JP2008249543A - Methods for preparing cells from tissue specimens - Google Patents

Abstract

A method for separating and preparing cells from a tissue specimen is provided.
A method for preparing cells from a tissue specimen, comprising the following steps 1) to 3):
1) A step of heat-treating a tissue specimen in a citric acid solution at 60 to 80 ° C,
2) a step of treating the heat-treated specimen with a proteolytic enzyme; and
3) A step of dispersing the enzyme-treated cells in the specimen in a surfactant solution.
[Selection figure] None

Description

  The present invention relates to a method for preparing cells from a tissue specimen.

  By using numerous valuable specimens such as paraffin-embedded tissue specimens and frozen tissue specimens that exist innumerably throughout the world, as well as various human and animal-derived cells, genetic and biochemical experiments, etc. Elucidation of pathological conditions such as cancer, clinical research, and therapeutic method development research have been made. In general, tissue block pieces and sections in which different types of cells are mixed without separation of the cells in the tissue specimen are used as experimental materials, and then nucleic acids such as DNA and RNA are used with various cell melting agents. Samples, protein samples and the like are extracted and prepared, and genetic analysis and biochemical analysis using the samples have been widely performed. However, samples extracted from materials with many different types of cells and tissues mixed together contain various cell-derived components, so accurate data cannot be obtained and abnormalities appear in specific cells. Such pathological conditions cannot be elucidated. Recently, it has been widely proposed that pathological specimens and tissues must be analyzed by distinguishing the pathology of each type of cell. Therefore, it is necessary to develop a method for isolating cells (non-patented). References 1 and 2).

  Therefore, in recent years, methods for selecting and separating target cells under a microscope from various stained tissues and cell clusters of tissue samples to be studied have been studied. The ability to collect target cells and cell masses from tissue specimens without deformation or destruction is important for confirming whether the target cells have been selectively acquired. In addition, if the cells can be separated while maintaining their form without being destroyed, the risk of contamination of foreign substances into the cells can be eliminated, and there is no outflow of cell contents from damaged cell membranes. For example, components such as nucleic acids and proteins possessed by cells and their ratio can be measured more accurately (Non-patent Documents 1 to 3).

  One such method for separating and collecting cells is a method of scraping only desired cells from a tissue with a needle or the like under a microscope. However, it is quite difficult to pinpoint micro-sized target cells manually, and the success or failure of cell separation largely depends on the judgment and ability of the experimenter. In general, this method tends to cause contamination of other cells and tissues, and as a result, it can be said that there is a possibility of causing a serious problem in the accuracy of the experimental study itself. In addition, the collected cells are often deformed or damaged by the force applied during curettage, and the type of cells obtained is often difficult to identify, making it difficult to prove the accuracy of the research. This is also a problem (Non-Patent Document 1 and Non-Patent Document 4).

  The other is a method of sorting cells by a laser microdissection method using a laser microscope. In this method, a target tissue or cell in a tissue specimen or cell specimen is displayed on a monitor, a range is selected on the image, and a target cell or tissue is selectively collected by cutting out with a laser beam. In addition to the microscope, this method requires at least a laser beam device, a device that controls the laser beam device, a device that mechanically operates the microscope, a digital camera device, a monitor screen, and a precise computer device that controls these functions. Therefore, the cost required for selective collection of cells is enormous. In addition, with this method, the allowable range of setting in various conditions such as the degree of dryness of the specimen and the type of slide glass is narrow, and the collection of tissues and cells from specimens that do not strictly meet the conditions required by the model used is often accurate. There is a problem that is not done. For this reason, the tissue specimen which can apply this method is limited, and cannot be widely used with respect to various specimens (Non-patent Documents 1 and 4).

  Furthermore, in this method, since the selection area to be cut out on the monitor image of the two-dimensional image is set, it is impossible to determine the state in which the cells are overlapped in three dimensions, and other cells are collected together and contaminated. Nation may occur. In addition, when collecting cells with protrusions, irregular cells, cells with a network structure such as the vascular structure, etc., it is difficult to accurately grasp the boundaries of the cells on the screen, so the entire cell is accurately isolated. It is difficult to do. Therefore, it is difficult to use for the overall functional analysis of those cells (Non-patent Document 2).

  Further, in the laser microdissection method, there is a process of skipping a cut-off portion when a target cell is collected, and the portion to be collected is also skipped, and cell deterioration is also a problem. Even if it is a method for dealing with it, there are problems that the procedure is complicated and the sample is wasted, such as an attempt to repeatedly peel and paste the cells from the glass slide or the substrate in many stages (non-patent) Document 5 and Patent Document 1).

  On the other hand, a method for directly extracting a nucleic acid sample or a protein sample from a paraffin-embedded tissue specimen or a frozen tissue specimen is also known. For example, a paraffin-embedded tissue specimen is deparaffinized and then treated in a solution containing a surfactant and a proteolytic enzyme at room temperature to 50 ° C. for 4 to 48 hours to destroy tissues and cells, and then phenol or chloroform After adding and mixing organic solvents such as, and separating them into an aqueous phase containing nucleic acid and an organic phase containing denatured protein, the aqueous phase is collected, and nucleic acid is obtained from the aqueous phase by alcohol precipitation. (Non-Patent Document 6). However, since this method uses a proteolytic enzyme, the cell destruction treatment is performed under relatively mild conditions, so that there is a problem that the treatment must be performed for a long time in order to obtain a sufficient nucleic acid recovery rate. . In contrast, deparaffin-treated paraffin-embedded tissue specimens are rapidly destroyed in the presence of a surfactant at a high temperature close to the boiling point, and then the cells are destroyed in a short time, followed by treatment with a protein hydrolase, There is also a method for obtaining a nucleic acid sample by denaturing a protein with an organic solvent and then precipitating the nucleic acid (Patent Document 2). However, since this method uses a technique for efficiently destroying cells in a short time, the cells themselves cannot be isolated.

  By the way, immunohistological staining is often used for tissue analysis of formalin-fixed paraffin-embedded tissue specimens. In immunohistochemical staining, antigens on tissues and cells to be detected by the primary antibody are masked by tissue-derived components that have been denatured or cross-linked during immobilization or paraffin embedding, thereby allowing reaction with the primary antibody. A phenomenon that decreases and the detection signal weakens is frequently observed. In order to solve this problem, a method of activating antigenicity by placing a tissue sample in a citrate buffer and heating it at a high temperature of about 100 ° C. or higher with an autoclave or microwave is known (Non-patent Document). 7). However, in this method, cell denaturation and nucleic acid damage occur at high temperatures.

  Thus, the development of a method suitable for intact isolation of cells from tissue specimens as described above has not yet been successful.

Japanese Patent No. 3820227 JP-A-8-70892 Simone N. L. et al., Trends in Genetics, (1998) 14: p.272-276 Hunt, J. L. and Finkelstein, S. D., Archives Pathology & Laboratory Medicine (2004) 128: p.1372-1378 Emmer-Buck et al., Science (1996) 274: p.998-1001 Curran, S. et al., J Clinical Pathology-Molecular Pathology (2000) 53: p.64-68 Banks, R.E. et al., Electrophoresis (1999) 20, p.689-700 Experimental Medicine, Yodosha, (1990) Vol. 8, No. 9, pp. 84-88 Shi, S-R. Et al., J Histochemistry & Cytochemistry (1991) 39: p.741-748

  It is an object of the present invention to provide a method for preparing cells from a tissue specimen in a state in which the cells can be isolated.

