JP2017181096A - Method for capturing rare cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for capturing rare cells at a high capture rate, from liquid containing the rare cells and blood.SOLUTION: A method for capturing a rare cell includes the steps of removing at least some of erythrocytes from liquid containing rare cells and blood, and filtering the liquid from which at least some of erythrocytes has been removed.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for capturing rare cells.

  Research and clinical significance of cancer cell enrichment is extremely large, and if cancer cells in blood can be enriched, that is, cancer cells can be selectively captured, they can be applied to cancer diagnosis. For example, the most important factor for the prognosis and treatment of cancer is the presence or absence of cancer cell metastasis at the first visit and treatment. When early diffusion of cancer cells reaches the peripheral blood, detecting circulating cancer cells (hereinafter also referred to as “CTC”) in the blood is useful for determining the progression of cancer pathology. Means. However, since blood components such as red blood cells and white blood cells are predominantly present in blood, it is difficult to detect an extremely small amount of CTC. Circulating Endothelial Cell (hereinafter also referred to as “CEC”) is also used as a biomarker for prognosis and treatment of cancer, but it is present in the blood only in a very small amount and is detected in the same manner as CTC. It is difficult.

  As a method for concentrating rare cells such as CTC and CEC from blood, there is a method using a filter (hereinafter also referred to as “filter method”). The filter method is a method of concentrating rare cells based on differences in cell size and deformability.

  As an example of the filter method, a method of detecting a small amount of CTC by using a resin filter using parylene has been proposed (Patent Document 1).

  As another example of the filter method, a method of improving the strength of the filter by using a metal filter and separating the leukocytes and the cancer cells depending on the deformability is proposed (Patent Document 2). ).

International Publication No. 2010/135603 JP 2013-42689 A

  The number of rare cells present in blood is extremely small, and in some cases, only one rare cell may be present in 7.5 mL of blood. In addition, rare cells are thought to have different properties such as the type and number of surface proteins for each cell, and in order to measure the heterogeneity, rare cell loss can be prevented as much as possible. Desired.

  This invention is made | formed in view of such a subject, and provides the method of capture | acquiring a rare cell with a high capture rate from the liquid containing a rare cell and blood.

  In the method for capturing rare cells of the present invention, a step of removing at least a part of red blood cells from a liquid containing rare cells and blood (removal step), and the liquid from which at least a part of red blood cells have been removed are filtered with a filter. Process (filtration process).

  When a rare cell and blood-containing liquid is filtered through a filter and the rare cell is captured on the filter, the red blood cell usually has a smaller cell diameter than the rare cell and white blood cell. It was thought that it would easily pass through the pores of this cell and did not affect the capture rate of rare cells. However, by removing at least some of the red blood cells from the liquid containing rare cells and blood as described above, the capture rate of the rare cells can be improved. Although not bound by a specific theory, the present inventors estimate the action of removing red blood cells in the present invention as follows. That is, by removing the red blood cells, the viscosity of the liquid containing the rare cells and blood is reduced, and the force for pushing the rare cells to the through-hole of the filter is reduced, so that the rare cells pass through the filter (rare cell loss). This can be mitigated.

  Here, in the removing step, the liquid containing rare cells and blood may be centrifuged to remove at least a part of the separated red blood cells. In the removing step, a hemolytic agent may be added to a liquid containing rare cells and blood.

  Further, the pore diameter of the filter may be 5 μm to 15 μm.

  Furthermore, the rare cell may be a blood circulating cancer cell or a blood circulating endothelial cell.

  According to the present invention, rare cells can be captured at a high capture rate from a liquid containing rare cells and blood.

It is a schematic diagram of a filter. It is a schematic diagram which shows the structure of a CTC acquisition apparatus.

  “Rare cell” means a cell to be captured, and the ratio of the number of cells is extremely small relative to the total number of all cells contained in a liquid containing rare cells and blood. Rare cells are, for example, cancer cells such as CTC or cancer stem cells or endothelial cells such as CEC.

  The liquid containing rare cells and blood can be blood, and can be obtained by diluting blood with a liquid such as a buffer solution.

  “Capture” means that the liquid containing the cells is filtered through, leaving the cells on the filter.

