WO2018211706A1

WO2018211706A1 - Rare cell capturing method

Info

Publication number: WO2018211706A1
Application number: PCT/JP2017/018898
Authority: WO
Inventors: 勝也遠藤; 明子伊藤
Original assignee: 日立化成株式会社
Priority date: 2017-05-19
Filing date: 2017-05-19
Publication date: 2018-11-22

Abstract

The present invention discloses a method for capturing a rare cell. This method comprises a step for filtering a blood sample with a filter in the presence of a polymer selected from the group consisting of homopolymers of 2-methacryloyloxyethyl phosphorylcholine and copolymers of 2-methacryloyloxyethyl phosphorylcholine. Said method is able to efficiently remove white blood cells.

Description

How to capture rare cells

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, it 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 tumor cells (Circulating Tumor Cell, hereinafter also referred to as “CTC”) 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 cancer prognosis and treatment, 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). ~ 4).

International Publication No. 2010/135603 JP 2013-42689 A JP 2011-163830 A International Publication No. 2015/012315

In recent years, advanced technologies for next-generation sequencers, digital PCR, and quantitative reverse transcription PCR (qRT-PCR), which can analyze genes from a small number of cells, can detect gene mutations and protein expression levels in rare cells. The research has shifted to “Evaluation”. This is because screening of novel marker proteins and genes is desired using rare cell gene analysis results.

When genetic analysis is performed in the presence of contaminating cells such as leukocytes, the gene information of rare cells is buried in the gene information of narrow cells, and the gene information of rare cells is not detected, or false negative May result. However, in the filter methods described in Patent Documents 1 to 4, the purity of the separated rare cells is not sufficient for the gene analysis and the like.

The present invention has been made in view of such problems, and provides a method for selectively capturing rare cells from a blood sample.

As a result of intensive studies, the present inventors have found that when a blood sample is filtered in the presence of a predetermined polymer, the leukocyte removal rate is improved as compared with the case where the blood sample is filtered in the absence of the polymer. It came to be completed.

That is, in the method for capturing rare cells in a blood sample of the present invention, the blood sample is obtained from a polymer selected from the group consisting of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine and a copolymer of 2-methacryloyloxyethyl phosphorylcholine. Filtering with a filter in the presence.

The filter may be coated with a polymer selected from the group consisting of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine and a copolymer of 2-methacryloyloxyethyl phosphorylcholine. If a filter coated with a polymer is used, the leukocyte removal rate is further improved.

The rare cells to be captured can be circulating blood cancer cells.

According to the present invention, rare cells can be selectively captured from a blood sample.

It is a schematic diagram of a filter. It is a schematic diagram which shows the structure of a CTC acquisition apparatus. 2 is a graph comparing leukocyte residual ratios of Comparative Example 1 and Example 1. FIG. It is the graph which compared the leukocyte residual rate of the comparative example 2, Example 2, and Example 3. FIG. FIG. 4 represents one embodiment of applying a blood sample to a reservoir.

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

“Contaminated cells” are cells other than rare cells contained in a blood sample. Usually, the ratio of the number of contaminating cells is extremely large relative to the number of rare cells. In the present specification, a “contaminating cell” may refer to a cell having the same diameter as a rare cell and having deformability among such cells, and more specifically, a leukocyte. There is a case.

Here, the “diameter” of the cell is the length of the longest line segment connecting two arbitrary points on the outline of the cell when observed with a microscope.

“Capture” means that the blood sample is filtered through a filter, and the cells remain on the filter. Further, “selectively capture” means that the ratio of the predetermined cells in the cell population remaining on the filter is higher than the ratio in the blood sample before filtration.

In the method for capturing rare cells in a blood sample of the present invention, the blood sample is selected from the group consisting of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine (hereinafter also referred to as MPC) and a copolymer of 2-methacryloyloxyethyl phosphorylcholine. Filtering with a filter in the presence of the selected polymer.