  As a result of intensive studies to solve the above problems, the inventors of the present invention caused a citric acid solution and a proteolytic enzyme in a suitable temperature range to act on a tissue specimen in two stages, and further included cells contained in the specimen. By dispersing in a surfactant solution, the cell-cell bonds in the tissue specimen are gently broken down, and the cells are well-separated from other types of cells and the shape is also kept well dispersed. The present inventors have found that a cell dispersion liquid can be prepared and have completed the present invention.

That is, the present invention includes the following.
[1] A method for preparing cells from a tissue specimen, comprising the following steps 1) to 3):
1) A step of heat-treating a tissue specimen in a citric acid solution at 60 to 80 ° C,
2) a step of treating the heat-treated specimen with a proteolytic enzyme; and
3) A step of dispersing the enzyme-treated cells in the specimen in a surfactant solution.

In this method, the heat treatment in a citric acid solution is more preferably performed for 5 to 30 minutes.
Preferably, the method further comprises tissue staining of the specimen.
The tissue specimen may be fixed.
A suitable example of the citrate solution is a citrate buffer.

The proteolytic enzyme is preferably at least one selected from the group consisting of trypsin, collagenase, dispase, papain, elastase, hyaluronidase, chymotrypsin, and pronase.
In this method, it is also preferable to treat the specimen using EDTA together with a proteolytic enzyme.

  The surfactant may be at least one selected from the group consisting of Tween 20, Tween 80, Triton X-100, Triton X-114, NP-40, Brij 35, octaglycoside, pluronic, and sucrose monolaurate. preferable.

[2] A method for analyzing cells in a tissue sample, comprising preparing a cell dispersion from a tissue sample by the method of [1] above, collecting cells from the sample, and using the cells for analysis.

[3] A kit for preparing cells from a tissue specimen, comprising a citric acid solution, a proteolytic enzyme, and a surfactant solution.

  The cell preparation method of the present invention can be prepared in a state in which many cells are well separated from a tissue specimen while minimizing damage to cells by a simple technique.

Hereinafter, the present invention will be described in detail.
In the present invention, a tissue specimen prepared from a biological sample is allowed to act in two stages with a citric acid solution in a suitable temperature range and then with a proteolytic enzyme, and then the cells contained in the specimen are dispersed in the surfactant solution. This provides a method capable of preparing cells in a well dispersed state while minimizing damage to the cells. In this method, for example, a target cell or cell mass can be selectively separated and collected while confirming dispersed cells under an inverted microscope.

  In the present invention, a “tissue specimen” refers to a biological sample containing cells such as collected organs, tissues, or cell populations, which has been subjected to any preservation treatment used in the fields of biology, histology, and the like. .

  The tissue specimen according to the present invention may be prepared from any biological sample. For example, the tissue specimen may be a tissue or cell mass collected for human pathological diagnosis or cytodiagnosis, a tissue or cell mass of an animal such as a mouse, a cell mass of a cultured cell derived from a human or other animal, or a cultured cell. It may be prepared from a tissue or a cell mass differentiated from a sample. The tissue specimen may be derived from any animal, for example, primates such as humans, gorillas, baboons, Japanese monkeys, pets such as dogs and cats, domestic animals such as horses, cattle, sheep, mice, rats Mammals including rodents such as guinea pigs and hamsters, fish such as carp, tuna and salmon, birds such as chickens, bunchos and ducks, and reptiles such as snakes and lizards. It is not something. The tissue specimen may be prepared from a normal tissue such as lymph node, brain, lung, thymus, kidney, bladder, stomach, muscle, intestine, mucous membrane, embryonic tissue, or a tumor originating from such tissue. It may be prepared from a tissue or cancerous tissue. Alternatively, the tissue specimen may be blood, lymph, semen, mucus, sputum, urine, ascites, pleural effusion, abscess, a gynecological organ, or a smear specimen from the bronchi. The tissue specimen may contain tumor cells, cancerous cells or pathogen-infected cells (virus-infected cells, bacterial-infected cells, etc.) in addition to normal cells.

  It is more preferable that the tissue specimen according to the present invention has been subjected to an arbitrary fixing process as is usually used in the field of histology. Fixation in the field of histology means that the whole or part of an organism (organ, tissue, cell, intracellular molecular structure, etc.) is prevented from breaking, self-degrading, decay, etc. To stop the dynamic changes that normally occur in living organisms so that they are preserved as close as possible to the state of life. The fixing process is generally performed by a water freezing process or a protein denaturing process. Fixation by freezing of water can be generally performed by rapid cooling or freeze drying. In addition, fixation by protein denaturation treatment can be performed by heating or treatment using a fixing agent. Fixing agents for tissue fixation are well known in the fields of biology and histology, and usually drugs known as protein coagulants and protein cross-linking agents are used. The fixing agent generally includes a solution (fixing solution) containing the fixing agent, for example, other components such as a pH adjusting agent, an osmotic pressure adjusting agent, a viscosity adjusting agent (such as sucrose) as appropriate in addition to the fixing agent. Applied in the form of a solution. Examples of fixatives include, for example, formalin (eg, neutral buffered formalin), glutar / aldehyde, formalin / alcohol, alcohol (ethanol, methanol), formaldehyde, paraformaldehyde, picric acid, Bouin, Zenker Liquid, Hely liquid, osmium liquid, or Carnoy liquid. The tissue specimen of the present invention may be dehydrated after being fixed. The scope of the present invention includes the use of either fixed or non-fixed tissue specimens.

  The tissue specimen of the present invention may be further embedded with an embedding agent (paraffin, methacrylic acid resin, etc.) (embedded tissue specimen), or may be frozen ( It may be a frozen tissue specimen) or a smear specimen. The tissue specimen of the present invention may be, for example, a paraffin-embedded tissue specimen, or in particular, a paraffin-embedded tissue specimen fixed with formalin or the like. In the case of an embedded tissue specimen, it is preferable to use the method of the present invention after removing the embedding agent to some extent in advance. For example, it is preferable to apply the method of the present invention after deparaffinizing a paraffin-embedded tissue specimen. Deparaffinization involves immersing a paraffin-embedded tissue specimen in a solvent (such as xylene, D-limonene, or octane) that can dissolve paraffin, and replacing the solvent several times with a new one. It can be performed by conventional methods such as methods. The amount of the solvent used is usually an amount that allows the tissue specimen to be sufficiently immersed. The tissue specimen after the deparaffinization treatment is preferably hydrophilized by a method of sequentially immersing in a water dilution series of ethanol.

  In the method of the present invention, the tissue specimen is preferably cut into 0.1 to 100 μm, more generally 1 to 50 μm, typically about 1 to 10 μm thick sections, blocks, and the like. In addition, the tissue specimen is not limited, but is preferably subjected to a citric acid solution treatment and a proteolytic enzyme treatment in a state mounted or accommodated on a support carrier for convenient observation. As the support carrier, substrates, containers, base materials and the like of various shapes and materials can be used, but those having transparency capable of microscopic observation are preferable, and heat resistance (preferably 60 ° C. to 90 ° C. or What has the heat resistance with respect to the temperature beyond it is preferable. The support carrier is not limited, but may be made of glass, plastic, or resin, for example. Specific examples of the support carrier include glass plates, slides such as slide glasses, cover glasses, chip substrates, petri dishes, test tubes, and the like. In order to prevent detachment of tissues and cells, it is more preferable to use a support with a cell detachment prevention coating such as a silane coating as these support carriers. For mounting on a support carrier, for example, a tissue section such as a paraffin-embedded section subjected to deparaffinization (paraffin removal) is placed on a support carrier such as a glass slide, pasted by progress and drying, and then citrated. It is preferable to use for the heat processing in a solution. A frozen tissue section, a smear sample, or the like attached on a slide glass can be subjected to a heat treatment in a citric acid solution as it is.

