JP2021019513A - Method for analyzing target particles - Google Patents

Abstract

To provide a method of easily and quickly recovering target cells contained in a sample with high efficiency without requiring special skills to accurately acquire the information of the collected target cells.SOLUTION: The problem is solved by introducing a sample containing target particles into a holding part, collecting the target particles held in the holding part together with contaminating particles contained in the sample after holding the target particles in the holding part, and analyzing the properties of the target particles in the presence of the recovered contaminating particles.SELECTED DRAWING: None

Description

The present invention relates to a method for analyzing target particles contained in a sample. In particular, the present invention relates to a method in which target particles contained in a sample are held in a holding portion, and then the held particles are recovered and analyzed.

In recent years, basic research on body fluids such as blood and cells constituting tissues such as organs, and research on application to clinical diagnosis and treatment by analyzing the cells have been promoted. For example, tumor cells (Circulating Tumor Cell, hereinafter referred to as CTC) are subjected to morphological analysis, histological analysis and genetic analysis from blood collected from cancer patients, and based on the findings obtained by the above analysis. Research is underway to determine treatment policies and to determine changes in pathological conditions during treatment. In particular, analysis of rare cells contained in blood, such as CTC, requires a technique that can individually clarify the information and functions of the rare cells in the presence of contaminating blood cells. There is.

As a means for recovering the target cells for individual analysis of the target cells, a method using FACS (Fluorescence-Activated Cell Sorting), optical tweezers, dielectrophoresis, micromanipulation and the like are known (Patent Documents 1 and 2, Non-Patent Documents 1 to 3). However, FACS makes it difficult to reliably select target cells, and the sorting / isolation operation damages the cells, which may affect the analysis results. In recovery using optical tweezers, dielectrophoresis, and micromanipulation, cells can be captured on a flat substrate or a micro-sized well, and the shape and optical information of the captured cells can be analyzed. Further, by collecting the captured target cells, property analysis such as gene analysis can be performed. However, even in these methods, recovery means such as a micropipette are operated, a precise position control mechanism is required, the recovery operation is complicated and technical skill is required, and recovery takes time. Such issues still exist.

JP-A-2009-002696 Japanese Patent Publication No. 2012-507733

Fu, AY. et al. , Nature Biotechnology, 17, 11091-1111 (1999) Hellmic, W. et al. et al. , Electrophoresis, 26, 3689-3696 (2005) Voldman, J. Mol. , Annual Review of Biomedical Engineering, 8,425-454 (2006)

An object of the present invention is to provide a method for recovering target cells contained in a sample easily, quickly and highly efficiently without requiring special skill, and accurately acquiring information on the recovered target cells. is there.

In order to solve the above problems, the present inventors have reached the present invention as a result of repeated diligent studies.

That is, the present invention includes the aspects described in [1] to [7] below:
[1] (1) A step of introducing a sample containing the target particles into the holding portion and holding the target particles in the holding portion, and (2) a step of recovering the held target particles by a recovery means, (3). ) In the step of analyzing the properties of the recovered target particles and the method of analyzing the target particles contained in the sample containing the target particles, the step (2) is the contamination contained in the target particles and the sample held in the holding portion. The analysis method, which is a step of simultaneously recovering the particles, and the step (3) includes a property analysis of the target particles in the presence of the recovered contaminant particles.

[2] The method according to the above [1], wherein the step (2) is a step of recovering the target particles so that the purity of the target particles is 20% to 80%.

[3] The method according to [1] or [2] above, wherein the holding portion is a recess capable of holding target particles and contaminant particles contained in the sample.

[4] The step (1) introduces a sample containing target particles into a particle holding device provided with a plurality of recesses capable of holding the target particles and contaminant particles contained in the sample, and the recesses provided in the holding device. The step is to hold a plurality of particles in at least 5% of the recesses, and the step (2) recovers from the recesses holding one or more target particles and one or more contaminant particles among the recesses holding the plurality of particles. The method according to the above [3], which is a step of collecting by means.

[5] The method according to [1] to [4] above, wherein the step (3) is a step of subtracting the property analysis result of the contaminant particle alone from the property analysis result of the target particle in the presence of the contaminant particle.

[6] The method according to the above [1] to [5], wherein the target particle is a particle made of a biomaterial.

[7] The method according to [6] above, wherein the property analysis in the step (3) is a base sequence analysis of nucleic acid contained in particles made of a biomaterial.

Hereinafter, the present invention will be described in detail.

In the present invention, the particles refer to insoluble substances that are dispersed alone or in an aggregated state in a solution. Specific examples include particles made of industrial materials such as beads, crushing balls, chromatographic separators, and adsorbents, and particles made of biomaterials such as cells, viruses, organelles, and vesicles. In particular, the present invention is a preferred method for the analysis of particles made of the biomaterial.