The MPC homopolymer and MPC copolymer are hereinafter collectively referred to as MPC polymer. Examples of the MPC copolymer include a copolymer of MPC and butyl methacrylate. As the MPC homopolymer or MPC copolymer, a commercially available product can be used. For example, LIPIDURE (registered trademark) -CM5206, LIPIDURE (registered trademark) -PC, LIPIDURE (registered trademark) -NH01, LIPIDURE (registered trademark) -BL103, LIPIDURE (registered trademark)-sold by NOF CORPORATION BL203, LIPIDURE (registered trademark) -BL206, LIPIDURE (registered trademark) -BL405, LIPIDURE (registered trademark) -BL502, LIPIDURE (registered trademark) -BL702, LIPIDURE (registered trademark) -BL802, LIPIDURE (registered trademark) -BL1002, LIPIDURE (registered trademark) -BL1201, LIPIDURE (registered trademark) -BL1301, LIPIDURE (registered trademark) -SF08 and LIPIDURE (registered trademark) -SF16 Etc. can be used.

Next, the structure of a filter that can be used in this step will be described with reference to FIG. The filter 200 has a structure in which a large number of through holes 110 are formed in a thin film 120 such as plastic or metal.

The opening shape of the through hole 110 can be, for example, a circle, an ellipse, a square, a rectangle, a rounded rectangle, or a polygon. From the viewpoint of efficiently capturing rare cells, the opening shape is preferably a circle, a rectangle or a rounded rectangle, and more preferably a rectangle or a rounded rectangle. A rounded rectangle is a shape having a rectangle and two semicircles that have the same radius as the short side of the rectangle and that are adjacent to each other and are adjacent to the two short sides of the rectangle. If the opening shape is a rectangle or a rounded rectangle, the through hole 110 is less likely to be clogged, and rare cells can be captured more selectively. The through holes 110 may be arranged as shown in FIG. 1, may be arranged in a staggered manner in which the arrangement is shifted for each column, or may be arranged randomly.

The hole diameter of the through hole 110 is set according to the diameter of the rare cell to be captured. When the rare cell has a diameter of 10 μm or more as in CTC, the diameter of the through hole 110 is preferably 5 μm to 15 μm, more preferably 6 μm to 12 μm, and even more preferably 7 μm to 10 μm. . 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. For example, when the opening shape is a rectangle, the diameter of the through hole 110 is the length of the short side of the rectangle. When the opening shape is a polygon, the hole diameter of the through hole 110 is the diameter of the inscribed circle of the polygon. When the opening shape is a rectangle or a rounded rectangle, a gap is formed in the long axis direction of the opening shape in the through-hole 110 even when rare cells are captured in the through-hole 110. Since contaminant cells can pass through the gap, the filter 200 can be prevented from being clogged.

The aperture ratio of the filter 200 is preferably 5 to 50%, more preferably 5 to 40%, from the viewpoint of maintaining a balance between the strength of the filter 200 and prevention of clogging of the filter 200. % Is more preferable. In this specification, the aperture ratio refers to the area occupied by the through hole 110 with respect to the entire area of the filter 200.

The thickness of the filter 200 is preferably 3 μm to 50 μm, more preferably 5 μm to 40 μm, and more preferably 5 μm to 30 μm from the viewpoint of maintaining a balance between the strength of the filter 200 and prevention of clogging of the filter 200. Is more preferable.

The filter 200 is preferably made of metal. Since metal is excellent in processability, the processing accuracy of the filter 200 can be increased, and the capture rate of rare cells can be further improved. In addition, since metal is rigid compared to other materials, its size and shape are maintained even when a force is applied from the outside. For this reason, even if a contaminated cell is slightly larger than the through-hole 110, the filter 200 does not deform | transform, a contaminated cell can deform | transform and can pass through the through-hole 110, and can capture a rare cell more selectively.

Examples of the metal include nickel, gold, silver, palladium, copper, iridium, ruthenium, chromium, and alloys thereof. Palladium and iridium have a high redox potential, are hardly soluble and have good characteristics, but are expensive. Nickel has a lower oxidation-reduction potential than hydrogen, so it is easily dissolved but is inexpensive. Silver and palladium are noble metals and are less expensive than palladium and iridium.

The manufacturing method of the filter 200 is not particularly limited, but for example, it can be manufactured as follows. First, a photoresist is placed on the substrate at a position that will later become the through-hole 110 of the filter 200, and a metal layer (thin film 120) having the through-hole 110 is formed by plating the substrate with a metal that becomes the filter 200. Is formed on the substrate. Then, the filter 200 is obtained by removing the substrate and the photoresist from the metal layer. As the substrate, for example, a substrate whose surface is plated with copper can be used. Copper is preferable as a substrate material because it can be easily removed by chemical dissolution with a chemical solution and has excellent adhesion to a photoresist.