  In the method of the present invention, the tissue specimen as described above is heat-treated in a citric acid solution at 60 to 80 ° C., more preferably 65 ° C. to 80 ° C., more preferably around 75 ° C. (73 to 77 ° C.). . Specifically, this heat treatment can be performed by immersing the tissue specimen in a citric acid solution maintained at the above heating temperature, for example, 60 to 80 ° C.

  The citric acid solution used here may be any aqueous solution containing citric acid. For example, a citric acid aqueous solution, a salt-containing aqueous solution to which citric acid is added, a citrate buffer, or the like can be used. Suitable citric acid solutions contain, for example, 0.1 mM to 100 mM, more preferably 1 to 30 mM, typically 10 mM citric acid. As the citric acid solution according to the present invention, it is preferable to use a solution in which acidity (pH) and osmotic pressure are adjusted so as not to damage cells and tissues. In this respect, the citrate buffer can be particularly preferably used in the method of the present invention. The pH of the citric acid solution used in the present invention can be varied in the range of pH 2 to 12 depending on the state of the tissue specimen, but is preferably pH 5 to 8, more preferably pH 6 to 7. For example, the citrate buffer used in the present invention can be prepared by mixing an aqueous citric acid solution and a citrate (sodium citrate, potassium citrate, etc.), and citric acid can be prepared with water (purified water, It can also be prepared by adding a hydroxide (NaOH, KOH, etc.) to adjust the pH to an aqueous citric acid solution dissolved in pure water, etc., but is not limited to these, and those skilled in the art will have an appropriate formulation. Can be prepared. Such a citric acid solution according to the present invention has a mild proteolytic action.

  The time for heat-treating the tissue specimen in the citric acid solution may vary to some extent depending on the concentration of the citric acid solution and the condition of the tissue specimen, but is usually 5 to 30 minutes, preferably 10 to 20 minutes, typically It may be 15 minutes.

  In this way, tissue samples are heat-treated in a citric acid solution at a temperature below 100 ° C., thereby suppressing the denaturation of nucleic acids and proteins in the cells and preventing the destruction of the cells. Can significantly weaken the binding between cells.

  In the method of the present invention, the tissue specimen that has been heat-treated in the citric acid solution as described above is further treated with a proteolytic enzyme. Degradation of intercellular bonds is promoted by allowing a proteolytic enzyme to act on a tissue specimen whose intercellular bonds have been weakened by heat treatment in a citric acid solution.

  The proteolytic enzyme used here is preferably an enzyme with a mild action that digests cell membranes and the like as much as possible while decomposing intercellular bonds. As such a proteolytic enzyme, those known to those skilled in the art as enzymes used in cell dispersing agents can be used. Suitable proteolytic enzymes include, but are not limited to, trypsin, collagenase, dispase, papain, elastase, hyaluronidase, chymotrypsin, pronase and the like. These enzymes are manufactured by invitrogen and can be obtained as commercial products.

  The proteolytic enzyme as described above is preferably applied to a tissue specimen as a proteolytic enzyme-containing solution contained in an aqueous solution such as water, a buffer solution, or a salt-containing solution. As the buffer solution, a phosphate buffer solution (for example, PBS), tris (hydroxymethyl) aminomethane (Tris) buffer solution, glycine buffer solution, good buffer solution and the like can be used. The concentration of the buffer such as phosphate in the proteolytic enzyme-containing solution is preferably 1 to 100 mM. The salt-containing solution is not limited, but is an aqueous solution containing any salt such as sodium chloride, calcium chloride, magnesium chloride, lithium chloride, for example, physiological salts such as physiological saline, Ringer's solution, Tyrode's solution, etc. A solution can be used. The concentration of the salt in the proteolytic enzyme-containing solution is preferably 1 to 500 mM. Such a proteolytic enzyme-containing solution preferably contains 0.01% to 10% (mass%), more preferably 0.1 to 1.0% (mass%) of the proteolytic enzyme with respect to the total amount of the solution. However, it is not limited to these. The pH of the proteolytic enzyme-containing solution can be appropriately adjusted between pH 2 and 12 depending on the working pH range of the proteolytic enzyme and other conditions, but is preferably pH 5 to 8.

  Proteolysis can be achieved by further adding an additional component such as a nuclease inhibitor or RNAase inhibitor for preventing nucleic acid degradation or a chelating agent for promoting dissociation of ionic bonds or protein-protein bonds to the above-mentioned proteolytic enzyme-containing solution The tissue specimen may be processed using the additional components along with the enzyme. Examples of the nuclease inhibitor include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), and salts thereof. As the additional component, EDTA is particularly suitable. EDTA may be added to the proteolytic enzyme-containing solution, for example, as a sodium salt of EDTA. EDTA not only functions as a nuclease inhibitor but also functions as a chelating agent, and can promote dissociation of ionic bonds and protein-protein bonds existing between cells or between cells and other coexisting substances. An additional component such as EDTA may be contained in the proteolytic enzyme-containing solution so as to have an appropriate concentration. For example, the EDTA concentration in the proteolytic enzyme-containing solution is usually 0.005 to 200 mM, preferably 0.01 to 10 mM, more preferably 0.1 to 5 mM.

  A preferred specific example of the proteolytic enzyme-containing solution according to the present invention is a solution containing trypsin and EDTA, for example, 0.25% trypsin-1 mM EDTA · 4Na solution (this may be an aqueous solution or a physiological saline solution). May be, but is not limited to).

  The proteolytic enzyme-containing solution as described above is preferably stored at −5 to −20 ° C. and thawed immediately before use in order to prevent inactivation due to self-digestion. Thereby, the proteolytic enzyme-containing solution can be used while maintaining the activity.

  Although the proteolytic enzyme treatment of the tissue specimen is not limited, for example, the above-described proteolytic enzyme-containing solution is added (upper layer) to the tissue specimen heat-treated in the citric acid solution, or the proteolytic enzyme It can be carried out by immersing the tissue specimen in the containing solution and incubating at the working temperature (preferably optimum temperature) of the proteolytic enzyme. The temperature of the proteolytic enzyme treatment can be appropriately set according to the enzyme used by those skilled in the art, but can be usually 30 to 40 ° C., typically 37 ° C. The time for the enzyme treatment may be appropriately set according to the enzyme used, but it is preferably 20 minutes to 120 minutes, more preferably 40 minutes to 90 minutes.

  By this proteolytic enzyme treatment, cell-cell bonds in the tissue specimen are further degraded, and cells are easily separated from the tissue specimen. In particular, the bonds between cells of different tissues, organs or cell types are broken down preferentially and better because they are weaker than the bonds between cells of the same organ, tissue or cell type. However, unless an extra external force is applied to the tissue specimen, the tissue specimen after the proteolytic enzyme treatment can still maintain the tissue structure before the citrate solution treatment and the proteolytic enzyme treatment. By observing under an optical microscope such as an inverted microscope, it is possible to identify cells constituting various biological structures (for example, blood vessel walls) in the tissue specimen.

