JP2019056678A - Target cell detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a target cell and a contaminating cell contained in a sample with high accuracy by accurately labeling the target cell and the contaminating cell. SOLUTION: Target cells and contaminating cells contained in a sample are permeated into a membrane by treatment with 0.03% (w / v) to 0.15% (w / v) of Triton X-100 (trade name). After labeling with a substance that recognizes a protein specifically possessed by the target cell and the contaminant cell, the target cell is detected based on the presence or absence of the label. [Selection diagram] None

Description

  The present invention relates to a method for detecting a target cell contained in a sample. In particular, the present invention relates to a method for detecting target cells with high accuracy by optimizing the membrane permeation process of target cells contained in a sample.

  In recent years, cells have been selectively separated and collected from samples such as body fluids such as blood, tissue suspensions obtained by suspending or dispersing tissues such as organs, and cell culture solutions. Research that applies to basic research, clinical diagnosis, and treatment is underway. For example, a tumor cell (Circulating Tumor Cell, hereinafter referred to as CTC) is collected from blood collected from a cancer patient, and morphological analysis, tissue type analysis or gene analysis is performed on the cell. Research to determine treatment policy based on this is underway.

  However, CTC has a very low probability of existence (in the case where the sample is whole blood derived from a cancer patient), and requires high-sensitivity detection. CTC is usually labeled with a nuclear labeling reagent such as DAPI (4 ′, 6-DiAmidino-2-PhenylIndole), and a protein (CK and p63) that is specifically contained in epithelial cells such as cytokeratin (CK) and p63 in the cell. Is labeled with a labeled antibody against the expression (expressed in the cell) and not labeled with a labeled antibody against a protein (marker) specific to leukocytes, such as CD45, as CTC. However, some CTCs are not labeled with a labeled antibody against the protein specifically possessed by the epithelial cells (Patent Document 1). In order to detect the CTCs, in order to eliminate false positives and false negatives, It is necessary to clearly and accurately label other contaminating cells (for example, leukocytes) and detect the cells with high accuracy.

WO2015 / 112955

  An object of the present invention is to provide a method for detecting a target cell with high accuracy by clearly and accurately labeling a target cell and a contaminated cell contained in a sample.

  In order to solve the above-mentioned problems, the present inventors have intensively studied to arrive at the present invention.

That is, the present invention can be exemplified as follows.
[1]
A method for detecting target cells and contaminating cells contained in a sample, comprising:
1) a step of permeating through a target cell and a contaminated cell membrane in a sample;
2) a step of labeling with a substance that recognizes the protein specifically possessed by the target cell and the contaminating cell, and 3) a step of detecting the target cell based on the presence or absence of these labels,
And a membrane permeation of target cells and contaminating cells is treated with 0.03% (w / v) to 0.15% (w / v) Triton X-100 (trade name).
[2]
[1] The target cell is a tumor cell, the protein specifically possessed by the target cell is an epithelial intracellular protein, and the labeling of the contaminated cell is performed with a substance that recognizes the protein specifically possessed by the contaminated cell membrane. The detection method described.
[3]
The sample is a blood sample, the contaminating cell is a leukocyte, the protein specifically possessed by the contaminated cell is a membrane protein of leukocyte, and the target cell is labeled with a substance that specifically recognizes the protein having the label in the target cell. [1] or [2].

  The present invention permeates the target cells and contaminated cells contained in the sample through a membrane, and labels them with substances that specifically recognize the target cells and the contaminated cells, respectively, and then based on the presence or absence of these labels, the target cells In the method for detecting urine, the membrane permeation of target cells and contaminating cells is treated with 0.03% (w / v) to 0.15% (w / v) of Triton X-100 (trade name). It is characterized by. The detection method of the present invention is particularly useful when the number of target cells contained in a sample is small and the number of contaminated cells is extremely large compared to the number of target cells. For example, the present invention is contained in blood. By applying to the detection of circulating tumor cells (CTC) in the blood, the reliability of the CTC detection result is improved, and cancer can be detected with high accuracy.

It is a figure which shows an example of the cell holding | maintenance apparatus which can be utilized with the detection method of this invention. It is a figure which shows another aspect of the cell holding | maintenance apparatus which can be utilized with the detection method of this invention. It is the figure which showed the holding | maintenance and detection of a cell using the cell holding | maintenance apparatus shown in FIG.

  Hereinafter, the present invention will be described in detail.

  As an example of the sample in the present invention, biological samples such as whole blood, diluted blood, serum, plasma, spinal fluid, umbilical cord blood, component blood collection, urine, saliva, semen, feces, sputum, amniotic fluid, ascites, peritoneal washing fluid, Tissue suspension obtained by suspending a piece of tissue such as liver, lung, spleen, kidney, skin, tumor, lymph node, or the biological sample obtained by separating from the biological sample or the tissue suspension Or the fraction containing the cell derived from the said tissue and the culture solution of the cell isolated previously are mention | raise | lifted. Among these, an example of a fraction containing a biological sample or tissue-derived cells is a fraction obtained by overlaying a biological sample or tissue suspension on a density gradient forming medium and then performing density gradient centrifugation.

