JP7413692B2 - Pretreatment method for samples containing cells - Google Patents

Description

The present invention relates to a method for preprocessing a sample containing cells. In particular, the present invention relates to a pretreatment method for removing contaminant cells from a sample containing target cells and contaminant cells while suppressing the loss of target cells.

In recent years, cells have been selectively separated and recovered from tissue specimens obtained by suspending or dispersing tissues such as blood and organs in solutions, cell culture media, etc., and the separated and recovered cells have been used for basic research. Research is underway to apply this to clinical diagnosis and treatment. For example, tumor cells (Circulating Tumor Cells, hereinafter referred to as CTCs) are collected from blood taken from cancer patients, and the cells are subjected to morphological analysis, tissue type analysis, and genetic analysis, and based on the findings obtained from these analyses, Research is underway to determine treatment strategies.

However, the number of CTCs contained in blood samples obtained from cancer patients is very small compared to blood cells (red blood cells, white blood cells, platelets), and pretreatment to remove the blood cells is essential. Furthermore, since the number of CTCs contained in a blood sample is very small, in order to detect CTCs with high sensitivity, it is necessary to minimize the number of missed CTCs due to the pretreatment. Furthermore, if there is a large amount of blood cells remaining as contaminants in the pretreatment, there is a risk of false positives occurring, so in order to accurately detect CTCs, it is necessary to reduce the amount of remaining blood cells as much as possible. be.

As a CTC detection method that minimizes missed CTCs due to pretreatment, Patent Document 1 discloses a method in which red blood cells contained in a blood sample are hemolyzed and removed using a hemolytic agent, and then all remaining cells are analyzed. A method for detecting CTCs is disclosed. However, the solution after the hemolytic treatment contains a large amount of contaminant cells such as white blood cells, and there is a problem that the detection time is extremely long. There was also the risk of false positives occurring.

As a method for removing contaminant cells other than CTCs contained in a blood sample, Non-Patent Document 1 discloses a method in which red blood cells contained in a blood sample are disrupted (hemolysed) using a hemolytic agent, and then the cells specifically bind to white blood cells, which are contaminant cells. A method of removing leukocytes using magnetic particles modified with an anti-CD45 antibody is disclosed. Although the method disclosed in Non-Patent Document 1 can remove leukocytes without losing most of the CTCs, the removal rate remained at about 80% (that is, about 20% remained).

Special Publication No. 2013-508729

Kalergi, Galatea et al. , Cellular Physiology and Biochemistry, 40, 411-419 (2016)

In order to detect target cells from a sample containing target cells and contaminant cells, pretreatment is necessary to remove the contaminant cells that cause false positives in the detection of target cells without missing any target cells. An object of the present invention is to provide a method in which the pretreatment is performed by replacing a solvent in a sample containing target cells and contaminant cells.

In order to solve the above problems, the present inventors have made extensive studies and have arrived at the present invention.

That is, the first aspect of the present invention is
(1) A step of centrifuging a sample containing target cells and contaminant cells;
(2) removing the supernatant obtained in step (1) to obtain a pellet containing target cells and contaminant cells;
(3) Adding a replacement solvent to the solution containing the pellet obtained in step (2) and centrifuging it;
(4) removing the supernatant obtained in step (3) to obtain a suspension containing the target cells;
A sample pretreatment method comprising:
The specific gravity of the target cells is greater than the specific gravity of the contaminant cells,
and the above method, wherein the addition of the replacement solvent is carried out under conditions such that the contaminant cells contained in the pellet are dispersed in the replacement solvent, but the target cells contained in the pellet are not dispersed in the replacement solvent.

A second aspect of the present invention is the method described in the first aspect, wherein the substitution solvent is added at a constant rate.

Further, a third aspect of the present invention is the method according to the first or second aspect, wherein the replacement solvent is an isotonic solution containing sugar.

Furthermore, the fourth aspect of the present invention is that the centrifugation conditions of (3) are that the centrifugal force is 200 x g or more and 2000 x g or less, and the centrifugation time is 1 minute or more and 30 minutes or less. The method described in any one of (3) to (3) above.

A fifth aspect of the present invention is a method for detecting target cells contained in a sample, which includes the steps shown in (I) to (IV) below:
(I) pretreating the sample by the method according to any one of the first to fourth aspects to obtain a suspension containing target cells;
(II) a step of introducing the suspension obtained in step (I) into a cell holding means having a holding section;
(III) a step of holding target cells and contaminant cells contained in the suspension in the holding section using dielectrophoretic force;
(IV) Detecting the target cells and/or contaminant cells retained in (III).

Furthermore, a sixth aspect of the present invention is a method for collecting cells contained in a sample, further comprising a step of collecting target cells and/or contaminant cells detected by the method described in the fifth aspect.

The present invention will be explained in detail below.

The sample in the present invention may be a solution or suspension containing at least the target cells and contaminant cells described below, and specifically includes whole blood, diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and blood component collection. samples that may contain blood-derived components such as urine, saliva, semen, feces, sputum, amniotic fluid, ascites, or pieces of tissue such as the liver, lungs, spleen, kidneys, skin, tumors, or lymph nodes. Examples include suspended tissue suspensions, and fractions containing sample- or tissue-derived cells obtained by separation from the aforementioned samples or tissue suspensions. Among these, an example of a fraction containing cells derived from a sample or tissue is a fraction obtained by layering a sample or tissue suspension on a density gradient forming medium and then performing density gradient centrifugation.