  Next, in the method of the present invention, the cells in the tissue specimen that have been treated with the citrate solution and treated with the proteolytic enzyme and in which the cells contained therein are easily separated are treated with the surfactant solution. Disperse in. For example, a surfactant solution is added to the tissue specimen (upper layer), and the tissue specimen is scraped with a needle or the like in the solution to easily float cells or cell masses that have been separated between cells in the solution. Can be made. Alternatively, cells or cell clusters scraped from the tissue specimen with a needle or the like may be added to the surfactant solution. At this time, it is also preferable to take out a part containing many target cells from the tissue specimen while observing under a microscope and disperse it in the surfactant solution. The surfactant solution containing the cells or cell mass obtained in this way is collected as necessary, and gently mixed by, for example, pipetting, and the bonds between the cells that have become very brittle further dissociate, As a result of the cells being isolated individually or released into solution as small cell clumps, the cells can be dispersed in the detergent. In the cell dispersion prepared as described above, a large amount of small cell clusters composed of isolated cells and cells of the same cell type are present in a good dispersion state.

Here, as the surfactant, it is preferable to use a nonionic surfactant or an amphoteric surfactant that has a low protein denaturation action and exhibits a mild action. Examples of such surfactants include, but are not limited to, Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Triton X-100 (Polyoxyethylene (10) octylphenyl ether), Triton X-114 (polyoxyethylene (8) octylphenyl ether), NP-40 (polyoxyethylene (9) octylphenyl ether), Brij 35 (polyoxyethylene ( 23) lauryl ether), octa glycosides (e.g., octyl glucopyranoside, and octyl thioglucopyranoside), Pluronic (Pluronic ^(R);. polyoxyethylene - polyoxypropylene block copolymers e.g., Pluronic F127, Pluronic F68, etc. Pluronic P105), Sucrose monolaurate is mentioned.

The surfactant solution used in the present invention is preferably one in which the above-described surfactant is contained in an aqueous solution such as a buffer solution or a salt-containing solution. As the buffer solution, a phosphate buffer solution (for example, PBS), tris (hydroxymethyl) aminomethane (Tris) buffer solution, glycine buffer solution, good buffer solution and the like can be used. The concentration of a buffering agent such as phosphoric acid in the surfactant solution is preferably 1 to 100 mM. The salt-containing solution is not limited, but is an aqueous solution containing any salt such as sodium chloride, calcium chloride, magnesium chloride, lithium chloride, for example, physiological salts such as physiological saline, Ringer's solution, Tyrode's solution, etc. A solution can be used. The concentration of the salt in the surfactant solution is preferably 1 to 500 mM. Such a surfactant solution may contain a surfactant in an amount of preferably 0.001% to 0.1% (mass%), more preferably 0.01 to 0.05% (mass%) based on the total amount of the solution. However, it is not limited to these. The pH of the surfactant solution can be appropriately adjusted between pH 2 and 12, but is preferably pH 5 to 8. Suitable examples of this surfactant solution include Tween20-containing buffer, for example, phosphate buffer containing 0.02% Tween20 (0.02% Tween20 / PBS; composition: 0.02% Tween20, 10 mM PBS, pH 7.4). It is done. The PBS used here can be easily prepared by those skilled in the art. For example, NaCl 8 g, Na ₂ HPO ₄ 1.1 g, KCL 0.2 g, KH ₂ PO ₄ 0.2 g are mixed with purified water, and the total is measured. A method of preparing to 1 L is also preferable.

  It is also preferable that the surfactant solution further contains an additional component such as a nuclease inhibitor or RNAase inhibitor for preventing nucleic acid degradation and a chelating agent for promoting dissociation of ionic bonds and protein-protein bonds. Examples of the nuclease inhibitor include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), and salts thereof.

  Dispersing the cells from the tissue specimen treated above in the surfactant solution prevents the cells from adhering to the container or the collection equipment or floating unstablely in the solution, and the dispersion state is good. A stable cell dispersion that is maintained can be obtained.

In the cell preparation method of the present invention as described above, before and after the citric acid solution treatment step and the proteolytic enzyme treatment step, and before the step of dispersing the cells in the surfactant solution, Alternatively, it may be washed or permeabilized with a salt-containing solution or the like. Examples of the buffer used here include, but are not limited to, a phosphate buffer (eg, PBS), a tris (hydroxymethyl) aminomethane (Tris) buffer, a glycine buffer, and a good buffer. , Phosphate buffer is preferred, phosphate buffered saline (PBS) is more preferred, 10 mM PBS (pH 7.4) (eg, NaCl 8 g, Na ₂ HPO ₄ 1.1 g, KCL 0.2 g, KH ₂ PO ₄ 0.2 g + More preferably, it is prepared to 1 L of purified water. Examples of the salt-containing solution include, but are not limited to, an aqueous solution containing any salt such as sodium chloride, calcium chloride, magnesium chloride, lithium chloride, for example, physiological saline solution such as physiological saline, Ringer's solution, Tyrode's solution, etc. Can be used.

  In the cell preparation method of the present invention, it is also preferable to tissue-stain a tissue specimen in order to facilitate cell observation. In the present invention, “tissue staining” means staining a specific site or structure of a cell or tissue using a staining method such as that used in the field of histology or the like. Tissue staining may be any histological staining method, for example, hematoxylin / eosin staining, Papanicolaou staining, Giemsa staining, May Giemsa staining, PAS staining, Alcian blue staining, mutical carmine staining, methyl green / pyronin staining, Sudan III staining , Grimelius staining, Gram staining, Grocott staining, immunostaining (ABC method, PAP method, LAB method, LSAB method) and the like. In hematoxylin / eosin staining, negatively charged biological sites are stained purple to light red with hematoxylin dye, for example, cell nucleus is purple to blue purple, lime, cartilage tissue, mucus, etc. are stained from red to light red On the other hand, negatively charged cytoplasm, cell stroma, various fibers, erythrocytes, keratinocytes, etc. are stained red to dark red by the eosin dye. This hematoxylin and eosin staining makes it possible to clarify the whole structure of the tissue structure under a microscope and to identify individual structures constituting the tissue and cells contained therein. In immunostaining, for example, antibodies against cell surface markers possessed by specific cells (for example, antibodies that specifically bind to cell surface marker CD34 possessed by vascular endothelial cells) are used as primary antibodies in tissue samples. The primary antibody is bound to a specific cell, and the primary antibody is captured using an antibody against the primary antibody (secondary antibody), and the binding reaction is detected by a conventional method to identify the presence of the specific cell. Will be able to. At the time of this immunostaining, for example, by using a common staining method such as hematoxylin staining, the specific cells can be distinguished from other cells in the tissue. For details on the various tissue staining methods as described above, for example, “All New Staining Methods [Medical Technology], separate volume” (published by Medical and Dental Medicine, 1999.4) can be referred to.

  The tissue staining step can be performed at any point in the method for preparing cells from the tissue specimen. For example, hematoxylin / eosin staining or hematoxylin staining (cell nucleus staining) is not limited, but can be performed after proteolytic enzyme treatment and before cell dispersion in a surfactant solution. The immunostaining is not limited, but is preferably performed after the citric acid solution treatment and before the proteolytic enzyme treatment. This is because the effect of antigen activation can also be obtained by heat treatment with a citric acid solution.

