JP2020096555A - Method for collecting cells - Google Patents

Abstract

To provide a method for accurately collecting target cells from a sample including a trace amount of target cells and a large amount of foreign cells.SOLUTION: A method for collecting cells includes: either one of a process of allowing a holding part to hold target cells after binding magnetic particles to the target cells included in a sample, or a process of allowing magnetic particles to bind the target cells after holding the target cells included in a sample by the holding part; and a process of allowing a magnetic substance to approach the target cells held by the holding part and collecting the target cells.SELECTED DRAWING: None

Description

The present invention relates to a method for accurately collecting target cells contained in a sample.

In recent years, cells have been selectively separated and collected from body fluids such as blood, tissue samples obtained by suspending or dispersing tissues such as organs in a solution, and cell culture solutions, and basic research on the separated and collected cells Research is being conducted to apply it to clinical diagnosis and treatment. For example, tumor cells (Circulating Tumor Cell, hereinafter CTC) are collected from blood collected from a cancer patient, and morphological analysis, tissue type analysis and gene analysis are performed on the cells, and based on the findings obtained by these analysis. Research is underway to determine treatment strategies.

Since the number of CTCs contained in a blood sample obtained from a cancer patient is very small compared to blood cells such as red blood cells and white blood cells, pretreatment for removing the blood cells is essential, and the magnetic force is easy to operate. Concentration (enrichment) using is performed. For example, Patent Document 1 discloses a method using magnetic particles to which an antibody against an epithelial marker whose expression is seen on the CTC surface is bound. However, according to this method, there is a risk that foreign cells other than CTC present in the blood sample may be sucked in a state of being involved.

JP, 2005-010177, A

An object of the present invention is to provide a method for accurately collecting the target cells from a sample containing a small amount of target cells and a large amount of foreign cells.

The present inventors have arrived at the present invention as a result of intensive studies to solve the above problems.
That is, the first aspect of the present invention is
After binding the magnetic particles to the target cells contained in the sample, holding the target cells in the holding part, or
After holding the target cells contained in the sample in the holding unit, one of the steps of binding magnetic particles to the target cells,
A step of collecting the target cells by bringing a magnetic substance close to the target cells held by the holding unit;
Is a method of collecting cells comprising.
In addition, a second aspect of the present invention is
The method described above further comprises a step of performing labeling before collecting the target cells and detecting the target cells based on the label.

Hereinafter, the present invention will be described in detail.

The sample may be a sample containing at least the target cells and contaminant cells described below, and specifically, blood (whole blood), diluted blood, serum, plasma, spinal fluid, umbilical cord blood, component blood samples, etc. Blood samples, biological samples such as urine, saliva, semen, feces, sputum, amniotic fluid, ascites, and suspensions of a piece of tissue such as liver, lung, spleen, kidney, skin, tumor, lymph node, Examples thereof include a fraction containing cells derived from the biological sample or the tissue, which is obtained by separating the biological sample or the tissue suspension, a culture solution of cells isolated in advance, cultured cells, and the like. Among them, the method of the present invention is suitable for collecting target cells from the above-mentioned blood sample.

The target cell is not particularly limited as long as it is a cell contained in the sample described above, but it is preferable that the target cell is a cell that is present in an extremely small number relative to the number of contaminating cells contained in the sample. For example, the number of contaminating cells contained in the sample is 0.1% to 10% or less than 0.1%, and the sample is a blood sample, and the contaminating cells are neutrophils and eosinophils. , White blood cells such as basophils, monocytes, and lymphocytes having specific characteristics, tumor cells such as blood circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), circulating fetal cells (CFC), various stem cells, and B cells can be exemplified as the target cells.

Examples of magnetic particles include particles that can be magnetized permanently, ferromagnetic particles, polymer particles that include or coat a magnetic material, and particles that exhibit magnetic behavior (superparamagnetism) only when placed in a magnetic field. Particles exhibiting superparamagnetism are preferred because the operation of collecting target cells is easy. An example of a material exhibiting superparamagnetism is iron oxide represented by magnetite. The size of the magnetic particles is preferably the same as or smaller than that of the target cells, because the influence of the magnetic particles on cell observation can be suppressed. For example, when the target cells are CTCs having a cell diameter of 5 μm to 25 μm, the average particle diameter of the magnetic particles is preferably 10 nm or more and 30 μm or less, more preferably 20 nm or more and 25 μm or less, and further preferably 30 nm or more and 5 μm or less.