  The method for capturing rare cells of the present invention includes a removal step of removing at least a portion of red blood cells from a liquid containing rare cells and blood, and a filtration step of filtering the liquid from which at least a portion of red blood cells have been removed with a filter. ,including. Hereinafter, these steps will be described in detail with reference to FIGS. 1 and 2.

  First, a removal process for removing at least a part of red blood cells in a liquid containing rare cells and blood will be described.

  Examples of a method for removing at least a part of red blood cells in a liquid containing rare cells and blood include a method by centrifugation. By centrifuging a liquid containing rare cells and blood, a layer is formed for each component of the blood. The layers are a plasma layer, a buffy coat layer, and an erythrocyte layer in order from the upper layer. The buffy coat layer is a layer containing leukocytes, and rare cells are also separated into this layer. By removing at least a portion of the red blood cell layer, at least a portion of the red blood cells in the liquid containing rare cells and blood can be removed. The method by centrifugation is preferable as a method for removing erythrocytes because it is easy to handle. Further, it is preferable to perform centrifugation after adding an anticoagulant to a liquid containing rare cells and blood because blood components can be sufficiently separated.

  The amount of the red blood cell layer to be removed is preferably 50% or more, more preferably 100%, based on the total amount of the red blood cell layer.

  Centrifugal force during centrifugation is preferably 100 × g to 1500 × g, more preferably 200 × g to 1300 × g, and even more preferably 300 × g to 1000 × g. If the centrifugal force is 100 × g or more, blood components can be sufficiently separated. If the centrifugal force is 1500 × g or less, the risk of rare cells being destroyed by centrifugal force can be reduced.

  The centrifugation time at the time of centrifugation is preferably 1 minute to 60 minutes, more preferably 3 minutes to 30 minutes, further preferably 5 minutes to 15 minutes. If the centrifugation time is 1 minute or longer, blood components can be sufficiently separated. A centrifugal time of 60 minutes or less is preferable from the viewpoint of working time.

  When the liquid containing rare cells and blood is blood, blood (blood sample) can be obtained by collecting blood from the subject by a normal method. In this case, additives conventionally added to blood samples such as anticoagulants and fixatives can be further added. Examples of the anticoagulant include disodium ethylenediaminetetraacetate (EDTA-2Na), dipotassium ethylenediaminetetraacetate (EDTA-2K), sodium citrate, sodium fluoride, and heparin. Examples of the fixing agent include paraformaldehyde (PFA), glutaraldehyde, and ethanol. These additives can be put in a blood collection tube before blood collection in advance, or can be added after blood collection. The blood sample can also be diluted with, for example, a buffer.

  As another method of removing at least a part of red blood cells in a liquid containing rare cells and blood, a method of adding a hemolytic agent to the liquid containing rare cells and blood can be mentioned. A hemolytic agent is a reagent that acts on red blood cells physically or chemically to lyse the red blood cells. As the hemolytic agent, those conventionally used can be used, and examples thereof include ammonium chloride, synthetic surfactants and alcohols. A method using centrifugation and a method using a hemolytic agent may be used in combination.

  Next, a filtration step of filtering the liquid from which at least a part of red blood cells has been removed with a filter will be described.

  By this step, the remaining blood components are removed from the liquid containing rare cells and blood, and the rare cells are selectively captured.

  Hereinafter, filters that can be used in this step will be described with reference to FIG. The filter 200 is not particularly limited as long as it has a structure in which a large number of through-holes 110 are formed in a thin film 120 such as plastic or metal, and a conventionally known filter can be used. The filter 200 removes components other than the rare cells from the liquid containing the rare cells and blood together with the filtrate, and captures the rare cells that have not passed through the through-hole 110 on the surface thereof.

  The filter 200 is preferably made of a metal, and is preferably made of a metal having gold plating on the surface from the viewpoint of low cytotoxicity. The filter 200 is preferably a filter whose biocompatibility on the filter surface has been improved by immersing in advance in mammalian serum or plasma. The filter 200 having the surface treated in this way can prevent blood cells from adsorbing on the surface of the filter 200 in the filtration step.

  When blood containing cancer cells is used as a liquid containing rare cells and blood, the filter through-hole 110 preferably has a pore diameter of 5 μm to 15 μm, more preferably 6 μm to 12 μm, and even more preferably 7 μm to 10 μm. By using the filter, it is possible to remove blood components such as red blood cells, white blood cells, and platelets as filtrate, and capture cancer cells that are rare cells in the filter 200. In this specification, the hole diameter of the through hole 110 (also simply referred to as “filter hole diameter”) refers to the maximum value of the diameter of a sphere that can pass through the through hole 110.