  Examples of target cells contained in the sample when the sample is a blood sample include tumor cells such as blood circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), and circulation. Examples include fetal cells (CFC) and various stem cells. On the other hand, the contaminating cells contained in the sample refer to cells other than the target cells. Specific examples of the contaminating cells when the sample is a blood sample include leukocytes, erythrocytes, platelets and small cells that are contained in the sample. And debris derived from these cells or the aforementioned target cells. The blood sample in the present invention is not limited to a blood sample such as whole blood, serum, plasma, umbilical cord blood, and component blood collection, but can be obtained by diluting the blood sample with a physiological saline solution or the like and separating it from the blood sample. A fraction containing cells derived from the blood sample is also included in the blood sample.

  In the present invention, membrane permeabilization of target cells and contaminated cells contained in a sample is performed. However, membrane permeation treatment may damage or elute membrane proteins. Therefore, when labeling intracellular proteins and membrane proteins, it is necessary to optimize the membrane permeation reagent and its concentration. As a result of studying optimization of the membrane permeation reagent, the present inventors have determined that 0.03% (w / v) to 0.15% (w / v) Triton X-100 (trade name, It was found that the cell membrane can be efficiently permeated while minimizing damage and elution of membrane proteins. The concentration of Triton X-100 may be within the above-described concentration range, but a range from 0.03% (w / v) to 0.1% (w / v) is more preferable, and 0.03% (w / v) A range from v) to 0.08% (w / v) is more preferred. The method of the present invention is particularly useful for labeling both target cells and contaminating cells without separating them. For example, when the protein specifically possessed by the target cell and the protein specifically possessed by the contaminated cell are present in the cell and in the cell membrane, respectively, the recognition substance can be permeabilized and damage to the membrane protein can be prevented. This is because it is necessary to suppress.

  In the present invention, after the membrane permeation treatment described above, the target cells and contaminating cells contained in the sample are labeled with a substance that recognizes the target cell and the protein specifically present in the contaminating cells, respectively, and based on the presence or absence of the labeling The target cell contained in the sample is detected.

In the present invention, a protein that is specifically present in a target cell (contaminated cell) is a protein that is specifically present in the target cell (contaminated cell) and that enables identification of the target (contaminated cell). Say. Here, it exists specifically in the target cell (contaminated cell).
It means that it exists in the target cell (contaminated cell) and does not exist in the contaminated cell (target cell), or the abundance in the target cell (contaminated cell) is larger than that of the contaminated cell (target cell). .

  In the present invention, a substance that specifically recognizes a protein specifically contained in a target cell (contaminated cell) is preferably a substance that can specifically bind to the protein (hereinafter also referred to as a specific binding substance) and optically. And a complex with a labeling substance capable of emitting a detectable signal. Examples of the specific binding substance include an antibody against the protein and a ligand / lectin that can specifically bind to the protein. The labeling substance is not particularly limited as long as it can emit an optically detectable signal (fluorescence, chemiluminescence, phosphorescence, etc.). For example, FITC (fluorescein isocyanate), PE (phycoerythrin) And enzymes that catalyze the reaction with fluorescent dyes such as APC (allophycocyanin) and rhodamine, and chemiluminescent substrates such as peroxidase, β-galactosidase, alkaline phosphatase, and luciferase.

The complex formation between the specific binding substance and the labeling substance may be performed by chemically bonding both substances directly by a known method, biotin (avidin or streptavidin) as the specific binding substance, and avidin as the labeling substance. Alternatively, streptavidin (biotin) may be bonded to each other, and then both may be indirectly bonded via a biotin-avidin (biotin-streptavidin) bond,
After binding a specific binding substance (antibody, ligand, lectin) with a specific binding substance to the labeling substance, both of them indirectly by the interaction (such as antigen-antibody reaction) between the specific binding substance and the substance. May be combined.

The membrane permeabilization of the target cells and contaminating cells contained in the sample, which is carried out in the present invention, is particularly suitable for the label shown in the following (I) or (II).
(I) Labeled with a substance that specifically recognizes a protein specifically contained in a target cell contained in a sample and a substance that recognizes a protein specifically contained in a contaminated cell membrane contained in the sample (II) A purpose contained in the sample Labeling with a substance that specifically recognizes a protein that has a cell membrane and a substance that specifically recognizes a protein that is contained in a contaminated cell contained in a sample Specific examples of the labeling performed in the present invention are shown below.

  A substance that recognizes cytokeratin (CK) or p63, which is a protein specifically contained in CTC, when the sample is the blood sample described above, the target cell is a tumor cell (CTC), and the contaminating cell is a leukocyte. And a substance that recognizes CD45 (Leucocyte Common Antigen: LCA) and CD50 (InterCellular Adhesion Molecule-3: ICAM-3), which are proteins specifically possessed in the leukocyte membrane. In addition, although 20 kinds of proteins from CK1 to CK20 are known as CK, any of them can be used as a protein specifically included in CTC.