In the present invention, target cells refer to cells contained in a sample that have a larger specific gravity than the contaminant cells to be removed by the pretreatment method of the present invention. Examples of target cells include tumor cells such as circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), circulating fetal cells (CFC), various stem cells, and B cells. When CTCs are used as target cells, the specific gravity of CTCs is generally about 1.040 to 1.065. Therefore, as an example of contaminant cells, platelets (general specific gravity: 1. Among white blood cells such as neutrophils, eosinophils, basophils, monocytes, and lymphocytes (general specific gravity: about 1.063 to 1.085), specific gravity is 1.063 to 1.085. The white blood cell count is about 1.065. Further, as long as the specific gravity conditions described above are met, the target cells may be leukocytes with specific characteristics, and the contaminant cells may be leukocytes without the above-mentioned characteristics.

The centrifugation step in the present invention is preferably carried out under conditions such that target cells contained in the sample are not damaged by the step. Specifically, the centrifugal force (Relative Centrifugal force) is preferably 200 x g or more and 2000 x g or less, more preferably 500 x g or more and 1000 x g or less. Further, the centrifugation time is preferably 1 minute or more and 30 minutes or less, more preferably 3 minutes or more and 10 minutes or less. The container used in this step is not particularly limited as long as it is a commercially available centrifuge tube. A preferred example of the container is a 50 mL centrifuge tube, and particularly preferred is a commercially available 50 mL disposable conical tube, such as the 50 mL Falcon Conical Tube (manufactured by Corning, product number 352070). By centrifuging the sample contained in the container described above under the conditions described above, target cells and contaminant cells contained in the sample are sedimented, and a pellet is formed at the bottom of the container. Since the specific gravity of the target cells is greater than the specific gravity of the contaminant cells, the target cells are mainly present in the lower layer (bottom) of the formed pellet, and the contaminant cells are mainly present in the upper layer.

After the sample (solution or suspension) is separated into a pellet and a supernatant by centrifugation, the supernatant is removed. If an attempt is made to completely remove the supernatant, there is a risk that the suction means (such as a pipette tip) may come into contact with the pellet, or when adding a replacement solvent, which will be described later, the added solvent may directly hit the pellet. Therefore, the supernatant is removed while leaving a small amount of the supernatant (hereinafter, this state is also referred to as "a solution containing pellets"). Specifically, if the container used in the centrifugation step is a tube with a capacity of 50 mL, the supernatant may be removed so that 0.5 mL or more and 20 mL or less remains; The following is preferable, and the remaining amount of supernatant is more preferably 2 mL or more and 10 mL or less.

After obtaining a solution containing pellets by centrifugation, a replacement solvent is added. The replacement solvent used in the present invention only needs to have a specific gravity that is at least lower than the specific gravity of the target cells. When the target cells are cells contained in a blood sample such as CTCs, examples of the replacement solvent include a hemolytic agent or buffer solution capable of disrupting red blood cells, a culture solution, and an aqueous solution containing sugar. Particularly when the collected cells are manipulated using dielectrophoresis, it is preferable to use a sugar-containing aqueous solution (saccharide-containing isotonic solution) that is isotonic to physiological osmotic pressure as the replacement solvent. Examples of sugars to be included in the aqueous solution include monosaccharides such as glucose and galactose, disaccharides such as sucrose and trehalose, sugar alcohols such as mannitol and xylitol, and polysaccharides such as dextran. Preferred examples of isotonic solutions containing sugar include xylitol or sucrose aqueous solutions of 230 mM or more and 330 mM or less.

Note that before adding the replacement solvent, a treatment to remove contaminant cells may be performed in advance. Specifically, if the contaminant cells are red blood cells or white blood cells, they may be hemolyzed, subjected to density gradient centrifugation, or a carrier that can bind to the contaminant cells (for example, if the contaminant cells are white blood cells, an anti-CD45 antibody immobilized carrier) The contaminant cells may be removed by binding them to the cells. Note that these removal operations may be performed alone or in combination.

In the present invention, when adding a replacement solvent, contaminant cells contained in the pellet are dispersed in the replacement solvent, while target cells contained in the pellet are not dispersed in the replacement solvent. As mentioned above, since the specific gravity of the contaminant cells contained in a sample is smaller than the specific gravity of the target cells contained in the same sample, the contaminant cells are located in the upper layer and the target cells are located in the lower layer (bottom) of the pellet. . That is, the above-mentioned conditions refer to conditions under which only the upper layer of the pellet is dissolved and the contaminant cells present in the upper layer are dispersed in the replacement solvent when the replacement solvent is added. Specifically, after fixing the container used in the centrifugation step diagonally, the supernatant after centrifugation is left behind (into the solution containing the pellets), and the replacement solvent is heated at a constant speed so as not to directly hit the pellets. It is preferable to add it at The fixing angle of the container is preferably 20 degrees or more and 70 degrees or less, more preferably 30 degrees or more and 60 degrees or less, when the open part of the tube is 90 degrees. The addition rate of the replacement solvent may be determined as appropriate depending on the type of replacement solvent, but if the replacement solvent is a solution containing xylitol, it is preferably 3.5 mL/s or more and 7.5 mL/s or less, and the replacement solvent contains sucrose. When it is a solution, it is preferably 1 mL/s or more and 3 mL/s or less. When adding the replacement solvent, it is preferable to add the solvent at a constant rate; however, it is preferable to use an electric pipetter to add the replacement solvent, since the replacement solvent can be added accurately and easily at a constant rate. Furthermore, when adding the replacement solvent, it is preferable to add it so that it flows along the wall surface of the container used in the centrifugation step, since the flow of the replacement solvent can be stabilized. After adding the replacement solvent, centrifuge the container again to pellet the cells of interest. After removing the supernatant, the pellet may be suspended in the remaining replacement solvent to obtain a suspension containing the target cells. Note that before recovering the pellets (suspending them in a replacement solvent), the above-described solution replacement operation with the replacement solvent may be repeated.