  After capturing the rare cells, the captured rare cells can be collected to obtain a rare cell-containing solution. Examples of the method for collecting rare cells include backwashing, micromanipulation, and pipetting. Backwashing means that the liquid is allowed to flow from the surface of the filter opposite to the surface where the cells are captured, and the cells captured on the filter are peeled off. Micromanipulation refers to collecting individual cells from a filter using a pipette. Pipetting means that the liquid on the filter is mixed using a micropipette, and the cells captured on the filter are peeled off.

  EXAMPLES Hereinafter, although an invention is concretely demonstrated based on an Example, this invention is not limited to a following example.

(Preparation of filter)
Photosensitive resin composition (PHOTEC RD-1225: thickness 25 μm, manufactured by Hitachi Chemical Co., Ltd.) 250 mm square substrate (MCL-E679F: substrate in which peelable copper foil is bonded to the surface of MCL, manufactured by Hitachi Chemical Co., Ltd.) Laminated on one side. Lamination was performed at a roll temperature of 90 ° C., a pressure of 0.3 MPa, and a conveyor speed of 2.0 m / min.

  A glass mask having an opening with an aperture ratio of 6.7% was allowed to stand on the surface of the substrate laminated with the photosensitive resin composition. The opening of the glass mask is a light transmitting part, and the rounded rectangular holes of 7.8 μm × 100 μm are arranged in the same direction at a constant pitch along the major axis and minor axis directions of the glass mask. Aligned facing. The size of the rounded rectangle is defined by the short side length (7.8 μm) and the long side length (100 μm) of the rectangle forming the rounded rectangle. Moreover, an aperture ratio means the area which a through-hole occupies with respect to the area of the whole filter.

Under a vacuum of 600 mmHg or less, the substrate on which the glass mask was placed was irradiated with ultraviolet rays at an exposure amount of 30 mJ / cm ² using an ultraviolet irradiation device.

  The substrate after the ultraviolet irradiation was developed with a 1.0% aqueous sodium carbonate solution to form a resist layer in which rounded rectangular photoresists were arranged on the substrate. This substrate with a resist layer was treated with a nickel plating solution having a pH of 4.5 at a temperature of 55 ° C. for about 20 minutes to form a nickel plating having a thickness of about 20 μm on the exposed copper foil. Table 1 shows the composition of the nickel plating solution.

  The obtained nickel plating layer was peeled from the substrate together with the peelable copper foil. The copper foil with the nickel plating layer is stirred for about 120 minutes in an etching chemical solution (MEC BRIGHT SF-5420B, manufactured by MEC Co., Ltd.) at a temperature of 40 ° C., and the copper foil portion is removed by chemical dissolution, thereby removing the metal filter. A nickel self-supporting film (size 20 mm × 20 mm) was obtained.

  A metal filter having fine through-holes by removing the photoresist remaining in the free-standing film by ultrasonically treating the free-standing film in a resist stripping solution (P3 Poleve, manufactured by Henkel) at a temperature of 60 ° C. for about 40 minutes. Got. The completed metal filter was free from damage such as wrinkles, breaks, scratches and curls, and had through holes with sufficient accuracy.

  The metal filter was immersed in acidic degreasing solution Z-200 (manufactured by World Metal Co., Ltd.) at a temperature of 40 ° C. for 3 minutes to remove organic substances on the metal filter.

  After washing the metal filter with water, the metal filter was removed at 80 ° C. by removing gold sulfite as an Au supply source from HGS-100 (manufactured by Hitachi Chemical Co., Ltd.), a non-cyan substitutional electroless Au plating solution. It was immersed for a minute and a pretreatment for displacement gold plating was performed. Next, the metal filter was immersed in HGS-100 at 80 ° C. for 20 minutes to perform displacement gold plating. The thickness of the displacement gold plating was 0.05 μm.

  After washing the plated metal filter with water, the metal filter was immersed in HGS-5400 (manufactured by Hitachi Chemical Co., Ltd.), which is a non-cyan reduced electroless Au plating solution, at 65 ° C. for 10 minutes to perform gold plating. Thereafter, the metal filter was washed with water and dried. The total thickness of the gold plating was 0.2 μm.