  When the sample is a suspension of tumor tissue, the target cells are tumor-associated fibroblasts (CAF), and the contaminating cells are leukocytes, α-SMA (α-SMA, which is a protein specifically contained in CAF) It can be labeled with a substance that recognizes smooth muscle actin) and a substance that recognizes CD45 and CD50, which are proteins specifically possessed by leukocyte membranes.

  When the sample is a solution after the iPS cells are differentiated into endoderm, the target cell is an undifferentiated iPS cell, and the contaminating cell is the endoderm, SSEA which is a protein specifically included in the iPS cell membrane -3 (Stage-specific Embryonic Antigen-3) and TRA-1-60, and a substance that recognizes SOX17 and HNF-3 (Hepatocyte Nuclear Factor-3), which are specific proteins in the inner lobe Can be labeled with.

  In the present invention, in addition to the detection based on the presence or absence of the label described above, additional detection may be performed. Examples of additional detection include detection of nucleated cells using nuclear labeling reagents such as DAPI (4 ′, 6-Diamidino-2-phenylIndole), hematoxylin, Hoechst 33342 (trade name), orange G, light green, eosin Cell detection using a cytoplasmic labeling reagent such as the above, and detection based on differences in cell size / shape / pattern based on bright-field images.

  When practicing the present invention, if a phenomenon occurs in which the shape of a deteriorated cell collapses or a gene or protein such as DNA or RNA in the deteriorated cell is degraded before cell detection, the target cell from the solution and Detection / analysis ability using proteins contained in contaminated cells is reduced. Therefore, it is preferable to perform a fixing treatment for suppressing the deterioration of the cells before the membrane permeation treatment described above or simultaneously with the membrane permeation treatment described above. Examples of the fixing treatment reagent include organic solvents such as aldehydes, acids, and alcohols, and heavy metal elements, but the present invention is not limited thereto.

  Aldehydes dissociate existing binding modes of proteins (for example, disulfide bonds and hydrogen bonds) to form new bonds. The bond may be a covalent bond with an amino group terminal such as lysine or arginine, an aromatic activated carbon such as tryptophan or thyroxine, or a methylene bridge formed by linking with another amino acid terminal residue. These new bonds can change the higher-order structure of the protein, and free molecules that are not involved in such bonds and cross-links can also hold the polypeptide chain to prevent further denaturation, thereby stabilizing the protein structure. And the cytoplasm can be gelled to suppress enzyme activity. As aldehydes that can be used as a reagent for fixing treatment, formaldehyde is representative, and glutaraldehyde, glyoxal, and the like are also included. In addition, although not directly acting, formaldehyde donors (formaldehyde donors) that liberate stabilizers such as formaldehyde by themselves undergoing hydrolysis or the like can also be used as fixing treatment reagents. Examples of formaldehyde donors include imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, bronopol (2-bromo-2-nitropropyne-1,3-diol) , Diazolidinyl urea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quaternium-15 (mesenamine 3-chloroallylochloride), hydroxymethylglycine sodium, amines and amides Examples include methylol, hydroxymethyl derivatives, methylol, methenamine, and paraformaldehyde.

  Since acids have a strong protein coagulation action, they can be used as a reagent for immobilization treatment. Examples include picric acid, tannic acid, osmic acid, acetic acid, glacial acetic acid, trichloroacetic acid (trichloroacetic acid: TCA), chromic acid, and salts thereof.

  Alcohols have the effect of insolubilizing and denaturing proteins through powerful dehydration and lipid dissolution, and therefore can be used as fixing treatment reagents. Of these, ethanol, methanol, and acetone are very often used. An organic solvent such as chloroform has the same effect as alcohols and can be used as a reagent for immobilization treatment.

  As the heavy metal element, chromium, manganese, zinc or the like can be used as a fixing treatment reagent.

  When the present invention is applied to a system in which the number of target cells contained in the sample is small and / or the number of contaminated cells is extremely large, such as detection of CTC contained in a blood sample, for example, it is contained in the sample in advance. Performing a step of concentrating target cells and / or reducing contaminating cells (hereinafter referred to as target cell concentration step) is preferable in that the target cells can be detected efficiently. The target cell concentration step is not particularly limited as long as the target cells can be collected more selectively by reducing components other than the target cells (contaminated cells and the like) contained in the sample. For example, a filter method that separates and concentrates using the difference in size between the target cell and a component other than the target cell (such as contaminated cells), and a magnetic particle that binds to the antibody using the difference in the antibody expression profile on the cell surface. Examples thereof include a magnetic bead method for separating and concentrating target cells using, and a specific gravity difference separation method using a specific gravity difference between cells. Of these, the specific gravity difference separation method is particularly preferable as the target cell concentration step because the target cells can be selectively concentrated in a short time.

  Hereinafter, details of the target cell concentration step by the specific gravity difference separation method will be described.