When the target particle is a particle made of the above-mentioned biological material, examples of the sample containing the target particle in the present invention include whole blood, diluted blood, serum, plasma, spinal fluid, umbilical cord blood, component blood sampling, urine, and the like. Biological samples such as saliva, semen, feces, sputum, sheep water, ascites, and peritoneal lavage fluid, and tissue suspensions in which pieces of tissue such as liver, lung, spleen, kidney, skin, tumor, and lymph nodes are suspended. Examples thereof include a fraction containing cells derived from the biological sample or tissue obtained by separating from the biological sample or tissue suspension, and a culture solution of cells isolated in advance. Among these, as an example of a fraction containing cells derived from a biological sample or tissue, there is a fraction obtained by layering a biological sample or tissue suspension on a medium for forming a density gradient and then centrifuging the density gradient.

When the sample containing the target particles is a blood sample, examples of the target particles include tumor cells such as blood circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), and circulating fetal cells. Examples include cells (CFC), antigen-specific T cells, and various stem cells. On the other hand, the contaminating particles are particles other than the above-mentioned target particles, and specifically, leukocytes, erythrocytes, platelets and vesicles, which are cells contained in the blood sample, and debris derived from these cells or the above-mentioned target particles. can give. The blood sample in the present specification is not limited to blood samples such as whole blood, serum, plasma, umbilical cord blood, and component blood sample, but the blood sample is diluted with physiological saline or the like, or separated from the blood sample. The obtained fraction containing cells derived from the blood sample is also included in the blood sample.

The holding portion in the present invention may have a shape that can hold the target particles and the contaminating particles contained in the sample, and may be simply flat or uneven. The shape (shape in the plan view) when the holding portion is uneven is not particularly limited as long as it can hold the target particles and the contaminating particles, and may be circular, elliptical, square, rhombus, or the like. It may be a polygon such as a parallelogram, a hexagon, or an octagon. Further, a concave polygon and a convex polygon are also included in the polygon.

When the holding portion is a recess capable of holding the target particles and the contaminant particles, the size of the recess may be appropriately selected according to the size and shape of the target particles and the contaminant particles to be collected, but the size of the recess is large. It is preferable that the target particles and the contaminated particles have a size that can hold at least one of the target particles, because the particle group containing the target particles can be easily recovered. For example, when the target particles are CTC (diameter: about 10 to 25 μm) and the contaminating particles are leukocytes (diameter: about 5 to 20 μm), it is preferable that the recesses have a diameter of 30 μm to 100 μm. The depth is preferably such that the retained cells can be observed, for example, 5 to 100 μm, more preferably 5 to 30 μm.

In the present invention, the method for introducing the sample containing the target particles into the holding portion is not particularly limited, and the sample containing the target particles may be simply introduced into the holding portion, or centrifugal force is used after introducing the sample. The particles may be forcibly held by the holding portion. Above all, the method of holding the particles in the holding portion by using the dielectrophoretic force after introducing the sample is preferable in that the particles can be held efficiently.

In the present invention, when a sample containing target particles is introduced into a particle holding device provided with a plurality of recesses (holding portions), the number of particles held in the recesses is not particularly limited. That is, the recesses provided in the particle holding device after the sample is introduced may include recesses in which no particles are held, or recesses in which a plurality of particles are held. However, from the viewpoint of efficient and / or rapid particle detection and recovery, the higher the particle density, that is, the larger the number of recesses that hold the particles and the larger the number of recesses that hold multiple particles. preferable. In particular, it is preferable that a plurality of particles are held in 5% or more of the total number of recesses provided in the particle holding device, preferably 40% or more, and particularly preferably 60% or more.

The target particles and contaminant particles held in the holding portion may be recovered by using recovery means such as optical tweezers, a means for generating dielectrophoretic force, and micromanipulation. The recovery is not particularly limited as long as it is a method for simultaneously recovering target cells and contaminating cells. As an example, when a micromanipulator is used, the particles may be collected by fixing the micromanipulator, or may be continuously collected while moving the micromanipulator. Further, when the holding portion is a recess that can hold the target particles and the contaminating particles, it may be recovered from a single recess that holds one or more of the target particles and the contaminating particles, and one that holds only the target particles. It may be continuously recovered from the above recesses and one or more recesses holding only the contaminant particles, or these recoverys may be combined. However, from the viewpoint of ease of operation, it is preferable to collect from a single recess, and from the viewpoint of achieving both convenience of operation and accuracy of property analysis, the ratio of target particles contained in the collected particles ( It is preferable to recover the target particles so that the purity) is 20% to 80%.

The method for analyzing the properties of target particles in the present invention is not particularly limited, and as an example, a mass spectrometry method using MALDI-TOF / MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer), a microarray, or the like is used. There is a hybridization method that has been used. When the target particle is a particle made of a biological material such as a cell, as a method for analyzing the properties of the target particle, base sequence analysis using a next-generation sequencer or the like, proteomics analysis / metabolomics analysis using a mass spectrometer can be exemplified. .. In the present invention, since the contaminating particles are recovered together with the target particles, when analyzing the properties of the target particles, there is a possibility that a result overlapping with the property analysis result of the contaminating particles collected at the same time may be obtained depending on the analysis method. Therefore, it is preferable to use an analysis method in which the property analysis result of the contaminated particles can be subtracted because the properties of the target particles can be clearly analyzed.