A suspension obtained by pretreating a sample by the method of the present invention and concentrating target cells can be used to detect the target cells by applying it to a slide, observing with a microscope or optical detector, or using flow cytometry. can be detected. Note that when detecting target cells by observing with a microscope or an optical detector, a suspension containing the cells is introduced into a cell holding means having a holding part capable of holding the cells, and the cells are placed in the holding part. After holding the cells, it is best to observe them using a microscope, optical detector, etc. Examples of the holding portion include a hole capable of storing the cells and a surface covered with a material capable of fixing the cells (eg, poly-L-lysine). Note that it is preferable that the size of the holding portion is large enough to hold only one cell, since the collection and analysis (morphological analysis, tissue type analysis, genetic analysis, etc.) of specific cells can be easily performed. Further, when holding cells in the holding part, it is preferable to use dielectrophoretic force because the cells can be held in the holding part efficiently. When using dielectrophoretic force, specifically, dielectrophoresis may be generated by applying an alternating current voltage, and cells may be introduced into the holding section. The AC voltage to be applied is preferably an AC voltage having a waveform in which charging and discharging of the cells in the holding part are repeated periodically, the frequency is between 100 kHz and 3 MHz, and the electric field strength is between 1 x 10 ^and 5 x 10. ⁵ V/m is particularly preferable (see WO2011/149032 and JP2012-013549).

Examples of cell holding devices that can be used in the detection method of the present invention include the cell holding devices shown in FIGS. 1 to 3.

The cell holding device 100 shown in FIGS. 1 and 3 is
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 part 131 and an outlet part 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 that applies signals to the electrodes 141 and 142;
It is equipped with The through-hole 111 of the insulating film 110 and the through-hole 121 of the light-shielding film 120 have the same size and shape, and the insulating film 110 and the light-shielding film 120 are provided so that the positions of the through-holes coincide with each other. .

The through hole 111, the through hole 121, and the electrode 141 provided in close contact with the lower part of the light shielding film 120 constitute a holding section 170 capable of holding cells in the cell holding device 100, and a liquid containing cells is introduced from the introduction section 131. Upon introduction, cells are introduced into the holding section 170. The light shielding film 120 can reduce optical noise such as background noise caused by autofluorescence of the insulating film 110 itself and crosstalk noise caused by light leakage from the adjacent holding parts 170, Only cell-derived light can be detected with high sensitivity and precision. The electrode 142 is provided in close contact with the upper surface of the spacer 130 to prevent the sample containing target cells introduced from the introduction section 131 from scattering or evaporating.

Note that the electrode 142 has a structure that is removable from the spacer 130 in order to facilitate recovery of the cells held in the holding part 170. Furthermore, it is preferable to use transparent electrodes such as ITO (indium tin oxide) for the electrodes 141 and 142 because the cells held in the holding part 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 with an insulating film 110, a light shielding film 120, and a spacer 130 sandwiched in the vertical direction as in the device shown in FIG. 1, or as in the device shown in FIG. As shown, the electrode 141 may be provided in the form of a comb-shaped electrode in which the + electrode 141a and the − electrode 141b are provided only on the lower surface of the light shielding film 120.

It is preferable that the size of the holding part 170 is large enough to hold only one target cell, since this makes it easier to detect the target cell in the detection step. Note that if the number of cells (sum of target cells and contaminant cells) contained in the sample to be expanded into the cell retention device 100 is expected to be greater than the number of holding sections 170 provided in the cell retention device 100, It is recommended that the sample be diluted so that an appropriate number of cells are developed, or that the sample to be subjected to development be weighed in advance.

Hereinafter, as an example of the detection method and collection method of the present invention, a method for detecting and collecting tumor cells (CTC) contained in a blood sample will be described in detail, but the present invention is not limited to the contents of this explanation. It's not something you can do.

(1) Collect a blood sample from a patient suspected of having cancer. Note that when collecting a blood sample, it is preferable to add an anticoagulant typified by a chelating agent such as citric acid or ethylenediaminetetraacetic acid (EDTA).

(2) A formaldehyde donor compound, an antiplatelet agent, and polyethylene glycol, which are preservatives for stabilizing CTCs, are added to the blood sample (or diluted blood sample) collected in (1). Note that if the blood sample is collected without adding an anticoagulant in (1), an anticoagulant is also added. At the time of the addition, all the substances constituting the preservative may be added at once, or solutions containing each component may be added individually.

(3) Red blood cells contained in a blood sample to which a preservative has been added (preserved blood sample) are disrupted (hemolyzed) using ammonium chloride. Disruption (hemolysis) of red blood cells with ammonium chloride is a method of disruption (hemolysis) that takes advantage of the difference in ion uptake ability between red blood cells and other cells, and has the advantage of being able to disrupt red blood cells while minimizing damage to other cells. This is a preferred method of disrupting red blood cells.