When binding the magnetic particles to the target cells, the magnetic particles may be bound to both the target cells and the contaminating cells, or the magnetic particles modified with a substance capable of specifically binding to the target cells may be bound only to the target cells. .. When magnetic particles are bound to both target cells and foreign cells, if the target cells are labeled, the target cells can be detected based on the label. The addition of the step of labeling the target cells is preferable in that the presence or absence of the target cells before and after the collection can be confirmed, and the existence position of the target cells can be specified. There is no problem as to the timing of labeling the target cells before the step of collecting the target cells described below, that is, before binding the magnetic particles to the target cells, before holding the target cells in the holding part, and holding the target cells. It may be any time from the time when the target cells are held to the time when the target cells are collected.

The method for labeling the target cells is not particularly limited, and the target cells may be directly labeled with a coloring reagent or a fluorescent reagent, or may be labeled with a labeled antibody against a protein contained in the target cells, You may label using the dye which can couple|bond with the nucleic acid contained in a target cell. Among them, the labeling method using a labeled antibody against the protein is preferable because the protein can be detected easily, with high sensitivity, and specifically. The substance that labels the antibody is not particularly limited, and examples thereof include fluorescent substances such as fluorescein isothiocyanate (FITC) and Alexa Fluor (trade name). The labeled cells may be detected using a means suitable for labeling, such as a microscope or an optical detector. For example, when labeling is performed using a fluorescent dye, a fluorescence microscope may be used.

In order to bind the magnetic particles to both the target cells and the contaminating cells, it is sufficient to modify the magnetic particles with a substance capable of binding to proteins, sugars, lipids and functional groups common to both cells. In order to bind magnetic particles only to cells, substances that can bind to proteins, sugars, lipids, and functional groups that are highly expressed in target cells compared to contaminated cells (substances that can specifically bind to target cells) May be modified into magnetic particles. As a specific example, when the target cell is CTC contained in a blood sample, cytokeratin (CK) or EpCAM (Epithelial cell adhesion molecule) can be exemplified as a substance capable of specifically binding to the target cell, and the target cell is a leukocyte. In this case, the leukocyte marker CD45 can be exemplified as the substance capable of specifically binding to the target cell.

The holding unit may be any mode capable of holding and storing the target cell, and may be a recess or through hole capable of storing the cell, or a material capable of fixing the substance (for example, poly-L-lysine or BAM (Biocompatible). Anchor for cell Membrane)). In addition, it is preferable that the holding portion is in the form of a recess or a through hole in which the target cells can be stored, because the target cells can be easily collected. When the holding portion is a recess, it is preferable that one end has an opening having a size capable of holding all or part of cells and the other end forms a bottom portion. In the case of a hole, one end has an opening with a size that can hold all or part of the cell and the other end is an opening narrower than the opening, or both ends hold part of the cell. It is advisable to adopt an aspect in which the opening has the largest possible size. It is preferable that the size (diameter) of the holding portion is such that only one cell contained in the sample can be held, because single or specific cells can be easily collected. For example, when the target cell is a CTC having a cell diameter of 5 μm to 25 μm, the holding portion is preferably a recess having a diameter of 30 μm and a depth of 30 μm.

In the cell holding device having the holding part, the region containing the sample containing the target cells may be hydrophilized for the purpose of preventing the target cells from adsorbing to other than the holding part. Examples of the method for hydrophilizing include corona discharge treatment, plasma treatment, photocatalytic coating, chemical modification with silane coupling agent, protein modification, etc. Protein modification that can be hydrophilized is preferred. Hydrophilization by protein modification may be performed by immersing or contacting the substrate with a protein-containing solution. The protein used for hydrophilization is not particularly limited, but generally, BSA (bovine serum albumin), OVA (ovalbumin) and the like which are soluble proteins contained in serum, milk, egg white and the like are preferable.