  The concentration of the target cells by the specific gravity difference separation method (reduction of contaminating cells) is not particularly limited as long as the target cells and components other than the target cells (such as contaminating cells) can be separated by the specific gravity difference. For example, a sample containing target cells is layered on a centrifuge tube containing a density gradient solution, and then subjected to centrifugation, whereby a fraction containing target cells and a fraction containing components other than target cells (contaminated cells, etc.) And collecting the fraction containing the target cell to concentrate the target cell (reducing contaminated cells). The density gradient solution used here is a liquid substance that forms a density gradient by itself or by centrifugation. The density (specific gravity) of the target cell is specified, and an appropriate one for the separation is selected and used. That's fine. Examples of selection indicators include nutrient components, pH, and isotonicity. Specific examples of density gradient solutions include sucrose, glycerol, dextran, metrizamide, iodixanol, a copolymer of sucrose and epichlorohydrin, colloidal silica particles with a polyvinylpyrrolidone coating, sucrose polymer, diatrizo Acids, iohexol, and the like are commercially available, such as Ficoll, Ficoll-Paque, Percoll (manufactured by GE Healthcare), Lymphoprep, Polymorphprep, OptiPrep, Nycodenz (manufactured by Axis-Shield).

  Centrifugation is generally performed at a low speed of about 1000 × g to 2000 × g, but the target cells are placed on the density gradient solution in consideration of the density of the target cells and the density gradient solution to be used. What is necessary is just to select the conditions maintained. For example, when the target cell is a tumor cell such as CTC, and centrifugation is performed under the above conditions, the density of the density gradient solution is 1.060 to 1.105 g / mL depending on the type of tumor cell to be concentrated. Can be set to a range. Among these, from the viewpoint of increasing the concentration rate of tumor cells, the density of the density gradient solution is preferably 1.075 g / mL or more, and more preferably 1.080 g / mL or more. On the other hand, for the same reason, the density of the density gradient solution is preferably 1.100 g / mL or less, more preferably 1.096 g / mL or less, and even more preferably 1.093 g / mL or less. The most preferred density gradient solution density for tumor cell enrichment ranges from 1.082 to 1.091 g / mL.

  The osmotic pressure of the density gradient solution may be appropriately set in the range from 200 mOsm / kg to 450 mOsm / kg, but more preferably in the range from 300 mOsm / kg to 400 mOsm / kg. The pH of the density gradient solution can be arbitrarily selected as long as the target cells are not damaged. In the case of normal cells, the pH may be set in the range from pH 6.8 to pH 7.8.

  When performing the target cell concentration step by the specific gravity difference separation method, the target cell is further added by adding a substance that specifically binds to the target cell or a substance that specifically binds to components other than the target cell (such as contaminated cells). It can be separated efficiently. The apparent density can be reduced by combining the substance that specifically binds with a substance having a relatively low density such as porous silica particles. Examples of the substance that specifically binds include biopolymers such as antibodies, antigens, peptides, polypeptides, growth factors, cytokines, and lectins that can specifically bind to target cells (or components other than target cells such as contaminated cells). It can be illustrated. In addition to the porous silica particles described above, substances that can be used for the purpose of adjusting the density described above include polyvinyl compounds such as polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polyacrylate, polymethacrylate, and polycarbonate. Examples include organic polymers, copolymers such as polystyrene latex, nylon, and polyterephthalate, inorganic materials such as glass, silica, and zirconia, biological polymers such as cellulose, dextran, agarose, cellulose, and sepharose, and biological samples such as red blood cells. it can.

  When the target cell concentration step by the specific gravity difference separation method is performed, an operation of positively removing contaminating cells may be added. For example, a hemolysis operation that positively removes red blood cells may be added when a CTC concentration operation is performed from a blood sample by a specific gravity difference separation method. The operation of positively removing the contaminating cells may be performed before the centrifugation operation or after the centrifugation operation. In addition, when the operation which removes a contaminated cell actively is performed before centrifugation operation, it is preferable to add centrifugation operation after that.

  In order to detect a target cell according to the present invention, for example, a sample containing the target cell (or a sample obtained by concentrating the sample in advance through the above-described target cell concentration step) is diluted, suspended, and then applied to the slide. Alternatively, the cells spread on a substrate having a holding portion, coated, or held in the holding portion may be detected using an optical means such as a microscope. Among the detection methods described above, the method of developing a sample on a substrate having a holding unit and detecting the cells held in the holding unit is capable of observing / analyzing each cell with high sensitivity and high accuracy, and later. When the cells are relabeled by detection, it is preferable in that the possibility that the retained cells are detached is reduced.

