 minutes (30 rpm / min).

(7) Wash once with room temperature cytosol removal Buffer.

(8) Add room temperature cytosol removal buffer at 3 ml / dish and shake at room temperature for 10 minutes (30 rpm / min).

(9) Wash twice with PBS (-) at room temperature.

3. Creating semi-dry state Place the semi- intacted dish on the sample stage of a fluorescence microscope (fluorescence microscope IX71 / OLYMPUS) equipped with a laser microdissection device (Laser System HCL-2100 / HOYA) The image under the microscope was visually judged until.

4). Laser Digestion When the above-mentioned semi-dry state was reached, the desired cells were moved to the observation field, and a fluorescent image was displayed on the display. In this state, mitochondrial aggregates shined with GFP in the cells were manipulated in the same manner as described with reference to FIG.

5. Collection by Manipulator The aggregate after the above-described digestion was collected by the method and apparatus described with reference to FIGS. The specific equipment used was (i) “Coarse Manipulator ONM-1 / OLYMPUS / NARISHIGE JAPAN”, (ii) “3D Joystick Hydraulic Micromanipulator MNO-202N / NARISHIGE JAPAN”, (iii) “Cell Tram Air 5176 (Used partially) / eppendorf ". The tapered glass rod is heat-processed so that a portion of about 2 cm from the tip is bent at an angle of about 120 degrees and becomes a linear needle downward, and the lateral position of the upper objective lens of the above LCM device In addition, the needle unit was attached to a commercially available manipulator operation block capable of three-dimensional fine movement manually and operated under microscope observation. When adhering to the tip of the thin glass rod and when removing it from the collection container, it is necessary to repeat the vertical movement of the glass rod as appropriate, and follow it while tracking on the image. It was. The collection container after the attachment to the glass rod is fixed to the fixing means (manufactured by the inventor) shown in FIGS. 6 and 7 for the cap portion of a commercially available PCR tube (BIO-BIK T-02). Then, 5 μl of PBS (SIGMA DULBECCO'S PHOSPHATE BUFFERED SALINE) was used. At this time, the strap part that connects the tube main body and the cap is fixed without being separated from the main body, and in the fixed state, the cap is opened and closed by pushing and returning only the tube main body with a finger or the like. The collection container was quickly sealed.

3. Results About 5 μm to 15 μm intracellular aggregates (including GFP-PrP ^c (Δ140-254) and mitochondria) formed in mouse neuroblastoma Neuro-2a that became semi-dry by semi-intacting, The laser microdissection was able to cut out accurately. The semi-dry state can be said to have an effect of effectively reducing water evaporation during ablation while leaving a liquid amount necessary for optically observing the intracellular structure. As a result, it was possible to isolate the target intracellular structure from the living cells with high accuracy while maintaining the live activity.

  The intracellular aggregates collected by collection with the manipulator could be subjected to subsequent molecular biological analysis. Further, in the solution stored in the collection container, even if substances other than the released intracellular structure C (for example, cell membrane pieces) adhere to the glass rod, they can be separated separately or easily separated. There was no problem. Furthermore, since it was confirmed that GFP fluorescence was emitted from the collected intracellular aggregates, it was proved that accurate isolation and / or collection was possible while maintaining live activity. It was. Since these methods are possible for intracellular structures, it is needless to say that they can be similarly applied to single cells having larger dimensions. In addition, the method of the present invention is advantageous for cells having complex shapes (neural cells, cancer cells, etc.) and various types of cells whose dimensions are sufficiently smaller than mammals.

  On the other hand, when the intracellular structure C after the LMD treatment was sucked using a manipulator-type suction device in a prior study, it might adhere to the suction tube and was difficult to collect. For this reason, it was possible to reliably and efficiently recover a desired substance by adopting a method of collecting by adhering to the tip of a thin rod made of glass or the like and performing the recovery operation as described above.