When the target cells are held in the holder, they may be held by gravity sedimentation or shaking, but it is preferable to use the dielectrophoretic force because the target cells can be efficiently held in the holder. When the dielectrophoretic force is used, specifically, an alternating voltage may be applied to generate dielectrophoresis and the target cells may be introduced into the holding unit. The AC voltage to be applied is preferably an AC voltage having a waveform in which charging/discharging of cells in the holding part is periodically repeated, the frequency is between 100 kHz and 3 MHz, and the electric field strength is 1×10 ⁵ to 5×10 5. ^It is particularly preferable to set it to ⁵ V/m (see WO2011/149032 and JP2012-013549A).

The cells retained in the retaining section may be subjected to storage and membrane permeation treatment. Examples of preservatives include formaldehyde, formaldehyde donor compounds (compounds capable of releasing formaldehyde by hydrolysis), aldehydes such as glutaraldehyde, alcohols such as methanol and ethanol, and solutions containing heavy metals. .. Examples of the cell membrane permeabilizing agent include alcohols such as methanol and ethanol, and surfactants such as saponin. In addition, in order to prevent non-specific reaction by the antibody, the retaining portion that retains the target cells after storage and permeabilization may be subjected to a blocking treatment with a protein, and then the target cells further modified with a fluorescent group. Alternatively, an antibody against a protein expressed by Escherichia coli or a labeling reagent (4',6-diamidino-2-phenylindole (DAPI) or Hoechst 33342 (trade name)) for fluorescently staining cell nuclei may be added.

The binding between the magnetic particles and the cells may be performed before the cells are retained in the retaining section or after the cells are retained in the retaining section. If the magnetic particles and the cells are bound before the cells are held in the holding part, the magnetic particles and the cells can be bound in the suspension of the sample and the magnetic particles, and thus the binding can be performed with high efficiency. On the other hand, when the magnetic particles and the cells are bound after the cells are held in the holding part, the cells are already held in the holding part, so that there is an advantage that the loss rate of the cells in the binding step with the magnetic particles is reduced. is there.

After holding the target cells in the holding unit, the target cells are collected by bringing the magnetic substance close to the holding unit holding the magnetic particles bound to the target cells. When the magnetic particles are also bound to the foreign cells, the target cells are labeled, the target cells are detected based on the label, and the magnetic cells are brought into close proximity to collect the target cells. In the method of the present invention, since there are no contaminating cells between the magnetic substance and the target cells, the target cells can be collected accurately without involving the contaminating cells.

The magnetic body may be a permanent magnet or an electromagnet having a magnetic force capable of attracting magnetic particles bound to target cells when brought close to each other. If it is a permanent magnet, a neodymium magnet having a strong magnetic force is preferable, but if it is desired to separate the attracted magnetic particles at an appropriate position, an electromagnet capable of controlling the generation of magnetic force depending on whether or not a current is applied is preferable. The size of the magnetic material may be changed as appropriate according to the application, and if you want to collect target cells in single cell units, set the size of the magnetic material to the same size as the cells or the holding part described below to achieve accurate collection. Is possible. On the other hand, when it is desired to collect a plurality of target cells at the same time, a magnetic substance having a larger diameter and/or magnetic force than that used when collecting a single cell unit may be used.

The properties of the target cells collected by the method of the present invention can be analyzed (including culturing of the collected cells). The characterization may be performed in a state where the magnetic substance remains bound to the magnetic particles to which the target cells are bound, or the characterization may be performed after peeling the magnetic particles from the magnetic substance. Examples of property analysis include morphological analysis, tissue type analysis, and gene analysis.

Hereinafter, an example of a method for preparing a sample will be described.
(1) Blood is collected from a patient suspected of having cancer or a cancer patient. When collecting blood, an anticoagulant such as citric acid, heparin, or ethylenediaminetetraacetic acid (EDTA) may be added. If necessary, the collected blood may be diluted with physiological saline or the like.
(2) A fraction containing the target cells is obtained by subjecting the collected blood (or diluted blood) to density gradient centrifugation. In addition, before performing the density gradient centrifugation, the collected blood (or diluted blood), a binding agent capable of binding to the contaminating cells (for example, when the contaminating cells are erythrocytes and leukocytes, RosetteSep (manufactured by StemCell Technologies)). You may add.
(3) The magnetic particles are bound to the target cells by adding magnetic particles modified with an antibody capable of specifically binding to the target cells to the fraction containing the target cells obtained in (2). It is preferable to centrifuge the fraction obtained in (2) and replace it with PBS (Phosphate buffered saline), and then add magnetic particles to increase the reaction efficiency between the target cells and the magnetic particles.
(4) A solution containing ammonium chloride is added to the fraction containing the target cells bound to the magnetic particles obtained in (3), and the mixture is stirred to lyse the red blood cells that are contaminating cells. By this operation, the observation of the separated and collected target cells becomes good.
(5) The solution containing the target cells after hemolytic treatment obtained in (4) is centrifuged to remove blood components that are contaminating cells, and the cells are pelleted and collected. If necessary, a step of resuspending the recovered pellet in the solution and centrifuging may be added. When the target cells are captured by the dielectrophoresis on the holding part, it is preferable to suspend the cells with an isotonic sugar solution.