In the present invention, as an example of a preferable apparatus for developing and detecting a sample containing target cells, the cell holding apparatus shown in FIGS. 1 to 3 can be mentioned.
The cell holding device 100 shown in FIG. 1 and FIG.
A flat insulating film 110 having a through-hole 111;
A flat light-shielding film 120 having a through-hole 121;
A flat spacer 130 having an inlet 131 and an outlet 132;
Electrodes 141 and 142 provided in close contact with the lower surface of the light shielding film 120 and the upper surface of the spacer 130;
A conductive wire 150 connecting the electrodes 141 and 142;
An AC power supply 160 for applying a signal to the electrodes 141 and 142;
It has. The through hole 111 provided in the insulating film 110 and the through hole 1221 provided in the light shielding film 120 have the same size and shape as each other, and are provided with the insulating film 110 and the light shielding film 120 so that the positions of the respective through holes coincide. . The electrode 141 provided in close contact with the lower part of the through-hole 111, the through-hole 121, and the light-shielding film 120 constitutes a holding unit 170 capable of holding cells in the cell holding device 100, and a liquid containing cells is introduced from the inlet 131. When introduced, the cells are introduced into the holding unit 170. The light shielding film 120 can reduce optical noise such as background noise due to autofluorescence of the insulating film 110 itself and crosstalk noise due to leakage light from the adjacent holding unit 170. Only the retained cell-derived light can be detected with high sensitivity and high accuracy. The electrode 142 is provided in close contact with the upper surface of the spacer 130, and prevents scattering and evaporation of the sample containing the target cell introduced from the inlet 131. Note that the electrode 142 has a structure that can be detached from the spacer 130 in order to facilitate the collection of the cells held in the holding unit 170. In addition, it is preferable that the electrodes 141 and 142 are transparent electrodes such as ITO (indium tin oxide) because the cells held in the holding unit 170 can be detected using a microscope or an optical detector.

  In the cell holding device 100 described above, the electrode substrate may be provided so as to sandwich the insulating film 110, the light shielding film 120, and the spacer 130 in the vertical direction as in the device shown in FIG. Thus, the electrode 141 may be provided in the form of a comb-shaped electrode in which the + pole 141a and the −pole 141b are provided only on the lower surface of the light shielding film 120.

  It is preferable that the size of the holding unit 170 be a size that can hold only one target cell because the target cell can be easily detected in the detection process. When the number of cells contained in the sample to be developed on the cell holding device 100 (the sum of target cells and contaminating cells) is expected to be larger than the number of holding units 170 provided in the cell holding device 100, It is good to dilute so that an appropriate number of cells may be developed, or to weigh a sample to be developed.

  The development of the sample containing the target cell (or the sample obtained by concentrating the sample in advance by the target cell concentration step) onto the cell holding device 100 can be performed by any method as long as it can be distributed on the substrate at intervals suitable for cell detection. The sample may simply be developed on the cell holding device 100, but after that, the cells are positively applied to the holding unit 170 provided in the cell holding device 100 by applying vibration or dielectrophoretic force. An operation to be held may be added. In particular, it is preferable to perform the holding operation by applying a dielectrophoretic force in that a living cell can be held in the holding unit 170 in a very short time of about several seconds. In order to cause the dielectrophoretic force 400 to act on the cell 300, an AC power source is used so that the lines of electric force are concentrated on the portion of the holding unit 170 while the space in the cell holding device 100 including the holding unit 170 is filled with liquid. 160 may be used to apply an AC voltage having a predetermined waveform to the electrodes 141 and 142 (FIG. 3). If the holding portions 170 are evenly arranged in an array, an electric field generated by the voltage applied between the electrodes is generated almost evenly in each holding portion 170, and the cells 300 can be induced and captured in the same manner with respect to each holding portion 170. Therefore, it is preferable.

  The magnitude of the AC voltage applied to the electrodes 141 and 142 using the AC power supply 160 may be a voltage sufficient to generate the dielectrophoretic force 400 that can move / hold the cell 300 in the holding unit 170. Specifically, an AC voltage having a peak voltage of about 1 V to 20 V and a frequency of about 10 kHz to 10 MHz such as a sine wave, a rectangular wave, a triangular wave, or a trapezoidal wave can be exemplified. In particular, when it is desired to hold only one cell in one holding part, it is preferable to use a rectangular wave having a frequency of 100 kHz to 3 MHz. Compared with other waveforms such as a sine wave, a triangular wave, and a trapezoidal wave, the rectangular wave reaches the peak voltage set instantaneously, so that the cells can be quickly moved to the holding unit, and two or more cells overlap. In this aspect, the probability of being held in the holding unit can be reduced (the probability of holding only one cell in one holding unit is increased). The cells can be regarded as electrical capacitors, but as long as the peak voltage of the rectangular wave does not change, it is difficult for current to flow to the cells held in the holding part and electric lines of force are less likely to occur. It is difficult for the dielectrophoretic force to be generated in the holding portion holding the cells. Therefore, once a cell is held in the holding unit, the probability that another cell is held in the same holding unit is reduced, and instead, another holding unit (where an electric field line is generated and a dielectrophoretic force is generated) The cells are sequentially held in an empty holding portion that does not hold cells.