Thus, it was confirmed that the method of the present invention can accurately isolate and collect not only cells but also intracellular structures while still maintaining the living activity. Cells or intracellular structures collected by the method of the present invention are subsequently pretreated (solubilization, centrifugation, etc.) in a predetermined container, and various analyzes (Western blotting, mass spectrometry, two-dimensional electrophoresis, PCR, etc.) Reaction etc.). In addition, since the mitochondria that form aggregates in cells as intracellular structures can be accurately isolated and collected, analysis of disease-related aggregated protein components can be realized for the first time by the present invention. Incidentally, the PrP ^c dealing in Example a prion disease associated protein (prion protein), the method of the present invention (and apparatus according to the above description) is maintained in an active state in which living studies neurological molecular level It is expected to contribute greatly to medical care such as research, drug discovery, and diagnosis.

As mentioned above, although this invention was demonstrated based on various embodiment and an Example, this invention is not limited to them, A various change etc. are possible within the range of the main point of the invention mentioned above. In addition, an apparatus for executing the method described above is also included in the present invention, and various changes and the like can be made based on the same gist. Furthermore, an operation method or a control program for operating the method described above using an apparatus for executing the method is also included in the present invention.
Hereinafter, the invention described in the scope of claims of the present application will be appended.
[1] A step of preparing a specimen containing living target cells or intracellular structures, a step of imparting liquid permeability to the specimen and controlling the water content in the specimen, and irradiating the specimen with laser And isolating the target living cell or intracellular structure from the specimen, and isolating the target living cell or intracellular structure.
[2] The method for isolating live target cells or intracellular structures according to [1], wherein the step of controlling the water content includes a step of obtaining a semi-dried specimen.
[3] The step of controlling the water content includes a step of performing semi-intacting such that a drug that forms a large number of micropores in a cell membrane is added to the specimen and a liquid component in the cell is taken out, and the semi-intacting step is performed. A method for isolating a target living cell or an intracellular structure according to [1] or [2], comprising a step of causing a liquid component in each cell to flow out of the cell.
[4] The step of separating includes a step of generating light from a photomarker substance previously incorporated in the target living cell or intracellular structure, and a step of determining a dissection location based on light emission of the photomarker substance And irradiating the specimen with a laser to separate the target living cells or intracellular structures from the specimen, and any one of [1] to [3] A method for isolating live target cells or intracellular structures as described.
[5] The live target cell or intracellular target according to [4], wherein the step of preparing the specimen includes a step of incorporating and visualizing a photomarker gene in the live target cell or intracellular structure. A method for isolating a structure.
[6] The method for isolating live target cells or intracellular structures according to [4], wherein the optical marker substance is a fluorescent marker substance.
[7] The target living cell or intracellular structure according to [4] or [5], wherein the step of emitting a light marker substance is an excitation light source having a wavelength different from that of the cutting-out step Isolation method.
[8] A step of preparing a specimen containing target cells or intracellular structures, a step of controlling the water content of the specimen, and a target living body in which a thin sampling rod having a flat tip is separated by a laser. A step of pressing the cell or an intracellular structure, and a step of releasing the target living cell or the intracellular structure attached to the flat tip portion in a collection container containing a solution. A method for collecting target living cells or intracellular structures.
[9] A step of preparing a specimen containing target cells or intracellular structures; a step of controlling the water content of the specimen; irradiating the specimen with a laser; A method for isolating a target cell or an intracellular structure, comprising a step of separating from a specimen.
[10] The specimen is pre-frozen or dried, and the step of controlling the water content increases the water content sufficiently once by using a melt or a solution for restoring from the dry state after freezing is thawed. The method for isolating a target cell or intracellular structure according to [9], wherein the water content is reduced to a low dry state after the treatment.