According to the present invention, the target cells can be accurately collected from a sample containing the target cells and a large amount of contaminating cells, and a plurality of target cells can be collected at a time via magnetic particles, so that the time required for collection can be shortened. ..

It is a figure which shows an example of the cell holding device which has a holding part and which can be utilized by the detection method of this invention. It is a front view of the apparatus shown in FIG. 5 is a diagram showing a procedure of Example 1. FIG. Of the results of Example 1, the results are obtained when one target cell (white blood cell) is retained in the retaining part. (A) is a bright field image, and (B) is a DAPI fluorescence image. The area surrounded by the dotted line is the area of the collection target holding unit. Of the results of Example 1, the results are obtained when two target cells (white blood cells) are held in the holding unit. The area surrounded by the dotted line is the area of the collection target holding unit.

Hereinafter, the present invention will be described in more detail with reference to examples relating to detection of white blood cells contained in a blood sample, but the present invention is not limited to the examples.

Example 1
(1) After collecting 3 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation), add 3 mL of physiological saline to the blood collection tube to dilute blood. A sample was prepared.
(2) The prepared diluted blood sample was overlaid on a density gradient solution having a density of 1.091 g/mL and centrifuged at 2000×g for 10 minutes at room temperature.
(3) After collecting the supernatant after centrifugation, it was centrifuged at 300×g for 5 minutes at room temperature to remove the supernatant. After removing the supernatant, the pellet containing the separated and collected cells was resuspended in 900 μL of PBS and 100 μL of anti-CD45 antibody-modified magnetic particle suspension (Dynabeads CD45, particle diameter 4.5 μm, Thermo Fisher Science), and at room temperature. Stir for 5 minutes. By this operation, the magnetic particles are bound to the white blood cells which are the target cells.
(4) A cell suspension containing magnetic particles was made up to 30 mL with hemolyzed blood containing 0.9% (w/v) ammonium chloride and 0.1% (w/v) potassium hydrogen carbonate, and 300 × Centrifuge at room temperature for 10 minutes at room temperature. By this operation, the erythrocytes mixed in the cell suspension are destroyed, and the separated and collected target cells are well observed.
(5) After removing the supernatant, the separated and collected cell-containing pellet was resuspended in 30 mL of a solution containing 300 mM mannitol, centrifuged at 300×g for 5 minutes at room temperature, and then the supernatant was removed. After resuspending it in 30 mL of a solution containing 300 mM mannitol again, centrifugation was carried out at room temperature for 5 minutes at 300×g, and the supernatant was removed. The operation is an operation for removing the contaminating cells contained in the diluted blood sample and concentrating the target cells.