  The AC power supply 160 provided in the cell holding device 100 shown in FIGS. 1 to 3 is preferably a power supply that generates an AC voltage having no DC component. When an alternating voltage having a direct current component is applied to the electrode, the cells move by receiving a biased force in a specific direction due to the electrostatic force (electrophoretic force) generated by the direct current component, making it difficult to retain the cells by the dielectrophoretic force. Because. In addition, when an AC voltage having a DC component is applied, ions contained in the cell-containing suspension generate an electric reaction on the electrode surface, generating heat, and the cells cause thermal motion, making it impossible to control movement due to dielectrophoretic force. Therefore, it becomes difficult to move / hold the holding unit. In this specification, an AC voltage having a DC component refers to a voltage whose frequency duty ratio is not 50%, a voltage having an offset, a voltage having an extremely long period (for example, 1 second or more), and the like.

  The target cells and the contaminated cells held in the holding unit 170 included in the cell holding device 100 are labeled with the target cells and / or the contaminated cells using substances that recognize the proteins specifically possessed by the target cells and the contaminated cells, respectively. Then, optical detection is performed using the detection unit 200 provided with the label separately. An example of the detection unit 200 is a fluorescence microscope. Cell fixation and membrane permeation treatment may be performed before labeling, but it is preferable to carry out the treatment after holding the cells in the holding portion because the possibility that the cells are detached from the holding portion is reduced.

  When a target cell is detected using a fluorescence microscope, a fluorescent dye such as FITC (fluorescein isocyanate), PE (phycoerythrin), APC (allophycocyanin) or rhodamine is used as the label, and excitation light corresponding to the fluorescent dye is used. May be detected based on the intensity of fluorescence derived from the labeled cells obtained by the irradiation. As a light source used for irradiation of labeled cells, a halogen lamp, a mercury lamp, a metal halide lamp, a laser, an LED, or the like can be used, and light from the light source can be an optical filter, a mirror, a lens, or the like as necessary. The observation area (the holding part holding the labeled cells) may be irradiated by the optical means constituted by As fluorescence signal information obtained from the observation region by the irradiation, fluorescence such as fluorescence intensity detected from fluorescence emitted by the fluorescent material, peak value of fluorescence intensity, minimum value of fluorescence intensity, average value of fluorescence intensity, integral value of fluorescence intensity, etc. A numerical value calculated from the intensity can be exemplified. Further, the signal information obtained from the observation region by the irradiation can be an image (bright field) composed of light intensity distribution by transmitted light and reflected light in a wide wavelength region, in addition to the information on the fluorescence. Among these, from the bright field information, information on the cell morphology such as the cell diameter, the cell area, the cell volume, the cell perimeter, and the roundness can be acquired. Therefore, in addition to the above-described detection based on the fluorescence intensity, the target cell can be detected with higher accuracy by combining the appearance information such as the size / shape / pattern of the target cell based on the bright field image.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example and a comparative example, this invention is not limited to the said example. In the present invention, both target cells and contaminating cells are labeled. In this embodiment, in order to confirm the luminance distribution, the labeling of target cells and the labeling of contaminating cells are performed separately.

Example 1
(1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5000, one end of which is a methoxy group and the other end is an N-hydroxysuccinimide ester group, and bovine serum albumin (BSA) (300 mg, 0.3 mmol) ) Was dissolved in sodium bicarbonate buffer (0.1 M, 15 mL), and the solution was stirred at room temperature for 3 hours to prepare BSA to which polyethylene glycol was bound (PEG-BSA). In the preparation, the molar ratio of mPEG-NHS to BSA (mPEG-NHS / BSA) was set to 2. After the preparation, the solution was replaced with pure water for 3 days using a dialysis membrane having a molecular weight cut off of 10,000.

  (2) 2 g of imidazolidinyl urea, 2 g of polyethylene glycol (PEG) having a molecular weight of 6000, 100 mg of ethylenediaminetetraacetic acid (EDTA), and 600 mg of sodium chloride were dissolved in 100 mL of ultrapure water, and the resulting solution was used as a stabilizer. .

(3) RPMI containing human prostate cancer cell line (LNCaP) as a target cell in 5% CO ₂ environment, 10% (v / v) FBS (Fetal bovine serum), 2 mM glutamine, 1.0 mM sodium pyruvate After culturing at 37 ° C. for 24 to 96 hours using -1640 medium, the medium was detached from the medium using 0.25% trypsin / 1 mM EDTA and collected in a tube. After collection, it was centrifuged at 1000 rpm for 5 minutes. The tumor cells (LNCaP) used as target cells in this example are cell lines that express cytokeratin (CK).

  (4) An equal amount of the stabilizer (2) was added to the suspension of LNCaP cells in (3), and the resulting solution was used as a preservation cell suspension.

  (5) The preserved target cell suspension is allowed to stand at room temperature for 10 minutes, centrifuged at 1000 rpm for 5 minutes at room temperature, and then the supernatant is removed to 0.9% (w / v) Five times the amount of hemolyzed blood containing ammonium chloride and 0.1% (w / v) potassium bicarbonate was added, and centrifuged at 300 × g for 10 minutes at room temperature.

  (6) After removing the supernatant after centrifugation, the cell pellet was resuspended in 1 mL of a solution containing (1) PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol.

  (7) After the resuspension was centrifuged at 300 × g for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was again added to PEG-BSA (0.1% (w / v) as BSA) and Resuspended in 1 mL of a solution containing 300 mM mannitol.