(4) After red blood cell disruption (hemolysis) treatment, CTCs and contaminant cells (white blood cells, platelets, etc.) contained in the blood sample are pelleted by centrifugation, and then the supernatant containing a hemolytic agent (ammonium chloride) is Remove.

(5) After dissolving the pellet containing CTCs and contaminant cells and suspending the cells by stirring the residual solution from (4) such as pipetting, add a solution containing at least sodium chloride and suspend again. let Examples of solutions containing at least sodium chloride include physiological saline and PBS (phosphate buffered saline), and PBS is preferably used because it can easily maintain physiological pH. Note that it is preferable to include a protein bound to a hydrophilic polymer (for example, BSA bound to polyethylene glycol) in the solution, since this improves the CTC recovery efficiency. The concentration of the protein bound to the hydrophilic polymer may be 0.01% (w/v) or more and 25% (w/v) or less as the final concentration of the protein in the resuspension liquid, and 0.02% (w/v) or more and 25% (w/v) or less. % (w/v) or more and 5% (w/v) or less, preferably 0.05% (w/v) or more and 2% (w/v) or less. Furthermore, by adding the protein to the solution, it is also possible to suppress nonspecific adsorption of CTC with a carrier modified with a binding site capable of binding to leukocytes.

(6) The CTC-containing suspension prepared in (5) is centrifuged again to collect a pellet containing CTCs and contaminant cells. This operation is performed for the purpose of increasing the suspension concentration of cells in the suspension and increasing the reactivity between leukocytes, which are contaminant cells, and a carrier capable of binding to leukocytes.

(7) Magnetic particles bound to anti-CD45 antibody are added as a carrier capable of binding to white blood cells that are part of the contaminant cells. The reaction time and reaction temperature with magnetic particles may be changed as appropriate depending on the characteristics of the antibody used.

(8) After binding the magnetic particles and white blood cells, the added magnetic particles are removed using a magnet (magnetic separation). The supernatant containing CTC and the remaining contaminant cells that could not be removed by the magnetic separation is collected, and added to a suitable replacement solvent at appropriate times to be suspended. The replacement solvent is not particularly limited as long as it is an isotonic solution with a specific gravity lower than that of CTC, but when the collected cells are manipulated using dielectrophoresis, a solution containing sugars such as xylitol, sucrose, and mannitol is used. Good. Note that the container used for suspension is not particularly limited as long as it is a tube for centrifugation, and for example, a commercially available 50 mL disposable conical tube may be used.

(9) After centrifuging the suspension obtained in (8), the supernatant is removed to obtain a pellet containing CTCs and contaminant cells contained in the suspension. Note that the centrifugation conditions are preferably such that the centrifugation strength is such that the target cells are not damaged by centrifugation. The centrifugal strength is preferably 200 x g or more and 2000 x g or less, more preferably 500 x g or more and 1000 x g or less. The centrifugation time is preferably 1 minute or more and 30 minutes or less, more preferably 3 minutes or more and 10 minutes or less. In addition, when removing the supernatant, it is sufficient to remove the supernatant so that the amount of residual liquid is 0.5 mL or more and 20 mL or less, but it is preferable that the residual liquid amount is 1 mL or more and 15 mL or less, and 2 mL or more and 10 mL or less. More preferred.

(10) After fixing the container containing the pellet containing CTCs and contaminant cells obtained in (9) and the residual solution after removing the supernatant (containing the solution containing the pellet) at an angle, the replacement solvent was kept constant. Add at speed. The angle at which the container is fixed is preferably 20 degrees or more and 70 degrees or less, and more preferably 30 degrees or more and 60 degrees or less, when the open part of the container is 90 degrees. Furthermore, since the specific gravity of contaminant cells is lower than that of CTCs, in the pellet, CTCs with a higher specific gravity are present in the lower layer (bottom) of the pellet, and contaminant cells with a lower specific gravity than CTCs are present in the upper layer of the pellet. Therefore, the replacement solvent may be added at a rate that only the upper layer of the pellet is dissolved, that is, at a rate that the contaminant cells present in the upper layer are dispersed in the replacement solvent. The addition rate may be determined as appropriate depending on the type of replacement solvent, but in the case of a solution containing xylitol, the addition rate is preferably 3.5 mL/s or more and 7.5 mL/s or less, and in the case of a solution containing sucrose. , the addition rate is preferably 1 mL/s or more and 3 mL/s or less. Note that the method of adding the replacement solvent is not particularly limited as long as the addition rate is constant, but it is preferable to use an electric pipettor because the liquid can be added accurately and easily at a constant rate. Furthermore, when adding the replacement solvent, it is preferable to add the liquid so that it flows along the wall surface of the container, since this can stabilize the flow of the liquid.

(11) Centrifuge the container after the operation in (10) again to pellet the CTCs and contaminant cells remaining in the operation in (10), remove the supernatant, and suspend the pellet in the remaining supernatant. As a result, a suspension containing CTC is recovered. Note that before recovering the suspension, the step (10) may be repeated to perform solvent replacement.