(CTC capture device)
Hereinafter, the CTC acquisition apparatus used in the present embodiment will be described with reference to FIG. A CTC capturing device 100 shown in FIG. 2 is a device that captures rare cells contained in a sample by filtering the blood sample with a filter. In addition, the captured rare cells can be stained with a staining solution to identify the rare cells and count the number of the rare cells.

  The CTC capturing device 100 is provided with a filter unit 1 having a filter therein, a processing liquid channel 3 for supplying a processing liquid to the filter unit 1, and a sample channel 4 for supplying a sample to the filter unit 1.

  A plurality of processing liquid storage containers 5 containing different processing liquids are provided on the upstream side of the processing liquid flow path 3. Examples of the processing liquid to be charged into the processing liquid storage container 5 include a staining liquid for staining rare cells and a cleaning liquid for cleaning rare cells captured by a filter. A soft tube is inserted into each processing liquid storage container 5 to form individual processing liquid flow paths 6. These flow paths are connected to the selection valve 8, and the processing liquid connected to the processing liquid flow path 3 is selected by rotating the selection valve 8.

  A reservoir 10 is connected to the sample flow path 4, and the sample is supplied to the reservoir 10. The filter unit 1 is configured to supply either the processing liquid or the sample, and control of which liquid of the processing liquid and the sample is supplied is attached to each of the flow paths 3 and 4. The pinch valves 12 and 13 are used for switching.

  The treatment liquid and the sample are supplied by suction by a peristaltic pump 14 provided downstream of the filter unit 1. The sample or the processing liquid flows through the processing liquid channel 3 or the sample channel 4 and is supplied to the filter unit 1, and then flows into the waste liquid tank 16. Rare cells in the sample are captured by a filter provided on the flow path in the filter unit 1 and stained with a staining solution.

  Each unit described above is controlled by the control unit 48. Specifically, the selection valve 8 is controlled by the selection valve driver 49 based on an instruction from the control unit 48. The pinch valves 12 and 13 are controlled by two valve drivers 50 connected to each other. The driving of the peristaltic pump 14 is controlled by a pump driver 51.

(Preparation of cancer cell suspension)
NCI-H358 cells, a non-small cell carcinoma cell line, were statically cultured in a culture flask at 37 ° C. in an RPMI-1640 medium containing 10% fetal bovine serum (FBS) under 5% CO ₂ conditions. . Cells are detached and collected by trypsin treatment, and the cells are washed with phosphate buffered saline (PBS), and then PBS containing 2 mM EDTA and 0.5% BSA (hereinafter also referred to as “washing solution”). ) To obtain a cancer cell suspension. As the PBS, a product code 166-23555 manufactured by Wako Pure Chemical Industries, Ltd. was used. For BSA, Albumin from bovine serum-Lyophilized powder, Bio Reagent for cell culture (manufactured by Sigma-Aldrich) was used. As EDTA-2Na, product code 345-01865 manufactured by Wako Pure Chemical Industries, Ltd. was used.

Example 1
The blood of a healthy human donor was collected in a vacuum blood collection tube containing EDTA-2Na, and after 48 hours from the blood collection, a cancer cell suspension was added in an amount that resulted in 500 cancer cells, and a 3 mL blood sample was prepared.

  The blood sample was centrifuged at a centrifugal force of 400 × g for 10 minutes, and the red blood cell layer was removed with a micropipette to obtain a blood sample with a red blood cell concentration of 0%.

  The following experiment was performed using the CTC capturing apparatus 100. First, the cleaning liquid was put into the reservoir 10 and the inside of the filter unit 1 was filled with the cleaning liquid. Subsequently, the blood sample was introduced into the reservoir 10 and fed at a flow rate of 600 μL / min for about 15 minutes. The filter was washed by feeding 6 mL of the cleaning solution from the processing solution storage container 5.

  A solution in which 4% PFA as a fixing agent was dissolved in PBS was sent to the filter unit 1, and the filter in which the cells were captured was immersed for 15 minutes. After washing the filter with a washing solution, a solution obtained by dissolving 0.2% Triton X-100 (manufactured by Sigma-Aldrich) in PBS was fed to the filter unit 1 and the filter was immersed for 15 minutes.