(8) After the cell suspension from which the supernatant was removed in (7) was held in the cell holding device 100 shown in FIGS. 1 and 3 by the method described below, tumor cells were detected. The cell holding device 100 is provided with a holding part 170 having a diameter of 30 μm and a depth of 40 μm.
(8-1) After introducing the cell suspension collected in (3) from the introduction unit 131, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) is applied from the AC power supply 260 to each electrode 141 and 142. Then, the cells were held in the holding part 270 by the dielectrophoretic force.
(8-2) A 300 mM mannitol aqueous solution containing 0.01% (w / v) poly-L-lysine is introduced from the introduction unit 131 while applying the AC voltage, and after standing for 3 minutes, the AC voltage Application was stopped, and the aqueous solution was removed from the discharge part 132 by suction.
(8-3) A 1% HCHO solution was introduced from the introduction part 131 and the cells were fixed by allowing to stand for 10 minutes, and then the reagent was removed by suction from the discharge part 132. Thereafter, PBS (Phosphate Buffered Saline) was introduced from the introduction part 131 to wash the remaining reagent.
(8-4) One of the following reagents was introduced from the introduction part 131 and allowed to stand for 10 minutes to permeate the cell membrane, and then the reagent was removed by suction from the discharge part 132. Thereafter, PBS (Phosphate Buffered Saline) was introduced from the introduction part 131 to wash the remaining reagent.
(Introduced membrane permeation reagent)
0.025% (w / v) Triton X-100 (trade name, hereinafter omitted) solution 0.05% (w / v) Triton X-100 solution 0.1% (w / v) Triton X-100 solution 0 .2% (w / v) Triton X-100 solution 0.5% (w / v) Triton X-100 solution (8-5) From the introduction part 131, the blocking solution is introduced and left to stand for 10 minutes. After passing through the cell membrane, the reagent was removed from the discharge part 132 by suction. Thereafter, PBS (Phosphate Buffered Saline) was introduced from the introduction part 131 to wash the remaining reagent.
(8-6) FITC (fluorescein isothiocyanate) -labeled anti-cytokeratin antibody (manufactured by Miltenyi Biotec) (hereinafter referred to as CK-FITC) and a nuclear staining reagent for labeling the target tumor cell from the introduction part 131 A cell staining solution mixed with DAPI (4 ′, 6-DiAmidino-2-PhenylIndole) (produced by Dojindo Laboratories) was introduced and cell labeling was performed (25 ° C., 30 minutes), and then from the introduction part 131 The remaining reagent was washed by introducing PBS (Phosphate Buffered Saline).
(8-7) In order to observe all cells held in the holding unit 170, a fluorescence microscope (IX71 manufactured by Olympus) equipped with a computer-controlled electric stage and a CMOS camera (ORCA-Flash 4.0 manufactured by Hamamatsu Photonics) Using this, bright field images and fluorescent images of all the holding parts were taken.
(8-8) The image taken in (8-7) is analyzed using analysis software LabVIEW (manufactured by National Instruments), cells not stained with DAPI (having no cell nucleus) are eliminated on LabVIEW, and CK -Cells labeled with FITC were detected as tumor cells (LNCaP) as target cells.
(8-9) The luminance distribution of CK in the target cells detected in (8-8) was analyzed with 256 gradations from 0 to 255, and the proportion of cells with high luminance (200 or more) in the detected cells was calculated. .

Comparative Example 1
(1) The CK luminance distribution was analyzed in the same manner as in Example 1 except that the membrane permeation reagents introduced in Example 1 (8-4) were changed to the following reagents, respectively.
(Introduced membrane permeation reagent)
1% (w / v) CHAPS (3-[(3-Cholamidopropylo) dimethylaminopropanosulfonate) solution 0.05% (w / v) Nonidet P-40 (trade name) solution 0.1% (w / v) Tween 20 (trade name) solution 0.1% (w / v) Saponin solution Table 1 summarizes the results of Example 1 and Comparative Example 1. When membrane permeabilization with 0.05% (w / v) and 0.1% (w / v) Triton X-100 solution, the ratio of target cells (tumor cells) with high CK brightness is 30% or more It was expensive. On the other hand, 0.025% (w / v), 0.2% (w / v) and 0.5% (w / v) Triton X-100 solution, and surfactants other than Triton X-100 are permeated through the membrane. When used as a reagent, the proportion of target cells (tumor cells) with high CK brightness was as low as 20% or less. From the above results, by treating cells using 0.03% (w / v) to 0.15% (w / v) Triton X-100 as a membrane permeation reagent, It turns out that the brightness | luminance fall by elution can be prevented.

Example 2
(1) After 3 mL of blood was collected from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 3 mL of the stabilizer prepared in Example 1 (2) was collected in the blood collection tube. And the obtained solution was stored as a diluted blood sample.