  In the present invention, the target cells can be easily separated and collected using the cell dispersion obtained by the method for preparing cells from the tissue specimen as described above. The cell dispersion may appropriately adjust the cell density by adding the above surfactant solution. By observing such a cell dispersion preferably under a microscope such as an inverted microscope, the cells of interest were isolated on a cell-by-cell basis, based on tissue staining as described above or based on cell morphology, etc. It can be found as a small cell mass composed of cells of the same or the same cell type. The found target cells can be selectively separated and collected from the cell dispersion liquid by a suction operation using a microneedle, a capillary tube or the like. Alternatively, when a small amount of unnecessary cells other than the target cells are mixed in the cell dispersion, only the target cells can be collected by selectively collecting and removing the unnecessary cells. it can. The target cell may be any cell that can be included in the tissue specimen. For example, but not limited to, vascular endothelial cells, Hodgkin cells, lymphoma cells, glandular epithelial cells, acinar cells, muscle cells, fibrocytes, neurons, adenocarcinoma cells, sarcoma cells, melanoma cells, macrophages, lymphocytes Can be suitably separated and collected from a tissue specimen by the method of the present invention.

  In the method of the present invention, the cells can be individually collected while dissociating the bonds between the cells and dispersing the cells individually or as small cell clumps in the solution and confirming the cell image with a microscope. For example, even when cells with protrusions or irregular cells are collected, they can be collected in a state of being reliably separated from surrounding cells.

  The target cells selectively separated and collected by the method of the present invention are used for any analysis using the cells, such as cytological, molecular biological, genetic, biochemical, or microbiological analysis. be able to. More specifically, for example, by extracting nucleic acids and proteins from the collected target cells by a conventional method and analyzing them, any properties such as genetic characteristics and biochemical characteristics of the cells are analyzed. Can be analyzed. Alternatively, in the case where the cell is infected with a pathogen such as a bacterium or virus, the pathogen can be extracted from the cell and analyzed. By analyzing such cells (including cytological, molecular biological, genetic, biochemical, or microbiological analysis), the characteristics of the cells present in the tissue specimen are examined in detail. Therefore, for example, detailed analysis can be performed on cancer cells contained in a tissue specimen of a specific cancer patient. The present invention also provides a method for analyzing cells by separating and collecting cells using the method for preparing cells from a tissue specimen as described above and using it for analysis. Such cell analysis methods can be easily and widely used for cytological, molecular biological, genetic, microbiological, biochemical analysis, etc., and can easily analyze many specimens and cases. Therefore, for example, it is possible to more easily carry out a large-scale detailed disease state analysis.

Two examples of specific experimental procedures for preparing a cell in a separated state from a tissue specimen and further separating and collecting the target cell by the method of the present invention are given below. However, the present invention is not limited to these examples.
1) For immunostaining (ABC method) (human tissue specimen)
1. Affix the paraffin-embedded section to a silane-coated glass slide with a thickness of 5 μm.
2. Deparaffinize with xylene and ethanol.
3. Wash with water and infiltrate PBS.
4. Soak in 15 mM 10 mM citrate buffer (PH 6.0) for 15 minutes.
5. PBS wash, 3 times 5 minutes
6. Perform blocking treatment if necessary. Guard around the tissue with Dako Pen.
7. Primary antibody reaction
8. PBS wash, 5 minutes 3 times
9. Secondary antibody (biotin labeling) reaction
10. PBS wash, 3 times 5 minutes
11. Peroxidase-labeled streptavidin reaction
12. PBS wash, 3 times 5 minutes
13. DAB coloring
14. Wash with water and infiltrate PBS.
15. Place a sufficient amount of 0.25% trypsin-1 mM EDTA · 4Na solution that maintains the activity immediately after thawing on the tissue and place on a hot plate set at 37 ° C for 60 minutes. At this time, in order to prevent drying, a cover is placed with damp paper. It can be left in the incubator for 60 minutes.
16. PBS wash, 5 minutes 3 times
17. Hematoxylin nuclear staining
18. Wash with water and infiltrate PBS
19. Place 0.02% Tween20 / PBS on the tissue.
20. While observing with an inverted microscope, gently scrape the site containing a large amount of the target stained cells with the tip of the needle and float on the supernatant in 0.02% Tween20 / PBS.
21. Pipette 0.02% Tween20 / PBS containing the cells, pipet it so that it does not foam several times in the tip, and transfer to a petri dish.
22. While observing with an inverted microscope, dilute with 0.02% Tween20 / PBS to a density that makes it easy to absorb cells with a syringe with a needle of 27G or less.
23. Wait for the cells to sink to the bottom, making it easier to observe and collect, and aspirate with a syringe with a needle of 27G or less.
24. If another cell is contaminated, transfer to another petri dish, dilute with 0.02% Tween20 / PBS, and selectively aspirate. Alternatively, unnecessary cells may be aspirated and discarded.
25. Refer to the number of cells recommended by the ready-made DNA or RNA extraction kit to be used.

2) For hematoxylin and eosin staining (human tissue specimen)
1. Affix the paraffin-embedded section to a silane-coated glass slide with a thickness of 5 μm.
2. Deparaffinize with xylene and ethanol.
3. Rinse with water and phosphate buffer (PBS).
4. Soak in 15 mM 10 mM citrate buffer (PH 6.0) for 15 minutes.
5. PBS wash, 3 times 5 minutes
6. Guard around the tissue with Dako Pen so that the solution does not spill from the glass slide in the next step 7.
7. Place a sufficient amount of 0.25% trypsin-1 mM EDTA · 4Na solution that maintains the activity immediately after thawing on the tissue, place on a hot plate set at 37 ° C, and place for 60 minutes. At this time, in order to prevent drying, a cover is placed with damp paper. You may leave it in the incubator for 60 minutes.
8. PBS wash, 5 minutes 3 times
9. Stain hematoxylin and eosin. It is easier to observe the cells until step 15 if the eye is stained with dark eyes.
10. After washing with water, infiltrate into PBS.
11. Place 0.02% Tween20 / PBS on the tissue.
12. Under an inverted microscope, gently scrape the site containing a large amount of the target cells with the tip of the needle and suspend it in the supernatant 0.02% Tween20 / PBS.
13. Pipette 0.02% Tween20 / PBS containing the cells, pipet them up to several times in the tip and transfer to a petri dish. For pipette tips, petri dishes, and microneedles, it is preferable that 0.02% Tween20 / PBS be flowed in advance in order to reduce cell adhesion.
14. Under an inverted microscope, dilute the cells with 0.02% Tween20 / PBS to obtain a density that can be easily absorbed with a syringe with a microneedle of 27G or less.
15. Wait for the cells to sink to the bottom and make it easier to observe and collect, then aspirate with a syringe with a needle of 27G or less.
16. If another cell is contaminated, transfer to another petri dish, dilute with 0.02% Tween20 / PBS, and perform selective aspiration. Alternatively, unnecessary cells may be aspirated and discarded.

  Furthermore, the present invention also provides a kit for preparing cells from a tissue specimen, comprising the citric acid solution, the proteolytic enzyme, and the surfactant solution described above.

  The citric acid solution, proteolytic enzyme, and surfactant solution may preferably be sealed in a purified and purified state, sterilized state, or nuclease removed state. In order to simplify and prevent contamination, these reagents may be dispensed and packaged in a predetermined amount in advance. Such a kit of the present invention may further include necessary equipment such as microneedles for separating and collecting cells. By using such a kit, a series of experiments can be performed simply, rapidly and precisely. This kit is much cheaper, can be used much more easily and easily than the price of a laser microdissection apparatus conventionally used for cell separation from tissue specimens.