(12) The cell suspension containing CTCs obtained in (11) is introduced into a cell holding device (for example, the device shown in FIGS. 1 and 2) having a holding part capable of holding cells, and After holding the cells in the blood sample, CTCs and/or contaminant cells (for example, blood components such as white blood cells) contained in the blood sample are detected by observing them with a microscope, an optical detector, or the like. CTCs may be detected, for example, based on the difference in size and shape between CTCs and contaminant cells in a bright field image, using labeled antibodies against proteins expressed in CTCs such as cytokeratin and EpCAM (Epithelial Cell Adhesion Molecule), and Alternatively, the cells may be stained with a labeled antibody against a protein expressed in the contaminant cells (such as CD45 when the contaminant cells are white blood cells), and detection may be performed based on the staining results.

(13) The CTCs and/or contaminant cells detected in (12) are collected using a collection means. An example of a collection means is a means for collecting by suction and discharge using a nozzle, and a specific example is a device disclosed in JP-A No. 2016-142616.

The present invention comprises a step of centrifuging a suspension containing target cells and contaminant cells, a step of removing the supernatant obtained by the centrifugation to obtain a pellet containing the target cells and contaminant cells, and after removing the supernatant. In the sample pretreatment method, the method includes the steps of adding a replacement solvent to a solution containing the obtained pellet and centrifuging the solution, and removing the supernatant after the centrifugation to obtain a suspension containing the target cells. The specific gravity of the target cells is greater than the specific gravity of the contaminant cells, and when the replacement solvent is added, the contaminant cells contained in the pellet are dispersed in the replacement solvent, while the target cells contained in the pellet are dispersed in the replacement solvent. It is characterized by being carried out under conditions that do not allow

According to the present invention, even if the number of target cells contained in a sample is very small compared to the number of contaminant cells, the contaminant cells contained in the sample can be removed and the target cells can be concentrated. In other words, when detecting target cells contained in a sample, the number of contaminant cells other than the target cells can be reduced, so the frequency of erroneously detecting contaminant cells as target cells (number of false positives) can be significantly reduced. Therefore, target cells can be detected with higher accuracy. Furthermore, since the method of the present invention can reduce the total number of cells to be detected, it also leads to a reduction in cell detection time.

As an example, when the present invention is applied to the detection of tumor cells (CTCs) contained in blood, CTCs can be detected with high accuracy by using the method of the present invention. Furthermore, when diagnosing cancer based on the presence of CTCs, the reliability of the determination result of the presence or absence of CTCs is improved, so that cancer can be diagnosed with high accuracy.

FIG. 2 is a diagram showing an example of a cell holding device that can be used in the detection method of the present invention. It is a figure which shows another aspect of the cell holding device which can be used with the detection method of this invention. FIG. 2 is a diagram showing the retention and detection of cells using the apparatus shown in FIG. 1.

Hereinafter, the present invention will be explained in more detail using Examples and Comparative Examples, but the present invention is not limited to these Examples. Note that the reference examples do not constitute the present invention.

Example 1
(1) Polyethylene glycol (mPEG-NHS) with a molecular weight of 5,000, which has a methoxy group at one end and an N-hydroxysuccinimide ester group at the other end, and bovine serum albumin (BSA) (300 mg, 0.5 mg). BSA bound to polyethylene glycol (PEG-BSA) was prepared by dissolving 3 mmol) in sodium hydrogen carbonate buffer (0.1 M, 15 mL) and stirring the solution at around 25° C. for 3 hours. During preparation, the mPEG-NHS and BSA molar ratio (mPEG-NHS/BSA) was set to 2. After preparation, the solution was replaced with pure water for 3 days using a dialysis membrane with a molecular weight cutoff of 10,000.

(2) Add 2.3 g of imidazolidinyl urea (preservative), 2.3 g of polyethylene glycol (PEG) with a molecular weight of 2000, 1.2 mg of tirofiban (antiplatelet agent), and 30 mg of ethylenediaminetetraacetic acid (EDTA) (anticoagulant) to a solution. The solution was dissolved in ultrapure water to a total volume of 30 mL.

(3) Human lung cancer cells (PC9 cells) were cultured at 37°C for 24 to 96 hours using RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO ₂ environment, and then 0.25% Cells were detached from the medium using trypsin/1mM EDTA and collected into a microtube. The supernatant was removed by centrifugation at 1000 rpm for 5 minutes, and the pellet was washed by resuspending in 1 mL of medium. The PC9 cells were used as the target cells in this example.

(4) Collect 3 mL of blood from a healthy individual who has given informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation), then add 0.45 mL of the solution prepared in (2) and (3 Approximately 100 PC9 cells prepared in ) were added to prepare a diluted blood sample.

(5) Add an aqueous solution containing 0.9% (w/v) ammonium chloride and 0.1% (w/v) potassium bicarbonate to 3.45 mL of the diluted blood sample prepared in (4) in a total volume of 90 mL. After addition, the red blood cells were disrupted (hemolysed) by standing at room temperature for 5 minutes. After hemolysis, the mixture was centrifuged at 900 xg for 5 minutes at 25°C.

(6) After removing 88 mL of the supernatant after centrifugation, a pellet containing PC9 cells and contaminant cells (white blood cells, platelets, etc.) was suspended in the remaining supernatant by pipetting.

(7) Add PBS (phosphate buffered saline) containing PEG-BSA (0.1% (w/v) as BSA) and 2% (w/v) BSA prepared in (1) to a total volume of 30 mL. After adding the mixture to the suspension obtained in (6) until the mixture was completely dissolved, it was centrifuged at 600 xg for 5 minutes at 25°C. 29 mL of the resulting supernatant was removed, and the pellet was resuspended in the remaining supernatant by pipetting.