  (2) The preserved diluted blood sample is allowed to stand at room temperature for 10 minutes, 75 μL of leukocyte / erythrocyte binding agent (Rosetep, manufactured by StemCell Technologies) is added, and then a density gradient solution having a density of 1.086 g / mL in the tube. Layered on top and centrifuged at 2000 × g for 10 minutes at room temperature.

  (3) After centrifugation, the supernatant was collected in a 50 mL container.

  (4) Within a few minutes after recovery, after measuring up to 30 mL with hemolyzed blood containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium bicarbonate, 10 × 300 × g Centrifuged at room temperature for minutes. By this operation, red blood cells mixed in the supernatant are destroyed.

  (5) After removing the supernatant after centrifugation, the cell pellet contains PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol prepared by the method described in Example (1). Resuspended in 30 mL of solution.

  (6) After the resuspension was centrifuged at 300 × g for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was again added to PEG-BSA (0.1% (w / v) as BSA) and Resuspended in 30 mL of a solution containing 300 mM mannitol. This operation is an operation for removing blood components.

  (7) As a staining reagent to be introduced in Example 1 (8-6), PE (phycoerythrin) -labeled anti-CD45 antibody (manufactured by Beckman-Coulter) for labeling leukocytes (hereinafter referred to as CD45-PE) and (6) In the same manner as in Example 1 (8) except that a reagent mixed with DAPI (4 ′, 6-DiAmidino-2-PhenylIndole) (made by Dojindo Laboratories), which is a nuclear staining reagent, was used. After detecting leukocytes remaining in the cell suspension from which the supernatant has been removed and analyzing the luminance distribution of CD45 in the detected leukocytes with 256 gradations from 0 to 255, high luminance (200 or more) in the detected cells The percentage of cells was calculated.

Comparative Example 2
(1) The CK luminance distribution was analyzed in the same manner as in Example 2 except that the membrane permeation reagent introduced in Example 1 (8-4) was changed to the reagent introduced in Comparative Example 1.

  Table 2 summarizes the results of Example 2 and Comparative Example 2. When membrane permeabilization was performed with 0.025% (w / v), 0.05% (w / v) and 0.1% (w / v) Triton X-100 solution, It was relatively high at over 20%. On the other hand, when a 0.2% (w / v) and 0.5% (w / v) Triton X-100 solution and a surfactant other than Triton X-100 were used as a membrane permeation reagent, CD45 high brightness The percentage of white blood cells was as low as about 10% or less. From the above results, leukocyte membrane protein damage and elution can be achieved by treating cells with 0.01% (w / v) to 0.15% (w / v) Triton X-100 as a membrane permeation reagent. It can be seen that a decrease in luminance due to can be prevented.

  When the results of Example 1 and the results of Example 2 were examined in combination, the membrane permeation treatment was performed with 0.05% (w / v) and 0.1% (w / v) Triton X-100. The ratio of target cells (tumor cells) with high CK brightness and the percentage of leukocytes with high CD45 brightness were both high. Therefore, by treating cells with 0.03% (w / v) to 0.15% (w / v) Triton X-100 as a membrane permeation reagent, the target intracellular protein and contaminating cell membrane protein It can be seen that both brightness reduction due to damage and elution can be prevented.

  In particular, when the membrane permeabilization treatment was performed with a 0.05% (w / v) Triton X-100 solution, the ratio of target cells (tumor cells) with high CK brightness was 35.5% (Example 1, Table 1), CD45 The ratio of high-intensity white blood cells is 43.8% (Example 2, Table 2), both of which are particularly high, so that the membrane permeation reagent is 0.03% (w / v) to 0.08% (w / v). It can be seen that treatment of cells with Triton X-100 in (1) can further prevent a decrease in luminance due to damage and elution of target intracellular proteins and contaminating cell membrane proteins.

DESCRIPTION OF SYMBOLS 100: Cell holding | maintenance apparatus 110: Insulating film 120: Light shielding film 121 * 122: Through-hole 130: Spacer 131: Inlet 132: Outlet 141 * 142: Electrode 141a: + pole 142b:-pole 150: Conductor 160: AC power supply 170: Holding unit 200: Detection unit 300: Cell 400: Dielectrophoretic force 500: Light

Claims (3)

A method for detecting target cells and contaminating cells contained in a sample, comprising:
1) a step of permeabilizing target cells and contaminating cells in a sample;
2) a step of labeling with a substance that recognizes the protein specifically possessed by the target cell and the contaminating cell, and 3) a step of detecting the target cell based on the presence or absence of these labels,
And a membrane permeation of target cells and contaminating cells is treated with 0.03% (w / v) to 0.15% (w / v) Triton X-100 (trade name).   The target cell is a tumor cell, the protein specifically possessed by the target cell is an epithelial intracellular protein, and the labeling of the contaminated cell is performed with a substance that recognizes the protein specifically possessed by the contaminated cell membrane. The detection method described.   The sample is a blood sample, the contaminating cell is a leukocyte, the protein specifically possessed by the contaminated cell is a membrane protein of leukocyte, and the target cell is labeled with a substance that specifically recognizes the protein having the label in the target cell. The method according to claim 1 or 2.

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