(6) The suspension containing cells from which the supernatant has been removed in (5) is introduced into the cell holding device 100 shown in FIGS. 1 and 2, and alternating current is applied from the signal generator 40 to the electrode substrates 21 and 22 via the lead wire 30. A voltage (1 MHz, 20 Vp-p) was applied for 3 minutes (the dielectrophoretic force 70 was applied) to hold the cells in the holding unit 50 of the device. The cell-retaining chip 10 included in the cell-retaining device 100 used in the present embodiment includes an insulator 12 having a plurality of through holes 12a having a diameter of 30 μm and a light-shielding chromium film (light-shielding member) having a plurality of through holes 11a having a diameter of 30 μm. 11) and the electrode substrate 21 are provided in close contact with each other in the order of the insulator 12-the light shielding member 11-the electrode substrate 21, and the sample inlet 13a, the outlet 13b and the penetrating portion 13c are provided on the upper surface of the insulator 12. This is a device in which a spacer 13 having a thickness of 1 mm and an electrode substrate 22 are provided in close contact with the upper surface of the spacer 13. The through holes 11a/12a and the electrode substrate 21 form a holding portion 50 having a diameter of 30 μm and a depth of 30 μm capable of holding the cells 60 (see FIG. 3(1)).
(7) While applying an alternating voltage under the conditions of (6), a 300 mM mannitol aqueous solution containing 0.01 (w/v)% poly-L-lysine (adhesive substance 80) was introduced, and the mixture was left standing for 3 minutes. The application of the AC voltage was stopped, and the aqueous solution was removed by suction (see FIG. 3(2)).
(8) An aqueous solution containing 50% (v/v) ethanol and 1% (w/v) formaldehyde (hereinafter referred to as a cell membrane permeation reagent) is introduced and allowed to stand for 10 minutes to allow the cell membrane to permeate to the holding portion. The introduced cells were sampled (see FIG. 3(3)).
(9) The residual cell membrane permeation reagent was washed by suction removing the cell membrane permeation reagent and introducing PBS.
(10) An aqueous solution (hereinafter, a labeling reagent) containing a fluorescent reagent (DAPI: 4′,6-diamidino-2-phenylindole) for labeling cell nuclei was introduced and allowed to stand for 30 minutes (see FIG. 3(4)).
(11) The labeling reagent was removed by suction and PBS was introduced to remove the remaining labeling reagent.
(12) The cell holding chip 10 containing the labeled cells 61 was placed on the stage of a fluorescence microscope (optical detector 200) and then imaged to observe the cells held in the holding holes. For this, a fluorescence microscope (IX71 manufactured by Olympus) equipped with a computer-controlled electric stage and an electron multiplying cooled CCD camera (EMCCD) (ADT-100 manufactured by FLOVEL) was used. LabVIEW (manufactured by National Instruments) was used as image acquisition and analysis software (see FIG. 3(5)).
(13) A target cell to be collected is determined based on the image captured in (12), and an iron wire having a diameter of 50 μm (magnetic property) magnetized by a neodymium magnet is provided directly above the holding unit 50 holding the target cell. The target cells were collected by lowering the body 300) (see FIG. 3(6)). After collecting the target cells, the periphery of the holding unit 50 where the cells were held was imaged.

The results are shown in Figures 4 and 5. FIG. 4 is a bright-field image (FIG. 4(A)) and a DAPI fluorescence image (FIG. 4(B)) before and after collection when one white blood cell, which is a target cell, is held in the holding portion (area surrounded by a dotted line). FIG. 5 is a DAPI fluorescence image before and after collection when two white blood cells, which are target cells, are held in the holding portion (area surrounded by a dotted line). From FIG. 4, it can be seen that white blood cells can be sucked and collected by bringing the magnetic substance close to the holding portion holding the target cells. From FIG. 5, even when a plurality of target cells are held in the holding unit, the plurality of target cells can be simultaneously sucked and collected by bringing the magnetic material close to the holding unit, while cells held in other holding units are collected. It can be seen that is not collected by suction. From the above results, it is understood that the method of the present invention can accurately collect target cells.

100: Cell holding device 200: Optical detector 300: Magnetic substance 10: Cell holding chip 11: Light-shielding member 12: Insulators 11a and 12a: Through hole 13: Spacer 13a: Inlet port 13b: Outlet port 13c: Through portion 21. 22: Electrode substrate 30: Conductive wire 40: Signal generator 50: Holding part 60: Cell 61: Labeled cell (target cell)
70: Dielectrophoretic force 80: Adhesive substance

Claims (4)

After binding the magnetic particles to the target cells contained in the sample, holding the target cells in the holding part, or
After holding the target cells contained in the sample in the holding unit, one of the steps of binding magnetic particles to the target cells,
A step of collecting the target cells by bringing a magnetic substance close to the target cells held by the holding unit;
A method for collecting cells, which comprises: The method according to claim 1, further comprising a step of performing labeling before collecting the target cells and detecting the target cells based on the label. The method according to claim 1 or 2, wherein the magnetic particles are particles modified with a substance capable of specifically binding to target cells. The method according to claim 1, wherein the sample is a blood sample and the target cells are tumor cells or white blood cells.