(8) 100 μL of anti-CD45 antibody modified magnetic particle suspension (Dynabeads CD45, manufactured by Thermo Fisher Scientific) and 100 μL of anti-CD15 antibody modified magnetic particle suspension (Dynabeads CD15, manufactured by Thermo Fisher Scientific) After mixing with the magnet The dispersion medium was removed and the magnetic particles were concentrated. After mixing the concentrated magnetic particles with the entire cell suspension obtained in (7), the mixture was stirred for 5 minutes at room temperature, and leukocytes bound to the magnetic particles were removed using a magnet.

(9) Transfer the entire cell suspension from which leukocytes were removed in (8) (some leukocytes and platelets remain as contaminant cells) to a 50 mL Falcon conical tube (Corning, product number 352070), and transfer the PEG- An aqueous solution containing BSA (0.1% (w/v) as BSA) and 280 mM xylitol (used as a replacement solvent) was added to a total volume of 30 mL, followed by centrifugation at 600 x g for 5 minutes at 25°C. Note that this operation is an operation for reducing the salt concentration in the solution and concentrating the target PC9 cells.

(10) After removing 25 mL of the supernatant after centrifugation, the conical tube was fixed at an angle of 45 degrees using a tilt stand. Decant the displacement solvent in a 100 mL beaker at an addition rate of 2.5 mL/s, 3 mL/s, 5 mL/s, or 10 mL/s so that the solvent flows along the wall of the tube. Added. After adding the mixture to a total volume of 30 mL, it was centrifuged at 600 xg for 5 minutes at 25°C.

(11) The same operation as (10) was performed again.

(12) After removing the supernatant after centrifugation to make the remaining liquid volume 850 μL, the pellet containing PC9 and remaining contaminant cells was resuspended by pipetting to obtain a cell suspension. After holding the suspension in the cell holding device 100 shown in FIGS. 1 and 3 by the method shown below, target cells were detected. Note that the cell holding device 100 is provided with a holding portion 170 having a diameter of 30 μm and a depth of 40 μm.
(12-1) After introducing the cell suspension from the introduction part 131, an AC voltage (voltage 20Vpp, frequency 1MHz, rectangular wave) is applied from the AC power supply 160 to each electrode 141, 142, and the dielectrophoretic force The cells were held in the holding part 170.
(12-2) A 280 mM xylitol aqueous solution containing 0.01% (w/v) poly-L-lysine is introduced from the introduction part 131 while applying the AC voltage, and after standing for 3 minutes, the AC voltage The application of was stopped, and the aqueous solution was removed by suction from the discharge part 132.
(12-3) A PBS solution containing 1% HCHO was introduced from the introduction part 131 and the cells were fixed by allowing it to stand for 10 minutes, and then the reagent was removed by suction from the discharge part 132. Thereafter, a PBS solution (hereinafter referred to as PBS-T) containing 0.05% (w/v) Tween 20 (trade name) was introduced from the introduction part 131 to wash away the remaining reagent.
(12-4) An aqueous solution containing 95% (v/v) ethanol was introduced from the introduction part 131 and allowed to stand for 10 minutes to permeate the cells through the membrane, and then the reagent was removed by suction from the discharge part 132. Thereafter, PBS-T was introduced from the introduction section 131 to wash away the remaining reagent.
(12-5) Introduce a PBS solution containing 10% (v/v) Goat Serum and 3% (w/v) BSA from the introduction part 131, and block the cells by leaving it for 10 minutes. Thereafter, the reagent was removed by suction from the discharge section 132.
(12-6) Introducing a cell labeling reagent containing a mixture of anti-cytokeratin mouse antibody (manufactured by Miltenyi Biotec), 10% (v/v) Goat Serum, and 3% (w/v) BSA from the introduction part 131, After labeling the PC9 cells by standing for 30 minutes, the reagent was removed by suction from the discharge part 132. Thereafter, PBS-T was introduced from the introduction section 131 to wash away the remaining reagent.
(12-7) From the introduction part 131, Alexa Fluor 488-labeled anti-mouse IgG1 antibody (manufactured by Thermo Fisher Scientific), PE (phycoerythrin)-labeled anti-CD45 antibody (manufactured by Miltenyi Biotec), DAPI (4',6- By introducing a cell staining reagent containing a mixture of DiAmidino-2-PhenylIndole (manufactured by Dojindo Laboratories), 10% (v/v) Goat Serum, and 3% (w/v) BSA, and leaving it for 20 minutes. Cells were stained. Thereafter, the cell staining reagent was removed by suction from the outlet 22. Thereafter, PBS-T was introduced from the introduction section 131 to wash away the remaining reagent.
(12-8) In order to observe all the cells held in the holding part 170, a fluorescence microscope (IX71 manufactured by Olympus) equipped with a computer-controlled motorized stage and a CMOS camera (ORCA-Flash 4.0 manufactured by Hamamatsu Photonics) was used. ) to take bright field and fluorescence images of all retention areas.
(12-9) The images taken in (12-8) were analyzed using the analysis software LabVIEW (manufactured by National Instruments), and the images were stained with DAPI (with cell nuclei) and Alexa Fluor 488 (with cell nuclei). Cells that expressed keratin) and were not stained with PE (do not express CD45) were detected.
(12-10) Among the cells detected in (12-9), the number of target cells, PC9 cells, was counted, and this was taken as the number of detections. The cancer cell detection rate was calculated by dividing the detected number by the number of PC9 cells added in (4).
(12-11) Among the cells detected in (12-9), cells that are clearly smaller than PC9 cells and about the same size as white blood cells (approximately 10 μm) are judged to be erroneously detected contaminant cells (false positives). and counted the number of false positives.