In the present invention, a step of introducing a sample containing target particles into a holding portion and holding the target particles in the holding portion, a step of recovering the held target particles by a recovery means, and a step of analyzing the properties of the recovered target particles. In the method for analyzing the target particles contained in the sample containing, the recovery step is a step of simultaneously recovering the target particles and the contaminating particles contained in the sample held in the holding portion, and the property analysis. The process is characterized in that the process includes property analysis of the target particles in the presence of the recovered contaminant particles. According to the present invention, when recovering the target particles to be subjected to property analysis, it is not necessary to recover the target particles in units of one particle, which is complicated and time-consuming as in the conventional case, so that no special skill for recovery is required. , The properties of the target particles can be analyzed.

It is an exploded view which shows one aspect of the particle holding apparatus provided with a plurality of holding parts, which can be used in this invention. It is a front view of the apparatus shown in FIG. It is a figure which shows the recovery of the particle held by the apparatus shown in FIG. 1 and FIG. 2 by the recovery means. It is a figure which shows the result of Example 4. FIG.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

FIG. 1 shows a particle holding device provided with a plurality of holding portions, which can be used in the present invention. A front view of the device shown in FIG. 1 is shown in FIG.

The particle holding device 100 shown in FIGS. 1 and 2 includes a flat plate-shaped light-shielding member 11 having a through hole 11a, a flat plate-shaped insulator 12 having a through hole 12a, an introduction port 21, a discharge port 22, and a through hole. The particle introduction and holding means composed of the flat plate-shaped spacer 20 having the portion 23, the electrode substrates 31 and 32 provided so as to sandwich the particle introduction and holding means in the vertical direction, and the electrode substrates 31 and 32 are connected to each other. A lead wire 40 and a signal generator 50 for applying a signal to the electrode substrates 31 and 32 are provided. The light-shielding member 11 and the insulator 12 are provided so that the through-hole 11a of the light-shielding member 11 and the through-hole 12a of the insulator 12 have the same dimensions and shapes, and the positions of the through-holes are the same. .. The holding portion 60 is formed by the electrode substrate 31 provided in close contact with the through hole 11a, the through hole 12a, and the lower part of the light-shielding member 11, and when a sample containing target particles is introduced from the introduction port 21, the holding portion 60 passes through the through portion 23. Particles are introduced into. The electrode substrate 32 is provided in close contact with the upper part of the spacer 20 to prevent scattering and evaporation of a sample containing target particles introduced from the introduction port 21. The electrode substrate 32 has a structure that can be removed from the spacer 20 in order to facilitate the recovery of the particles held in the holding portion 60.

Next, an example of the analysis method of the present invention using the particle holding device 100 shown in FIGS. 1 and 2 will be described.

(1) Target particle holding step A sample containing the target particles 70 is introduced into the particle holding device 100 from the introduction port 21, and the target particles 70 and the contaminating particles 80 contained in the sample are held in the holding portion 60. In the particle holding device 100 shown in FIGS. 1 and 2, when the target particles 70 and the contaminating particles 80 are held by the holding portion 60, the dielectrophoretic force 300 can be used for introduction. Specifically, an AC voltage is applied from the signal generator 50 to the electrode substrates 31 and 32 to generate a dielectrophoretic force 300, and the holding portion 60 holds the target particles 70 and the contaminating particles 80. When the target particle is a cell made of a biological material, the AC voltage applied from the signal generator 50 to the electrode substrates 31 and 32 is periodically charged and discharged by the target particle (cell) 70 held in the holding unit 60. It is preferable that the AC voltage has a waveform that repeats the above, and it is particularly preferable that the frequency is between 100 kHz and 3 MHz and the electric field strength is between 1 × 10 ⁵ and 5 × 10 ⁵ V / m (WO2011 / 149032). And Japanese Patent Application Laid-Open No. 2012-013549).

When the particles are held in the holding unit 60 by using the dielectrophoretic force 300, the sample containing the target particles 70 introduced into the particle holding device 100 is a dispersion medium in which the particles can be moved by the dielectrophoretic force 300. It is necessary to prepare a sample in which As an example of the dispersion medium, an aqueous solution containing saccharides such as mannitol, xylitol, glucose and sucrose, an electrolyte such as calcium chloride and magnesium chloride, and / or a protein such as BSA (bovine serum albumin) are further contained in the aqueous solution. An aqueous solution can be given. In particular, when the target particles are particles made of a biomaterial such as cells, it is preferable to use a sugar solution prepared to have a concentration of an isotonic solution in that damage to the particles is reduced.

The concentration of the target particles contained in the sample is not particularly limited, but one or more target particles 70 and one or more contaminant particles 80 are provided in at least a part of the holding portions 60 provided in the particle holding device 100. A retained concentration is preferable in that particles can be detected and recovered efficiently. In the case of the particle holding device 100 shown in FIGS. 1 and 2, the number of particles introduced into the particle holding device 100 may be 50% or more of the number of holding portions 60 provided in the particle holding device 100, and the number of holding portions 60. It is preferably twice or more, and more preferably three times or more the number of holding portions 60. For example, when the target particles are rare cells (CTC or the like) in which only a few to several tens of particles are present in 1 mL of a blood sample, 50% or more of the number of holding portions 60 provided in the particle holding device 100 is introduced. By doing so, it is possible to make a recess that holds at least one rare cell as a target particle and one or more leukocytes as a contaminating particle.