  By using the present invention, a target cell can be selected from a tissue specimen by using an inexpensive and simple process under a microscope without using a special precision instrument or the like, without mixing cells or tissues other than the target cell. Can be separated and collected.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1] Separation of cells from tissue specimen 1-Use of immunostaining (ABC method) In this example, formalin-fixed paraffin-embedded specimens of lymph nodes excised from human lymphoma patients were used for vascular endothelial cells (anti-CD34 antibody). And Hodgkin cells (mononuclear large spherical cells with large nucleolus and detectable with anti-VEGF antibody) were selectively separated and collected. FIG. 1 exemplarily shows a result of observing vascular endothelial cells as the dissociation of cells proceeds.

First, a 5 μm-thick paraffin-embedded section obtained from the above sample was placed on a silane-coated slide glass (manufactured by Matsunami), dried and pasted by a conventional method. Next, the paraffin-embedded slide glass was placed in xylene (about 150 μl), soaked at room temperature for 15 minutes, exchanged xylene, and soaked twice for 3 minutes for deparaffinization. Subsequently, the deparaffinized sample was immersed in 100% ethanol twice for 3 minutes and in 95% ethanol (diluted with purified water) twice for 3 minutes for hydrophilic treatment. After hydrophilization treatment, the sample was washed with purified water, and further PBS (phosphate buffered saline; here 10 mM PBS (pH 7.4) [NaCl 8 g, Na ₂ HPO ₄ 1.1 g, KCL 0.2 g, KH ₂ PO ₄ 0.2 g + prepared water prepared to 1 L in total]; The sample after permeabilization was permeabilized by incubating for 15 minutes in 10 mM citrate buffer (composition: 10 mM sodium citrate buffer, pH 6.0) maintained at 75 ° C. In addition, this 10 mM citrate buffer solution is 0.38 g of citrate monohydrate (C ₆ H ₈ O ₇ · H ₂ O), trisodium citrate dihydrate (C ₆ H ₅ O ₇ Na ₃ · 2H) in advance. ₂ O) 2.41 g and purified water to prepare a total of 1 L. The sample was then washed 3 times with PBS for 5 minutes. After washing, the sample was washed with 3% hydrogen peroxide-added methanol for 10 minutes and with PBS for 5 minutes three times, and then treated with 10% normal goat serum for 10 minutes for blocking treatment. Furthermore, in order to prevent the solution added in the subsequent steps from spilling from the glass slide, Dako Pen (DAKO) was used to surround the tissue section on the glass slide.

  Next, anti-CD34 antibody (Nichirei Co., Ltd.) for vascular endothelial cells and anti-VEGF antibody (Santa Cruz Biotechnology, Inc.) for Hodgkin cells were used as primary antibodies. A primary antibody reaction was performed.

  Specifically, discard the liquid from the sample after the blocking reaction (10 minutes) using the above normal goat serum, wipe the periphery of the section with a filter paper, and remove the primary antibody anti-CD34 antibody (mouse monoclonal antibody; Nichirei) It was placed on a section and reacted overnight at 4 ° C. The used anti-CD34 antibody has a concentration adjusted for immediate use.

  On the other hand, anti-VEGF antibody (rabbit polyclonal antibody; Santa Cruz Biotechnology, Inc.) was used after being diluted 400 times with PBS. Discard the solution from the sample after blocking reaction (10 minutes) using normal goat serum above, wipe the periphery of the section with filter paper, and place the prepared primary anti-VEGF antibody (Santa Cruz Biotechnology, Inc.) on the section. The mixture was allowed to react at room temperature for 3 hours.

  Next, the sample after the primary antibody reaction was washed with PBS for 5 minutes × 3 times, and further subjected to a secondary antibody reaction. Specifically, biotin-labeled anti-mouse IgG + anti-rabbit IgG antibody (goat antibody; concentration adjusted; Nichirei) was added as a secondary antibody and allowed to react at room temperature for 10 minutes.

  The sample after the secondary antibody reaction was washed with PBS for 5 minutes × 3 times and then subjected to peroxidase-labeled streptavidin reaction. First, peroxidase-labeled streptavidin (concentration adjusted; Nichirei) was added to the sample and reacted at room temperature for 5 minutes. The obtained sample was washed 3 times with PBS for 5 minutes, and then DAB color reaction was performed using a peroxidase substrate kit (Nichirei). A chromogenic substrate (reagent A) and a substrate buffer (reagent B) were mixed 1d each with 1 ml of purified water, and the chromogenic reagent (reagent C) 1d was added and mixed, and the solution was placed on the tissue section. While confirming the degree of color development, the reaction time was set to around 10 minutes.

  The resulting sample was then washed with purified water and then permeabilized by incubating in PBS for 3 minutes at room temperature. After permeabilization, place a sufficient amount of 0.25% trypsin-1 mM EDTA / 4Na solution (aqueous solution) that retains the activity immediately after thawing (about 200 to 300 μl depending on the size of the tissue section) on the tissue section of the sample. Then, it was placed on a hot plate heated to 37 ° C., and the cover was placed for 60 minutes together with wet paper to prevent drying. Alternatively, it was placed in an incubator kept at 37 ° C. for 60 minutes.

  After the reaction, wash the sample with PBS for 5 minutes x 3 times, then with purified water, and then infiltrate the slide glass with the section into about 150 ml of New Hematoxylin solution (Muto Chemical Co., Ltd.) for 30 seconds. Hematoxylin nuclear staining was performed.

  FIG. 1A shows a stained image of vascular endothelial cells after immunostaining (stained brown by DAB coloring) and hematoxylin nucleus staining (purple staining). As shown in FIG. 1A, only the inner wall of the blood vessel was immunostained brown, indicating that the vascular endothelial cells were specifically stained with the anti-CD34 antibody. In this tissue section, vascular endothelial cells were separated from other lymphoma cells and the like, and the tissue sections were in a favorable dissociated state. Similarly, for Hodgkin cells, Hodgkin cells were isolated from other lymphocytes and the like, and the dissociation state was shown to be good.

  The samples that have been immunostained and hematoxylin nuclear stained as described above are then washed with purified water, incubated for 3 minutes at room temperature in PBS, and permeabilized, with 0.02% Tween20 on the tissue section. About 200 to 300 μl of PBS solution (hereinafter 0.02% Tween20 / PBS) was placed depending on the size of the tissue section.

  While observing the stained image of this sample under an inverted microscope, the upper part of the tissue section was roughly scratched with the tip of the needle so that the region containing a large amount of immunostained cells (vascular endothelial cells or Hodgkin cells, respectively) was used. Cells at that site were dissociated and suspended in 0.02% Tween20 / PBS. When observed under an inverted microscope, cells and small cell clusters were contained in 0.02% Tween20 / PBS in a state of being well dissociated for each cell type or cell type. A photograph of the cell mass of vascular endothelial cells suspended in 0.02% Tween20 / PBS is illustrated in FIG. 1B.

  0.02% Tween20 / PBS containing the cells was sucked up with a pipette and pipetted several times so as not to be bubbled in the chip to promote cell dispersion, and then transferred to a petri dish. Cell-containing 0.02% Tween20 / PBS solution (cell dispersion) in a petri dish is diluted by appropriate addition of 0.02% Tween20 / PBS, and then the cells sink to the bottom of the petri dish so that the cells can be easily observed and collected. Left to stand. Thereafter, when observed under an inverted microscope, the dispersed state of the cells in the solution was good. FIGS. 1C and D show photographs of the vascular endothelial cells thus dispersed in the solution.