Table 1 shows the results of the cancer cell detection rate and the number of false positives. In Table 1, the number of false positives refers to the number of contaminant cells incorrectly determined as target cells due to non-specific adsorption of cancer cell marker (cytokeratin)-labeled antibodies to white blood cells and platelets with weak CD45 expression. refers to something. Under the condition of addition rate of 5 mL/s, the detection rate of cancer cells is relatively maintained (60% or more and less than 70%), while the number of false positives is relatively suppressed (10 or more and less than 30). I understand. Therefore, under the conditions of an addition rate of 5 mL/s, the steps shown in (10) and (11) can be carried out to disperse contaminant cells (platelets, white blood cells, etc.) contained in the pellet in the replacement solvent, while dispersing the target cells (such as It can be seen that the experiment was carried out under conditions in which the cells (cells) were not dispersed in the replacement solvent. On the other hand, when the substitution solvent is added at a rate slower than the addition rate of 5 mL/s (addition rate of 2.5 mL/s and 3 mL/s), the detection rate of cancer cells is high (more than 70%), but , the number of false positives also increased (more than 30). Furthermore, when the substitution solvent is added at a faster rate than the addition rate of 5 mL/s (addition rate of 10 mL/s), the number of false positives is small (less than 10), but the detection rate of cancer cells also increases. decreased (less than 60%).

In this example, the specific gravity of cancer cells, which are target cells, is about 1.040 to 1.065, which is higher than the specific gravity of platelets (about 1.032), which are contaminant cells. Further, the specific gravity of white blood cells, which are also contaminant cells, is about 1.063 to 1.085, and some of them have a specific gravity lower than that of cancer cells. When a cell suspension containing target cells (cancer cells) and contaminant cells (platelets, white blood cells, etc.) is centrifuged, platelets and some low-density white blood cells are cancerous in the pellet obtained after centrifugation. It accumulates on top of the cells (lower layer) (upper layer). When a replacement solvent is added after removing the supernatant, the addition of the solvent applies an impact to the pellet, thereby loosening the upper part of the pellet and dispersing the cells in the replacement solvent, thereby removing contaminant cells together with the supernatant. Therefore, the slower the addition speed of the replacement solvent, the smaller the impact on the pellets, making it difficult for the pellets to unravel (that is, the smaller the amount of dispersion in the replacement solvent), and the faster the addition speed, the larger the impact on the pellets, making the pellets It dissolves easily (that is, the amount of dispersion in the replacement solvent increases). From the above, the addition operation of the replacement solvent was carried out under the conditions that the contaminant cells mainly contained in the upper layer of the pellet were dispersed in the replacement solvent, while the target cells mainly contained in the lower layer (bottom) of the pellet were not dispersed in the replacement solvent. By performing this at an addition rate of 5 mL/s (in the example), only the contaminant cells contained in the upper layer of the pellet can be dispersed without losing the target cells (cancer cells), resulting in a lower cancer cell detection rate. It is possible to reduce false positives while maintaining the

Reference example 1
In Examples 1 (10) and (11), the same procedure as in Example 1 was carried out, except that the addition rate of the replacement solvent was 5 mL/s, and two different operators (operator A and worker B) performed the operations. The cancer cell detection rate and the number of false positives were calculated using the method.

Example 2
In Examples 1 (10) and (11), the addition rate of the replacement solvent was 5 mL/s, an electric pipetter was used for the addition of the replacement solvent, and two different operators (worker A and worker B) performed the addition. Otherwise, the cancer cell detection rate and the number of false positives were calculated in the same manner as in Example 1.

Comparative example 1
In Examples 1 (10) and (11), the same method as in Example 1 was used except that the replacement solvent addition rate was 1.5 mL/s or 10 mL/s and an electric pipetter was used for adding the replacement solvent. The cell detection rate and the number of false positives were calculated.

Table 2 summarizes the results of the cancer cell detection rate and the number of false positives in Reference Example 1, Example 2, and Comparative Example 1. In Example 1 and Reference Example 1 (Worker B), the detection rate of cancer cells was relatively maintained (60% or more and less than 70%), while the number of false positives was relatively suppressed (10 or more and less than 30%). However, in Reference Example 1 (Worker A), the number of false positives increased (more than 30). Therefore, it can be seen that when the replacement solvent is added by decantation in the pretreatment method of the present invention, there is a possibility that variations depending on the operator may occur. When an electric pipettor was used to add the replacement solvent (Example 2), the cancer cell detection rate was improved (more than 70%) and the number of false positives was also reduced compared to when the substitution solvent was added using a decant (Example 1). reduced (less than 10). Furthermore, there was no variation among operators, which occurred when adding the replacement solvent by decantation (Reference Example 1), and it was confirmed that stable results could be obtained. This is thought to be because, while it is difficult to add liquid stably with a decant due to hand shake, etc., it is easy to add liquid stably at a constant speed with an electric pipettor.