In order to prevent the target particles 70 and the contaminant particles 80 held in the holding portion 60 from re-flowing out, the particles may be fixed to the holding portion 60 using an adhesive reagent. Examples of the adhesive reagent when the particles are cells include poly-L-lysine, collagen, and BAM (cell membrane modifier, manufactured by Yuka Sangyo Co., Ltd.). The particles can be fixed to the holding portion 60 by coating the holding portion 60 with the adhesive reagent in advance, introducing a sample containing the target particles, holding the particles in the holding portion 60, and fixing the particles to the holding portion 60. Often, the adhesive reagent may be introduced after the particles are held in the holding portion 60, and the held particles may be fixed in the holding portion 60.

(2) Recovery Step of Retained Particles The target particles 70 and the contaminating particles 80 held in the holding unit 60 are simultaneously recovered by using the recovery means 400 (FIG. 3). In FIG. 3, after removing the electrode substrate 32 provided in the particle holding device 100, the particles held in the holding portion 60 are sucked by using the micromanipulator 400 which is a collecting means, and then discharged into a recovery tube or the like prepared in advance. Then, the particles are collected.

In collecting the particles, the target particles 70 and / or the contaminating particles 80 held in the holding unit 60 may be identified based on the characteristics of these particles. For example
When identifying based on the optical characteristics of the target particle such as a bright-field image, a fluorescence image, or a chemiluminescent image, the detection may be performed using an optical measuring instrument such as an optical detector or an optical microscope.
When identifying based on the characteristics such as elasticity and viscosity of the target particle, it may be detected using an ultrasonic measuring instrument such as an ultrasonic microscope.
When identifying based on the radiochemical characteristics of target particles such as target particles labeled with radioisotopes, detection may be performed using a radiation detector such as a scintillation detector.
When identifying based on the thermal responsiveness and thermal physical characteristics of the target particles, the thermal responsiveness and thermal physical characteristics may be detected using a detectable device.

Further, when identifying the target particles and / or the contaminating particles, the particles may be labeled based on the characteristics of these particles before detection. As an example, when the particle is a particle made of biological material, antigen-antibody, receptor-hormone, receptor-ligand, lectin-sugar chain (carbohydrate), Fc receptor or mouse IgG-Protein A, avidin-biotin, streptavidin Interactions such as-biotin and virus-receptors can be used to identify target and / or contaminant particles. As a specific example, when the target particle is a cell, the cell is bound with a nucleic acid labeling reagent such as DAPI (4', 6-DiAmidino-2-PhenylIndole) or a fluorescent substance such as PE (phycoerythrin). It may be labeled with a fluorescence microscope 200 and detected with a fluorescence microscope 200 (FIG. 2).

In the present invention, by simultaneously collecting the target particles 70 and the contaminating particles 80 held in the holding unit 60, the target particles can be easily and quickly recovered without requiring special skill or complicated operation. This step is not particularly limited as long as it is a method capable of simultaneously recovering the target cells 70 and the contaminating cells 80. As an example,
(I) As shown in FIG. 3, one or more target particles 70 and one or more contaminant particles 80 held in one holding portion may be simultaneously collected by using a micromanipulator 400.
(II) The target particles 70 and the contaminating particles 80 held by the plurality of holding portions 60 may be continuously collected while moving the micromanipulator 400.
From the viewpoint of ease of operation, the recovery method (I) described above is preferable. Further, from the viewpoint of property analysis of the target particles described later, the ratio of the target particles 70 to the contaminant particles 80 recovered in this step is 20% of the ratio of the target particles 70 to the total number of particles to be recovered (purity of the target particles 70). It is preferable to collect the particles so as to be 80%.

(3) Property Analysis Step of Target Particles The property analysis of the target particles 70 recovered from the holding unit 60 is performed in the presence of the contaminating particles 80 recovered at the same time. It is preferable to subtract the analysis result from the property analysis result of the contaminant particle 80 alone because the property of the target particle 70 can be clearly analyzed.

An example of a preferable analysis method when the sample containing the target particles is a blood sample containing blood circulating tumor cells (CTC) will be described below.
(I) Whole-genome amplification and library preparation are performed on a sample containing CTC (target particles) and leukocytes (contamination particles) collected from the holding unit 60, and gene mutations are detected using a next-generation sequencer.
(Ii) Separately, the gene mutation of leukocyte alone is detected by the same method as in (i).
(Iii) By subtracting the gene mutation detected in (ii) from the gene mutation detected in (i), the gene mutation of CTC alone is detected.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

Example 1 Comparison of gene mutations between leukocytes and cancer cell lines Gene mutations were detected in a population of healthy leukocytes and a population of cancer cell lines by the methods shown below.

(1) 7.5 mL of erythrocyte crushing solution containing ammonium chloride as a main component was added to 0.5 mL of healthy person blood obtained by obtaining informed consent, and the erythrocytes were crushed by allowing to stand at room temperature for 5 minutes. Then, the cells were washed by centrifugation at 900 × g for 5 minutes to remove the supernatant, and then resuspending the cell pellet in PBS (phosphate buffered saline).