  Next, from the solution in the petri dish, the target cells (vascular endothelial cells or Hodgkin cells) that were immunostained were collected by aspiration using a syringe with a needle of 27 gauge (G) or less under an inverted microscope. Whether it was a vascular endothelial cell or a Hodgkin cell was also judged from its morphological characteristics. When cells different from the target cells were mixed, the collected solution was transferred to another petri dish and diluted by adding 0.02% Tween20 / PBS, and the target cells were again selectively aspirated therefrom.

  When each of the aspirates of vascular endothelial cells and Hodgkin cells collected as described above was observed again under an inverted microscope, cells other than the target cells were hardly contained.

  Vascular endothelial cells are irregular in shape and form a network structure in the space between the surrounding cells and the back side of the tissue specimen, so it is difficult to separate them from adjacent cells. For example, when vascular endothelial cells are separated by the laser microdissection method, other cells are likely to be mixed. In addition, Hodgkin cells are isolated and dissociated in tissues, and are difficult to separate and collect from surrounding tissues. According to the above examples, such vascular endothelial cells and Hodgkin cells could be successfully dissociated from other tissues and isolated, so that the method of the present invention is advantageously used for cell separation preparation. It was shown that it can be done.

[Example 2] Cell separation from tissue specimen 2-Use of hematoxylin and eosin staining In this example, a cell separation method similar to that in Example 1 was used except that hematoxylin and eosin staining was used instead of immunostaining. Mucosal epithelial cells were selectively isolated and collected from formalin-fixed paraffin-embedded specimens of mouse intestinal tissue.

  First, a 5 μm-thick paraffin-embedded section obtained from the above sample was placed on a silane-coated slide glass (manufactured by Matsunami), dried and pasted by a conventional method. Next, the slide glass (sample) with the paraffin-embedded section attached is placed in xylene (150 μl), allowed to stand at room temperature for 10 minutes, the xylene is changed, and this standing step is repeated two more times to remove it. Paraffin treatment was performed. Subsequently, the sample subjected to the deparaffinization treatment was hydrophilized by immersing the sample in 100% ethanol twice for 3 minutes and in 95% ethanol (diluted with purified water) twice for 3 minutes. After the hydrophilization treatment, the sample was washed with purified water and further permeabilized by incubation in PBS at room temperature for 5 minutes. The permeabilized sample was permeabilized by incubating for 15 minutes in 10 mM citrate buffer (composition: 10 mM sodium citrate buffer, pH 6.0) maintained at 75 ° C. The sample was then washed 3 times with PBS for 5 minutes. After washing, a Dako Pen (DAKO) was used to surround the tissue section on the sample glass slide.

  Next, place a sufficient amount of 0.25% trypsin-1 mM EDTA · 4Na solution (aqueous solution) that retains the activity immediately after thawing (about 200 to 300 μl depending on the size of the tissue section) on the tissue section of the sample. It was placed on a hot plate heated to 37 ° C., and the cover was placed with wet paper to prevent drying, and left for 60 minutes. Alternatively, it was placed in an incubator kept at 37 ° C. for 60 minutes.

  After the reaction, the sample was washed 3 times with PBS for 5 minutes and then stained with hematoxylin and eosin. The sample was allowed to react with the added hematoxylin solution (Muto Chemical Co., Ltd.) for 15 minutes, lightly washed with water, immediately rinsed with 1% hydrochloric acid and 70% ethanol, immediately washed with water, and permeated into eosin solution (Mudo Chemical) for 5 minutes. I let you. By this hematoxylin / eosin staining, the cell nucleus is stained purple, and the cytoplasm and stroma are stained red.

  After washing the sample stained with hematoxylin and eosin with purified water and incubating in PBS for 3 minutes at room temperature, 0.02% Tween20 / PBS was added on the tissue section according to the size of the tissue section. About 200-300 μl.

  When the stained image of this sample was observed under an inverted microscope, it was found that the intestinal muscular layer, submucosa and mucosal epithelial cells were well dissociated from each other in the tissue section. Then, next, a site containing many mucosal epithelial cells on the tissue section was roughly scraped with the tip of the needle to dissociate cells in the upper layer of 0.02% Tween20 / PBS and float them. When observed under an inverted microscope, cells and small cell clusters were contained in 0.02% Tween20 / PBS in a state of being well dissociated for each cell type or cell type.

  0.02% Tween20 / PBS containing the cells was sucked up with a pipette and pipetted several times so as not to be bubbled in the chip to promote cell dispersion, and then transferred to a petri dish. Cell-containing 0.02% Tween20 / PBS solution (cell dispersion) in a petri dish is diluted by appropriate addition of 0.02% Tween20 / PBS, and then the cells sink to the bottom of the petri dish so that the cells can be easily observed and collected. Left to stand. Thereafter, when observed under an inverted microscope, the dispersed state of the cells in the solution was good.

  Next, from the solution in the petri dish, mucosal epithelial cells were aspirated and collected using a syringe with a needle of 27 gauge (G) or less under an inverted microscope. When cells different from the mucosal epithelial cells were mixed, the collected solution was transferred to another petri dish and diluted by adding 0.02% Tween20 / PBS, from which the mucosal epithelial cells were selectively aspirated again.

  The aspirated solution collected as described above was observed again under an inverted microscope, and almost no cells other than mucosal epithelial cells were contained.

[Example 3] Examination of various reaction conditions In this example, further examination was carried out on the various reaction conditions described in the above examples.
First, the same experiment as in Example 1 was performed using 10 mM citrate buffer (pH 7.0) instead of 10 mM citrate buffer (pH 6.0). As a result, even when a 10 mM citrate buffer (pH 7.0) was used, good dissociation between cells was confirmed in the stained image of the tissue section after trypsin treatment. However, it was shown that dissociation progressed more when 10 mM citrate buffer (pH 6.0) was used.

  On the other hand, an experiment was conducted in the same manner as in Example 1 except that the holding temperature of 10 mM citrate buffer (pH 6.0) was set to 65 ° C. or room temperature (about 20 ° C.) instead of 75 ° C. As a result, when the experiment was performed at room temperature, almost no cell dissociation was observed in the tissue section after trypsin treatment. On the other hand, when the experiment was performed at 65 ° C., good dissociation of cells was observed in the tissue section, but it was also shown that better dissociation was obtained at 75 ° C.

Furthermore, instead of 10 mM citrate buffer (pH 6.0), 2 × SSC (saline citrate buffer; sodium citrate dihydrate 8.8 g, NaCl 17.5 g, H ₂ O were mixed to prepare a total of 1 L When the same experiment as in Example 1 was performed except that the above-mentioned (pH 7.0) was used, the tissue section after trypsin showed a weaker dissociation level and slight cell atrophy, but the dissociation itself Was shown to be accepted.

  The experiment was performed in the same manner as in Example 1 except that the treatment time with 0.25% trypsin-1 mM EDTA · 4Na solution was changed from 60 minutes. As a result, when the reaction time was significantly longer than 60 minutes (100 minutes), dissociation of cells was observed in the tissue section after trypsin treatment, but deformation of cells and nuclei may be observed. there were.