Even if an electric pipettor is used to add the replacement solvent, a high cancer cell detection rate can be maintained (over 70%) at an addition rate of 1.5 mL/s, but the number of false positives will increase (over 30 cells) and the addition rate will be 1.5 mL/s. At a speed of 10 mL/s, the number of false positives could be reduced (less than 10), but the cancer cell detection rate was reduced (less than 60%) (Comparative Example 1). From this, when using a xylitol solution as the replacement solvent, if the addition rate is 3.5 mL/s or more and 7.5 mL/s or less, the number of false positives can be reduced while maintaining a high cancer cell detection rate. It can be said that it is preferable.

Comparative example 2
The replacement solvent used in Examples 1 (9) to (11) was a 280 mM sucrose aqueous solution, and the replacement solvent in Examples 1 (10) and (11) was added at an addition rate of 0.5 mL/s or 5 mL using an electric pipettor. The cancer cell detection rate and the number of false positives were calculated in the same manner as in Example 1, except that the addition was performed at a rate of /s.

Example 3
The cancer cell detection rate and the number of false positives were calculated in the same manner as in Comparative Example 2, except that the replacement solvent addition rate was 1.5 mL/s in Examples 1 (10) and (11).

The results of the cancer cell detection rate and the number of false positives in Comparative Example 2 and Example 3 are summarized in Table 3. When the substitution solvent addition rate is set to 0.5 mL/s, a high cancer cell detection rate can be maintained (more than 70%), but the number of false positives increases (30 or more), and when the addition rate is set to 5 mL/s, the number of false positives decreases. Although it was possible to reduce the number of cancer cells (to less than 10 cells), the cancer cell detection rate decreased (to less than 60%) (Comparative Example 3). On the other hand, when the addition rate was 1.5 mL/s, the number of false positives was reduced (less than 10) while maintaining a high cancer cell detection rate (70% or more) (Example 3). From this, when using a sucrose solution as the replacement solvent, it is preferable to set the addition rate to 1 mL/s or more and 3 mL/s or less, as this can reduce the number of false positives while maintaining a high cancer cell detection rate. I can say that.

100: Cell holding device 110: Insulating film 120: Light shielding film 111, 121: Through hole 130: Spacer 131: Introduction section 132: Exhaust section 141, 142: Electrode 141a: + pole 141b: - pole 150: Conductive wire 160: AC power supply 170: Holding section 200: Detection section 300: Cell 400: Dielectrophoretic force 500: Light

Claims (9)

(1) A step of centrifuging a sample obtained from blood containing target cells and contaminant cells;
(2) A part of the centrifuged supernatant obtained in step (1) is removed, and a pellet-like precipitate containing target cells and contaminant cells formed in a layer depending on the specific gravity of the cells , and the original sample obtaining a sample containing a centrifugal supernatant of
(3) adding an isotonic solution containing sugar to the sample obtained in step (2) at a constant rate ;
(4) centrifuging the sample obtained in step (3);
(5) removing the centrifuged supernatant obtained in step (4) to obtain a suspension containing target cells from which contaminant cells have been removed ;
A pretreatment method for obtaining a suspension containing target cells from which contaminant cells have been removed from a blood-derived sample containing target cells and contaminant cells, comprising:
The isotonic solution containing sugar is an isotonic solution containing sugar adjusted so that the specific gravity is smaller than the specific gravity of the target cell,
and the step of adding the sugar-containing isotonic solution at a constant rate disperses the contaminant cells contained in the upper layer of the pellet-like precipitate in the sugar-containing isotonic solution, while adding the sugar-containing isotonic solution to the pellet -like precipitate. The target cells contained in the lower layer are not dispersed in the sugar-containing isotonic solution , and when adding the sugar-containing isotonic solution, the pellet-like precipitates contained in the sample obtained in step (2) above are removed. This is done by adding at a constant rate using an electric pipettor, under the conditions that only the upper layer of the substance is suspended .
Said method. 2. The isotonic solution containing sugar adjusted to have a specific gravity lower than that of the target cells is an isotonic solution containing sucrose or xylitol adjusted to have a specific gravity lower than the target cells. Method described. The method according to claim 2, wherein the step of adding the sugar-containing isotonic solution at a constant rate is performed at a constant rate of 1 mL/s to 3 mL/s using an electric pipettor, and the sugar is sucrose. . 3. The method of claim 2, wherein the step of adding the sugar-containing isotonic solution at a constant rate is performed where the sugar is sucrose and is carried out using an electric pipettor at a constant rate of 1.5 mL/s. A claim in which the sugar is xylitol, and the step of adding the isotonic solution containing sugar at a constant rate is performed using an electric pipettor at a constant rate of 3.5 mL/s or more and 7.5 mL/s or less. Method of Section 2. The method of claim 2, wherein the step of adding at a constant rate the isotonic solution containing a sugar according to claim 2, wherein the sugar is xylitol, and is carried out at a constant rate of 5 mL/s using an electric pipettor. . A claim in which the centrifugation in step (4) according to claim 1 is carried out under conditions where the centrifugal force is 200 x g or more and 2000 x g or less, and the centrifugation time is 1 minute or more and 30 minutes or less. The method according to any one of Items 1 to 6 . A method for detecting target cells contained in a sample, including the steps shown in (I) to (IV) below:
(I) pretreating the sample by any of the methods described in claims 1 to 7 to obtain a suspension containing target cells;
(II) a step of introducing the suspension obtained in step (I) into a cell holding means having a holding section;
(III) a step of holding the target cells contained in the suspension in the holding section using dielectrophoretic force;
(IV) Detecting the target cells retained in (III). A method for collecting cells contained in a sample, further comprising the step of collecting target cells detected by the method according to claim 8 .