The flowchart figure which showed the method in the 1st embodiment The figure which showed typically the apparatus for performing the semi-interacting process in 1st Embodiment The figure which showed typically the procedure of the semi-intacting process using the apparatus of FIG. The figure which showed typically the LMD apparatus for performing the laser microdissection (LMD) process in 1st embodiment. The figure which showed typically the mode of LMD performed using the LMD apparatus of FIG. The figure which showed typically the collection system for performing the collection process in 1st embodiment Sectional view along line AA of the fixing means 65 of FIG. The figure which showed typically the collection process of the target site | part using the collection system shown in FIG. 6 and FIG. The figure which showed typically the collection process of the target site | part using the collection system shown in FIG. 6 and FIG. The figure which showed the situation of collection of the target part on the display The figure which showed the situation of collection of the target part on the display

Explanation of symbols

11 ... Preparation of specimen, 12 ... Semi-intacting treatment, 13 ... Laser microdissection, 14 ... Collection of target site, 21 ... Injector, 22 ... Dish, 23 ... Micropipetta, 24, 25 ... Recovery container, 26 ... CPU, 51 ... Laser spot, 62 ... Recovery container, 63 ... Glass rod, 64 ... Operating arm, 65: Fixing means, 71: Fixing table, 72: Cavity, 73: Fixing plate, 81: Laser irradiation means, 91: Tip of glass rod, 101 ... Illumination light source, 102 ... Field stop, 103 ... Aperture stop, 104 ... Dichroic mirror, 105 ... Laser objective lens, 106 ... Stage, 108 ... Dish, 109 ... Stage drive unit, 110 ... objective lens for observation, 111 Dichroic mirror, 112 ... UV barrier filter, 113 ... imaging lens, 114 ... half mirror, 115 ... CCD camera, 116 ... image processing and stage control device, 117 ... display , 118 ... Operation unit, 119 ... Mirror, 120 ... Relay lens, 121 ... UV cut filter, 122 ... Eyepiece lens, 123 ... UV laser head, 123a ... Laser light source 124 ... UV imaging lens, 125 ... variable slit, 126 ... LED, 127 ... dichroic mirror, A ... target living cell, B ... specimen, C ... intracellular Structure.

Claims (8)

Producing a specimen containing live target cells or intracellular structures;
Providing liquid permeability to the specimen and controlling the water content in the specimen;
Irradiating the specimen with a laser to separate the target living cells or intracellular structures from the specimen;
Only including,
The step of controlling the water content includes
Adding a drug that forms a large number of micropores in the cell membrane to the specimen, and performing semi-intacting such that a liquid component in the cell is extracted;
Allowing the liquid component in each semi-intacted cell to flow out of the cell;
A method for isolating live target cells or intracellular structures , comprising:   The method for isolating target living cells or intracellular structures according to claim 1, wherein the step of controlling the water content includes a step of obtaining a semi-dried specimen. The detaching step comprises:
Generating light from a photomarker substance pre-incorporated into the target living cell or intracellular structure;
Determining a dissection location based on light emission of the light marker material;
Irradiating the specimen with a laser to separate the target living cells or intracellular structures from the specimen;
The method for isolating live target cells or intracellular structures according to claim 1 or 2 , wherein The step of preparing the specimen includes
The method for isolating a target living cell or intracellular structure according to claim 3 , comprising a step of incorporating and visualizing a photomarker gene in the target living cell or intracellular structure. 4. The method for isolating live target cells or intracellular structures according to claim 3 , wherein the optical marker substance is a fluorescent marker substance. The method for isolating live target cells or intracellular structures according to claim 3 or 4 , wherein the step of causing the light marker substance to emit light is an excitation light source having a wavelength different from that of the cutting step. Producing a specimen containing live target cells or intracellular structures;
Imparting liquid permeability to the specimen and controlling the water content of the specimen;
Irradiating the specimen with a laser to separate the target living cells or intracellular structures from the specimen;
Pressing a small collection rod having a flat tip against target live cells or intracellular structures separated by a laser;
Releasing the target living cells or intracellular structures attached to the flat tip in a collection container containing a solution;
Only including,
The step of controlling the water content includes
Adding a drug that forms a large number of micropores in the cell membrane to the specimen, and performing semi-intacting such that a liquid component in the cell is extracted;
Allowing the liquid component in each semi-intacted cell to flow out of the cell;
How to collect the characterized by containing Mukoto, target cells or subcellular structures. The agent for forming a large number of micropores in the cell membrane is selected from the group consisting of bacterial toxins, bacterial exotoxins and toxins derived from eukaryotic cells. For isolation of target living cells or intracellular structures of

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