(2) The cell suspension obtained in (1) was centrifuged at 900 × g for 5 minutes, and the supernatant was removed. The remaining cell pellet was suspended in 1 mL of PBS containing 1% (w / v) formaldehyde (hereinafter, also referred to as "cell fixation reagent"), and the cells were fixed by allowing to stand for 10 minutes. Then, the cells were washed by centrifugation at 600 × g for 5 minutes to remove the supernatant, and then resuspending the cell pellet in PBS.

(3) A lung cancer cell line (H1975 strain) cultured on a 24-well microplate was exfoliated with trypsin and collected in a microtube. After centrifuging at 600 xg for 5 minutes to remove the supernatant, the cell pellet was washed by resuspending in PBS.

(4) Genomic DNA was extracted from the leukocyte suspension obtained in (2) and the cancer cell line suspension obtained in (3) using NucleoSpin Tissue (manufactured by Takara Bio Inc.).

(5) For each genomic DNA obtained in (4), Ion AmpliSeq Cancer Hotspot Panel v2 (hereinafter, CHPv2) and Ion AmpliSeq Library Kit Plus (both prepared by Thermo Fisher Scientific) were used. , Ion S5 (manufactured by Thermo Fisher Scientific) was used for gene mutation analysis.

Table 1 shows the mutations of Frequency 10% or more detected in the population of healthy leukocytes and the population of cancer cell lines, respectively. A / G mutations at positions 55,141,055 (PDGFRA gene) on chromosome 4 (chr4) and G / A mutations at positions 112,175,770 (APC gene) on chromosome 5 (chr5) are caused by leukocytes. It was commonly detected in both cell lines. On the other hand, the C / T mutation at positions 55,152,040 (PDGFRA gene) on chromosome 4 (chr4) and the G / A mutation at positions 55,249,063 (EGFR gene) on chromosome 7 (chr7) are It is detected only in the chromosomal cell line, and it can be seen that it is a gene mutation peculiar to the cancer cell line (H1975 strain).

Example 2 Gene analysis from a mixed heterologous cell sample (Part 1)
Gene mutations in cancer cell lines were detected in a mixed sample of healthy leukocytes and cancer cell lines by the method shown below.

(1) 3 mL of healthy person blood obtained by obtaining informed consent was scalpel-up to 90 mL with an erythrocyte crushing solution containing ammonium chloride as a main component, and allowed to stand at room temperature for 5 minutes to crush erythrocytes. Then, after centrifuging at 900 × g for 5 minutes to remove the supernatant, the cell pellet was subjected to a solution containing PEG-BSA (0.1% (w / v) as BSA) and 280 mM xylitol (hereinafter, “PEG-BSA”). It was washed by suspending it with "xylitol solution"). The cleaning operation was repeated 3 times.

(2) A lung cancer cell line (H1975 strain) cultured on a 24-well microplate was exfoliated with trypsin and collected in a microtube. The supernatant was removed by centrifugation at 600 xg for 5 minutes, and the cell pellet was washed by resuspending in 1 mL of cell culture medium.

(3) The cancer cell line suspension obtained in (2) was centrifuged at 600 × g for 5 minutes to remove the supernatant, and then the cell pellet was suspended in 1 mL of an erythrocyte crushing solution containing ammonium chloride as a main component. The erythrocytes were crushed by allowing them to stand at room temperature for 5 minutes. Then, after centrifuging at 900 × g for 5 minutes to remove the supernatant, the cell pellet was washed by suspending it in a PEG-BSA xylitol solution. The cleaning operation was repeated 3 times.

(4) The leukocyte suspension obtained in (1) and the cancer cell line suspension obtained in (3) were introduced into the particle retention device 100 shown in FIGS. 1 and 2, respectively. By applying an AC voltage (1 MHz, 20 Vp-p) from the signal generator 50 to the electrode substrates 31 and 32 for 10 minutes, the dielectrophoretic force 300 causes the holding portion 60 of the device to hold the leukocyte or cancer cell line. , A retention device that retained white blood cells, and a retention device that retained cancer cells were prepared. The particle holding device 100 used in this embodiment includes an insulator 12 having a plurality of through holes 12a having a diameter of 30 μm, a light-shielding chrome film (light-shielding member 11) having a plurality of through holes 11a having a diameter of 30 μm, and an electrode substrate 31. A spacer 20 having a thickness of 1 mm, which is provided in the order of the insulator 12-the light-shielding member 11-the electrode substrate 31 from the top, and further has a sample introduction port 21, a discharge port 22 and a penetration portion 23 on the upper surface of the insulator 12. Is a device in which an electrode substrate 32 is provided in close contact with each other on the upper surface of the spacer 20. The through holes 11a / 12a and the electrode substrate 31 form about 300,000 holding portions 60 capable of holding the target particles 70 and the contaminant particles 80 having a diameter of 30 μm and a depth of 40 μm.

(5) A 280 mM xylitol aqueous solution containing 0.01% (w / v) poly-L-lysine is introduced from the introduction port 21 while applying the AC voltage, allowed to stand for 3 minutes, and then the AC voltage is applied. Was stopped, and the aqueous solution was sucked and removed from the discharge port 22.