  After washing the filter with the washing solution, 2 mL of cell staining solution (FITC-labeled anti-Cytokeratin antibody, PE-labeled anti-CD45 antibody, 1 μg / mL Hoechst 33342) was sent to the filter unit 1, and the cells on the filter were fluorescently stained for 30 minutes. Thereafter, the cells were washed with 3 mL of washing solution.

  The filters were observed with a fluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy) equipped with a computer-controlled electric stage and a cooled digital camera (AxioCam 506 mono, Carl Zeiss Microscopy). In order to observe the FITC-derived fluorescence, a filter set 38 (manufactured by Carl Zeiss Microscopy) was used for the fluorescence microscope, and an image of the observation field was obtained. ZEN (manufactured by Carl Zeiss Microscopy) was used for image acquisition and analysis. The number of cancer vesicles (FITC positive cells) was counted, and the capture rate (%) of cancer cells was calculated as cancer fine capture rate = number of captured cancer cells / number of cancer cells added to blood sample × 100.

  The above experiment was performed on the blood of three healthy donors, and the average value of the obtained cancer cell capture rate was determined.

(Example 2)
Instead of a blood sample with an erythrocyte concentration of 0%, after centrifuging the blood sample, an amount of erythrocyte layer corresponding to 50% by volume of the total amount of erythrocyte layer, and an amount of plasma layer corresponding to 50% by volume of the total amount of erythrocyte layer, , Was removed by a micropipette, and the experiment was performed in the same manner as in Example 1 except that the blood sample (red blood cell concentration was 50% of the red blood cell concentration of the blood sample before removal of red blood cells) was used. The average value of the capture rate of cancer cells was determined.

(Comparative Example 1)
Except that the erythrocyte layer was not removed, and the number of blood samples used was equal to 2 healthy donors, the experiment was carried out in the same manner as in Example 1, and the capture rate of cancer cells captured by the filter The average value of was obtained.

(Evaluation)
The results of Examples 1 and 2 and Comparative Example 1 are shown in Table 2. As shown in Examples 1 and 2, if the red blood cells in the blood sample were removed before filtering the blood sample with a filter, the cancer was captured at a higher capture rate than in Comparative Example 1 in which the red blood cells were not removed. Cells could be captured.

(Example 3)
In the same manner as in Example 1, blood of a healthy donor was collected into a vacuum blood collection tube containing EDTA-2Na, and after 48 hours from the blood collection, a cancer cell suspension was added in an amount such that 500 cancer cells were added. A blood sample was prepared.

  To the blood sample, 30 mL of VersaLyse Lysing Solution (manufactured by Beckman Coulter) was added as a hemolytic agent, mixed with a vortex mixer, and reacted at room temperature for 10 minutes to hemolyze the blood sample. The sample after hemolysis was centrifuged at 400 × g for 10 minutes, and the supernatant was removed with a micropipette to obtain a blood sample with an erythrocyte concentration of 0%.

  Experiments were performed in the same manner as in Example 1 except that the blood sample obtained by hemolysis and having a red blood cell concentration of 0% was used as a blood sample, and the capture rate of cancer cells captured by the filter was determined. By adding the hemolytic agent, the cancer cell capture rate was improved as in Example 1.

DESCRIPTION OF SYMBOLS 1 ... Filter unit, 3, 6 ... Processing liquid flow path, 4 ... Sample flow path, 5 ... Processing liquid storage container, 8 ... Selection valve, 10 ... Reservoir, 12, 13 ... Pinch valve, 14 ... Peristaltic pump, 16 ... Waste liquid Tank, 48 ... control unit, 49 ... selection valve driver, 50 ... valve driver, 51 ... pump driver, 100 ... CTC capture device, 110 ... through hole, 120 ... thin film, 200 ... filter.

Claims (5)

Removing at least a portion of red blood cells from a liquid containing rare cells and blood;
Filtering the liquid from which at least some of the red blood cells have been removed with a filter;
A method for capturing rare cells, comprising:   The method according to claim 1, wherein in the removing step, the liquid containing rare cells and blood is centrifuged to remove at least a part of the separated red blood cells.   The method according to claim 1 or 2, wherein, in the removing step, a hemolytic agent is added to a liquid containing rare cells and blood.   The method according to claim 1, wherein the filter has a pore size of 5 μm to 15 μm.   The method according to any one of claims 1 to 4, wherein the rare cell is a blood circulating cancer cell or a blood circulating endothelial cell.

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