  In addition, 1) treatment with 10 mM citrate buffer (pH 6.0) but no treatment with 0.25% trypsin-1 mM EDTA · 4Na solution, and 2) 10 mM citrate buffer (pH 6.0) The same experiment as in Example 1 was performed for each of the cases where the treatment with 0.25% trypsin-1 mM EDTA · 4Na solution was performed without performing the above treatment. Specifically, for Experiment 1), the experiment described in Example 1 except for the reaction step with 0.25% trypsin-1 mM EDTA · 4Na solution and the subsequent washing step with PBS for 5 minutes × 3 times was performed and stained. When the tissue section was scraped with a needle and observed under an inverted microscope at a low magnification, almost no cell dissociation was observed (FIG. 2A). Similarly, for Experiment 2), the experiment described in Example 1 was conducted except for the permeabilization step with 10 mM citrate buffer (pH 6.0) at 75 ° C. and the subsequent washing step with PBS for 5 minutes × 3 times. When the stained tissue section was scraped with a needle and observed at a low magnification under an inverted microscope, almost no cell dissociation was observed (FIG. 2B). From these results, it is difficult to disperse cells well in the solution because it is difficult to disperse the cells well in the solution by citrate solution treatment and trypsin solution treatment. It was shown that the final separation is not possible.

  Further, instead of 0.02% Tween20 / PBS, PBS and PBS containing various concentrations of Tween20 (0.01% to 0.1% Tween20 / PBS) were used, respectively, and the same experiment as in Example 1 was performed. As a result, when PBS is used, the cell dispersion is remarkable because of the remarkable adhesion of the cells to the instrument, the fact that the collected cells cannot be separated from the supernatant well when centrifuging, and the cells are not stable in the liquid. Problems such as difficulty in separating and collecting the cells from the cells occurred. Cell collection was possible using 0.01% to 0.1% (mass%) Tween20 / PBS, but at low concentrations, adhesion to the instrument was observed, and at high concentrations, the solution was prone to foam. PBS was shown to be the most suitable concentration for cell harvesting.

[Example 4] Extraction and analysis of DNA from isolated and collected cells Compared with tissue specimens that were simply permeated with PBS, cells treated with citrate buffer, trypsin, EDTA, and Tween20 were separated from tissue specimens. For the purpose of confirming whether or not the DNA of the cells was damaged, cells were separated and collected from the paraffin-embedded tissue specimen as follows, DNA was extracted from the collected cells, and the DNA was analyzed.

  First, cultured cells of a human malignant lymphoma cell line (Raji cells (provided by Tohoku University Cell Center) with formalin-fixed paraffin-embedded specimens in RPMI (Roswell Park Memorial Institude) -1640 (10% FBS) medium) Three samples pasted on glass (Matsunami) were prepared and subjected to deparaffinization treatment and hydrophilization treatment in the same manner as in Example 1. For each of the obtained samples, the following 1) to 3) Except for each condition, basically the same experiment as in Example 1 was performed, and malignant lymphoma cells were collected.

1) After permeabilization in PBS at room temperature, permeation with 10 mM citrate buffer (PH6.0), PBS washing step, 0.25% trypsin-1 mM EDTA, 4Na solution treatment, PBS washing step Without immunostaining, malignant lymphoma cells were collected from the specimen sections. (Control experiment)
2) Permeabilization with 10 mM citrate buffer (PH 6.0) (65 ° C., 15 minutes), treatment with 0.25% trypsin-1 mM EDTA · 4Na solution (37 ° C., 60 minutes), and then in 0.02% Tween 20 / PBS Harvest the cells.
3) Permeabilization with 10 mM citrate buffer (PH6.0) (75 ° C, 15 minutes), treatment with 0.25% trypsin-1 mM EDTA · 4Na solution (37 ° C, 60 minutes), and then in 0.02% Tween20 / PBS Harvest the cells.

Next, genomic DNA was extracted from about 2 × 10 ^{6 of} each of the collected cells by a conventional method, and PCR was performed using this as a template to amplify the GAPDH gene. The primers used for this PCR are as follows.
Forward primer: 5'-GAAGGTGAAGGTCGGAGT-3 '(SEQ ID NO: 1)
Reverse primer: 5'-GAAGATGGTGATGGGATTTC-3 '(SEQ ID NO: 2)

  The PCR reaction was carried out under the following conditions: 30 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds.

  When the obtained PCR product was electrophoresed on a 2% agarose gel, clear bands were detected in the samples derived from the above three conditions. That is, even when the DNA contained in the cells obtained by the methods 2) and 3) above was used as a template, inhibition of the PCR reaction was not observed, and no difference was observed between the three bands.

  From this result, it was shown that the DNA extracted from the cells separated and collected from the tissue specimen by the cell preparation method of the present invention has a quality that can be used for DNA analysis.

  The cell preparation method of the present invention can promote good dissociation between cells constituting a tissue by satisfactorily decomposing cell-cell bonds while minimizing damage to cells. By using this method, it is possible to selectively separate and collect target cells from a tissue specimen without destroying them. In this method, cells can be dispersed well in a solution, so that cells having a shape and structure that are usually difficult to separate and collect from other cells and tissues can be easily separated and collected. Moreover, since the method of the present invention uses only simple techniques and does not use expensive equipment or reagents, it is simple and economical. The kit of the present invention can be advantageously used for cell preparation, for example, as a histopathological examination kit.

FIG. 1 is a photograph showing how dissociation of vascular endothelial cells from tissue proceeds in the method of the present invention. A is a tissue section after citrate solution and trypsin treatment, B is a cell mass containing vascular endothelial cells derived from a tissue section after citrate solution and trypsin treatment, and C and D are blood vessels dispersed in 0.02% Tween20 / PBS. The stained image of endothelial cells (C and D are separate visual fields under a microscope) is shown. FIG. 2 is a photograph showing the dissociated state of cells when only 10 mM citrate buffer (pH 6.0) treatment or 0.25% trypsin-1 mM EDTA · 4Na solution treatment is performed. A shows a stained image of a curettage fragment derived from a tissue section treated only with 10 mM citrate buffer (pH 6.0), and B shows a stained image of a curettage fragment derived from a tissue section treated only with 0.25% trypsin-1 mM EDTA · 4Na solution.

  The sequences of SEQ ID NOs: 1 and 2 are primers.

Claims (10)

A method for preparing cells from a tissue specimen, which comprises the following steps 1) to 3).
1) A step of heat-treating a tissue specimen in a citric acid solution at 60 to 80 ° C,
2) a step of treating the heat-treated specimen with a proteolytic enzyme; and
3) Step of dispersing the cells in the specimen treated with the enzyme in a surfactant solution   The method according to claim 1, wherein the heat treatment in a citric acid solution is performed for 5 to 30 minutes.   The method according to claim 1, further comprising tissue staining the specimen.   The method according to any one of claims 1 to 3, wherein the tissue specimen is fixed.   The method according to claim 1, wherein the citrate solution is a citrate buffer.   The method according to any one of claims 1 to 5, wherein the proteolytic enzyme is at least one selected from the group consisting of trypsin, collagenase, dispase, papain, elastase, hyaluronidase, chymotrypsin, and pronase.   The method according to claim 1, wherein the specimen is treated with EDTA together with a proteolytic enzyme.   The surfactant is at least one selected from the group consisting of Tween 20, Tween 80, Triton X-100, Triton X-114, NP-40, Brij 35, octaglycoside, pluronic, and sucrose monolaurate. Item 8. The method according to any one of Items 1-7.   A method for analyzing cells in a tissue specimen, comprising preparing a cell dispersion from a tissue specimen by the method according to any one of claims 1 to 8, collecting the cells therefrom, and using the cells for analysis. .   A kit for preparing cells from a tissue specimen, comprising a citric acid solution, a proteolytic enzyme, and a surfactant solution.

Images