(6) The cell-fixing reagent was introduced from the introduction port 21 and allowed to stand for 10 minutes to fix the cells. Then, the cell-fixing reagent was aspirated and removed from the discharge port 22, and PBS-T (Phosphate buffered saline with Tween 20) was introduced from the introduction port 21 to wash the remaining cell-fixing reagent.

(7) An aqueous solution containing 95% (w / v) ethanol (hereinafter referred to as a membrane permeation reagent) was introduced from the introduction port 21 and allowed to stand for 10 minutes to perform membrane permeation treatment of cells. Then, the cell-fixing reagent was aspirated and removed from the discharge port 22, and PBS-T was introduced from the introduction port 21 to wash the residual membrane permeation reagent.

(8) 850 μL of a cell staining reagent mixed with DAPI (4', 6-DiAmidino-2-Phenyl Industry) (manufactured by Dojin Chemical Research Institute) was introduced from the introduction port 21, and the cells were labeled by allowing to stand for 20 minutes. .. Then, the cell staining reagent was aspirated and removed from the discharge port 22, and PBS-T was introduced from the introduction port 21 to wash the remaining cell staining reagent.

(9) After detecting DAPI-positive cells (white blood cells or cancer cell lines) held in the holding portion 60 using a fluorescence microscope 200 (IX71 manufactured by Olympus Corporation), as shown in FIG. 3, micro is a suction means. Using the manipulator 400, the leukocyte or cancer cell line held in the holding portion 60 of the particle holding device 100 from which the electrode substrate 32 on the upper surface was removed was aspirated. A heterologous cell mixed sample is prepared by discharging the aspirated leukocyte or cancer cell line into a 0.2 mL microtube so that the ratio of cancer cell number: leukocyte count is 1: 1 or 1: 2 and mixing them. did.

(10) Whole genome amplification of cells in a mixed sample was performed using an Ampli1 WGA kit (manufactured by Silicon Biosystems). Then, for the obtained whole genome amplification product, a library was prepared by CHPv2 and Ion AmpliSeq Library Kit Plus (both manufactured by Thermo Fisher Scientific), and Ion S5 (manufactured by Thermo Fisher Scientific) was used. Analyzed.

(11) From the gene mutation of the mixed sample detected in (10), the gene mutation detected by the gene mutation analysis from the healthy subject leukocyte population in Example 1 (that is, in Table 1, "presence or absence of mutation (leukocyte)" column By subtracting the mutation with a circle in, a mutation peculiar to the cancer cell line (H1975 strain) was detected.

Table 2 shows the detection results of the gene mutation (Frequency 10% or more) peculiar to the cancer cell line (H1975 strain) found in Example 1. In this example, the heterologous cell mixed sample was prepared 6 times each, and the results for each preparation are shown in columns a to f. In the sample in which the cancer cell line and the leukocyte were recovered 1: 1 (cancer cell purity 50%), the cancer cell line (H1975 strain) found in Example 1 was prepared 3 times out of 6 times. A peculiar gene mutation could be detected. Further, even in a sample in which a cancer cell line and leukocytes were recovered at a ratio of 1: 2 (cancer cell purity 33%), the cancer cells found in Example 1 were used once out of six preparations. A gene mutation peculiar to the strain (H1975 strain) could be detected.

From the above results, when analyzing the target particles (cancer cells) contained in the sample, even if the target particles are collected in a state of being mixed with the contaminating particles (white blood cells) contained in the sample, the mixed state is obtained. It can be seen that the target particles (cancer cells) can be analyzed by subtracting the property analysis results of the contaminating particles (white blood cells) alone from the property analysis results of.

Example 3 Gene analysis from a mixed heterologous cell sample (Part 2)
From Example 2, it was found that the target particles could be analyzed from the property analysis results of the mixed sample of the target particles and the contaminating particles. Therefore, even if the sample has a lower purity than that of Example 2, the target particles can be used. It was verified whether it could be analyzed.

(1) 7.5 mL of erythrocyte crushing solution containing ammonium chloride as a main component was added to 0.5 mL of healthy person blood obtained by obtaining informed consent, and the erythrocytes were crushed by allowing to stand at room temperature for 5 minutes. The cells were then washed by centrifugation at 900 xg for 5 minutes to remove the supernatant and resuspending the cell pellet in PBS.

(2) A lung cancer cell line (H1975 strain) cultured on a 24-well microplate was exfoliated with trypsin and collected in a microtube. The supernatant was removed by centrifugation at 600 xg for 5 minutes, and the cell pellet was washed by resuspending in 1 mL of cell culture medium.

(3) The leukocyte suspension obtained in (1) and the cancer cell line suspension obtained in (2) were centrifuged at 900 × g for 5 minutes, respectively, and the supernatant was removed. Each of the remaining cell pellets was suspended in 1 mL of the cell fixation reagent and allowed to stand for 5 minutes to fix the cells. The supernatant was then removed by centrifugation at 600 xg for 5 minutes and the cell pellet was washed by suspension in PBS.

(4) After each cell suspension of (3) is centrifuged at 600 × g for 5 minutes, the supernatant is removed, suspended in 1 mL of a membrane permeation reagent, and allowed to stand for 5 minutes to permeate the cell membrane. Processing was performed. The cells were then washed by centrifugation at 600 xg for 5 minutes at room temperature to remove the supernatant and suspending the cell pellet in PBS.

(5) After centrifuging each cell suspension of (4) at 600 × g for 5 minutes, the supernatant is removed, suspended in 1 mL of a cell stain mixed with DAPI, and allowed to stand for 20 minutes. The cells were labeled. The cells were then washed by centrifugation at 600 xg for 5 minutes at room temperature to remove the supernatant and suspending the cell pellet in PBS.

(6) Each cell suspension of (5) was centrifuged at 600 × g for 5 minutes, the supernatant was removed, and the suspension was suspended in a 300 mM mannitol solution. A part of the obtained leukocyte suspension and cancer cell line suspension was collected, and the number of cells was calculated based on the cell count using a fluorescence microscope. Based on the calculation result, a heterologous cell mixed sample is prepared by mixing the white blood cells in each suspension with the cancer cell line so that the number of cancer cells: the number of white blood cells is 1: 1 or 1: 5. did.

(7) According to the methods described in (10) and (11) of Example 2, mutations specific to the target cell (cancer cell line (H1975 strain)) were detected from the heterologous cell mixed sample of (6).

Table 3 shows the detection results of the gene mutation (Frequency 10% or more) peculiar to the cancer cell line (H1975 strain) found in Example 1. In this example, the heterologous cell mixed sample was prepared twice, and the results for each preparation are shown in columns a and b. Cancer cell number: In the sample mixed so that the white blood cell count was 1: 1 (purity of the cancer cell line 50%), one of the two prepared samples was the cancer cell line found in Example 1 (cancer cell line). A gene mutation peculiar to the H1975 strain could be detected. On the other hand, in the sample mixed so that the number of cancer cells: the number of white blood cells was 1: 5 (purity of the cancer cell line 17%), the gene mutation peculiar to the cancer cell line (H1975 strain) found in Example 1 was found. Could not be detected.

From the above results, when the target particles (cancer cells) are analyzed in a state of being mixed with the contaminating particles (white blood cells), if the purity of the target particles in the mixed sample is less than 20%, the analysis of the target particles can be performed. It turns out that it can be difficult.

Example 4 Relationship between the number of particles introduced and the number of particles held in the holding unit When a sample containing particles is introduced into the particle holding device 100 shown in FIGS. 1 and 2, the number of particles to be introduced and the number of particles held in the holding unit 60 are introduced. The relationship with the number of retained particles was confirmed.

(1) 3 mL of healthy person blood obtained by obtaining informed consent was scalpel-up to 90 mL with an erythrocyte crushing solution containing ammonium chloride as a main component, and allowed to stand at room temperature for 5 minutes to crush erythrocytes. Then, the cells were centrifuged at 900 × g for 5 minutes to remove the supernatant, and the cell pellet was resuspended in 30 mL of PEG-BSA washing solution.

(2) The resuspension was centrifuged at 600 × g for 5 minutes to remove the supernatant, and the cell pellet was washed by resuspending with 30 mL of PEG-BSA xylitol solution. The cleaning operation was repeated 3 times.

(3) The cell suspension obtained in (2) was prepared so that the number of introduced cells was 100,000 to 2 million, and the prepared cell suspensions are shown in FIGS. 1 and 2. It was introduced into the particle holding device 100. After the introduction, an AC voltage (1 MHz, 20 Vpp) was applied from the signal generator 50 to the electrode substrates 31 and 32 for 10 minutes, so that the cells were held in the holding portion 60 of the device by the dielectrophoretic force 300. The particle holding device 100 used in this embodiment is the same as the device described in Example 2 (4).

(4) Cell fixation, membrane permeation and labeling were carried out according to the methods described in Examples 2 (5) to (8).

(5) Using a fluorescence microscope 200 (IX71 manufactured by Olympus Corporation), all the cells held in each holding part 60 were detected, and the number of cells held in each holding part 60 was measured.

FIG. 4 shows the relationship between the number of cells introduced into the particle holding device 100 and the number of cells held in each holding portion 60. It can be seen that if 200,000 or more cells (that is, 50% or more of the number of holding units 60 (300,000)) are introduced into the particle holding device 100, a plurality of cells can be held in 5% or more of the holding parts 60. .. Further, when 680,000 or more cells (that is, twice or more the number of the holding parts 60) are introduced into the particle holding device 100, 1 million or more cells (that is, the holding part 60) are introduced into 40% or more of the holding parts 60. It can be seen that a plurality of cells can be retained in 60% or more of the retention unit 60 by introducing (three times or more of the number of cells).

When 200,000 or more cells (that is, 50% or more of the number of holding portions 60 (300,000)) are introduced into the particle holding device 100, the ratio of the recesses that hold one or more cells is about 50%. From this, it can be seen that a plurality of cells are retained in 10% or more of the recesses in which the cells are retained. For example, when CTC contained in a blood sample is used as a target particle, the number of CTCs generally ranges from several to several tens per mL, and therefore 10% of the recesses in which cells are retained. If a plurality of cells are retained, there may be recesses that retain one or more of CTC, which is a target particle, and leukocyte, which is a contaminating particle.

Example 5 Detection rate of target cells An attempt was made to detect target cells from a mixed sample of healthy leukocytes (contamination cells) and cancer cell lines (target cells) by the method shown below.

(1) Approximately 200 lung cancer cell lines (PC9 strain) were added to 3 mL of healthy subject blood obtained by obtaining informed consent to prepare a spike sample. The spike sample was scalpel-up to 90 mL with an erythrocyte crushing solution containing ammonium chloride as a main component, and allowed to stand at room temperature for 5 minutes to crush the erythrocytes. Then, the cells were centrifuged at 900 × g for 5 minutes to remove the supernatant, and the cell pellet was resuspended in 30 mL of PEG-BSA washing solution.

(2) The resuspension was centrifuged at 600 × g for 5 minutes, the supernatant was removed, and the cell pellet was washed by resuspending with 30 mL of PEG-BSA xylitol solution. The cleaning operation was repeated 3 times.

(3) The cell suspension obtained in (2) was prepared so that the number of introduced cells was 25,000 or 1 million, respectively, and the prepared cell suspension was used in Example 4 (3). ), The holding unit 60 was used to hold the cells.

(4) Cell fixation and membrane permeation were performed according to the methods shown in (5) to (7) of Example 2.

(5) 850 μL of a cell labeling reagent mixed with an anti-cytokeratin mouse antibody (manufactured by Miltenyi Biotec) was introduced from the introduction port 21, and the cells were allowed to stand for 30 minutes to label cancer cells. Then, the cell labeling reagent was aspirated and removed from the discharge port 22, and PBS-T was introduced from the introduction port 21 to wash the residual cell labeling reagent.

(6) From the inlet 21, Alexa Fluor 488-labeled anti-mouse IgG1 antibody (manufactured by Thermo Fisher Science), PE (phycoerythrin) -labeled anti-CD45 antibody (manufactured by Beckman-Coulter) and DAPI (manufactured by Dojin Chemical Research Institute). 850 μL of the cell staining reagent mixed with the above was introduced, and the cells were stained by allowing to stand for 20 minutes. Then, the cell staining reagent was aspirated and removed from the discharge port 22, and PBS-T was introduced from the introduction port 21 to wash the remaining cell staining reagent.

(7) Using a fluorescence microscope 200 (IX71 manufactured by Olympus Corporation), all the target particles 70 and the contaminant particles 80 held in the holding unit 60 are detected, and the target particles held in the holding unit 60 are based on the following criteria. And the number of contaminant particles was counted.
Target particles (cancer cell line): Cell contaminating particles (leukocytes) stained with DAPI (having cell nuclei), stained with Alexa Fluor 488 (expressing cytokeratin), and not stained with PE (not expressing CD45). ): Cells stained with DAPI (having cell nuclei), stained with PE (expressing CD45) and not stained with Alexa Fluor 488 (not expressing cytokeratin), introduced into particle retention device 100 on Table 4. The relationship between the number of cells and the detection rate of target particles (cancer cell lines) is shown. Here, the detection rate is the number detected in (7) with respect to the number of cancer cell lines added to the blood of healthy subjects in (1) (the number including the dilution in the suspension preparation of (3)). Represents the percentage of cells. It can be seen that rare target cells can be detected with a high detection rate regardless of the number of cells introduced into the particle retention device.

100: Particle holding device 11: Light-shielding member 12: Insulators 11a and 12a: Through hole 20: Spacer 21: Introductory port 22: Discharge port 23: Penetrating part 31 and 32: Electrode substrate 40: Conductor 50: Signal generator 60: Holding part 70: Target particles 80: Contaminating particles 200: Fluorescence microscope 300: Dielectrophoretic force 400: Aspiration means (micromanipulator)

Claims (7)

(1) A step of introducing a sample containing target particles into a holding portion and holding the target particles in the holding portion.
(2) A step of recovering the retained target particles by a recovery means and
(3) The process of analyzing the properties of the recovered target particles and
In the method of analyzing the target particles contained in the sample containing
The step (2) is a step of simultaneously recovering the target particles and the contaminating particles contained in the sample held in the holding portion.
The step (3) is a step including property analysis of the target particles in the presence of the recovered contaminant particles.
The analysis method. The method according to claim 1, wherein the step (2) is a step of recovering the target particles so that the purity of the target particles is 20% to 80%. The method according to claim 1 or 2, wherein the holding portion is a recess capable of holding target particles and contaminant particles contained in the sample. In the step (1), the sample containing the target particles is introduced into a particle holding device provided with a plurality of recesses capable of holding the target particles and the contaminating particles contained in the sample, and among the recesses provided in the holding device. It is a step of holding a plurality of particles in a recess of at least 5%.
The method according to claim 3, wherein the step (2) is a step of recovering the target particles and the contaminating particles from the recesses holding one or more of the plurality of particles by the collecting means. The method according to claims 1 to 4, wherein the step (3) is a step of subtracting the property analysis result of the contaminating particles alone from the property analysis result of the target particles in the presence of the contaminating particles. The method according to claims 1 to 5, wherein the target particle is a particle made of a biomaterial. The method according to claim 6, wherein the property analysis in the step (3) is a base sequence analysis of nucleic acid contained in particles made of a biomaterial.