The filter 200 may be covered with an MPC polymer. When the blood sample is filtered with the filter 200 covered with the MPC polymer, the leukocyte removal rate can be further improved. The MPC polymer that coats the filter 200 is as described above. The MPC polymer that coats the filter 200 may be the same type as the MPC polymer that coexists with the blood sample, or may be a different type. The filter 200 can be coated by a known method. For example, the filter 200 covered with the MPC polymer is obtained by immersing the filter 200 in a solution containing the MPC polymer and drying it.

The method for filtering the blood sample and the apparatus used for the filtration are not particularly limited, but it is preferable to control the flow rate during liquid passage. From the viewpoint of minimizing the damage to rare cells, the viewpoint of pushing out contaminated cells, and the prevention of clogging of the filter 200, the flow rate during liquid passage is preferably 50 μL / min to 3000 μL / min, preferably 100 μL / min. Minute to 1000 μL / min is more preferable, and 200 μL / min to 800 μL / min is more preferable.

The method for filtering a blood sample in the presence of MPC polymer is not particularly limited. In one embodiment, the blood sample and MPC polymer may be mixed and the resulting mixture may be filtered. In another embodiment, the blood sample may be filtered and then washed with a solution containing MPC polymer. For example, as shown in FIG. 5, the blood sample 310 is gently applied using a micropipette so that the lower layer of the buffer solution 300 held in the reservoir, and the upper layer is the buffer solution 300 and the lower layer is the blood sample 310. A two-layered mixture can be prepared.

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples.

(Preparation of filter)
A metal filter having a shape shown in FIG. 1 and having 3000 holes of 8 μm × 100 μm in 6 mm × 6 mm, a thickness of 18 μm, and nickel-based and gold-plated on the surface was produced.

Photosensitive resin composition (PHOTEC RD-1225: thickness 25 μm, manufactured by Hitachi Chemical Co., Ltd.) 6 mm square substrate (MCL-E679F: substrate obtained by bonding peelable copper foil to the surface of MCL, manufactured by Hitachi Chemical Co., Ltd.) Laminated on one side. Lamination conditions were performed at a roll temperature of 90 ° C., a pressure of 0.3 MPa, and a conveyor speed of 2.0 m / min.

Next, a glass mask having a light-transmitting portion with a rounded rectangular shape, a size of 8 × 100 μm and a pitch of 75 μm on the short axis and 160 μm on the long axis was left on the photoresist laminate surface of the substrate. In this embodiment, a glass mask in which rounded rectangles facing in the same direction are arranged at a constant pitch in the major axis and minor axis directions was used.

Subsequently, under a vacuum of 600 mmHg or less, an ultraviolet ray irradiation device was used to irradiate ultraviolet rays with an exposure amount of 30 mJ / cm ² from above the substrate on which the glass mask was placed.

Next, development was performed with a 1.0% aqueous sodium carbonate solution to form a resist layer with a rectangular photoresist standing vertically on the substrate. The exposed copper portion of the resist-coated substrate was plated at about 18 μm for about 20 minutes at a temperature of 55 ° C. in a nickel plating solution adjusted to a pH of 4.5. Table 1 shows the composition of the nickel plating solution.

Next, the obtained nickel plating layer is peeled off together with the peelable copper foil of the substrate, and this peelable copper foil is chemically dissolved by a chemical solution in a stirring process at a temperature of 40 ° C. for about 120 minutes (MEC BRIGHT SF-5420B, MEC shares). The self-supporting membrane (6 mm × 6 mm) to be a metal filter was removed by removing by the company.

Finally, the photoresist remaining in the free-standing film was removed by resist stripping (P3 Poleve, Henkel) by ultrasonic treatment at a temperature of 60 ° C. for about 40 minutes to produce a metal filter having fine through holes.

This produced a metal filter having through holes with sufficient accuracy without damage such as wrinkles, creases, scratches, and curls.

Next, the metal filter was immersed in an acidic degreasing solution Z-200 (manufactured by World Metal: trade name) to remove organic substances on the metal filter (40 ° C. for 3 minutes).

After washing with water, pretreatment of replacement gold plating under conditions of 80 ° C for 10 minutes with a solution obtained by removing gold sulfite as a gold supply source from HGS-100 (trade name, manufactured by Hitachi Chemical Co., Ltd.), a non-cyan electroless Au plating Went.

Next, it was immersed in HGS-100 (trade name, manufactured by Hitachi Chemical Co., Ltd.), which is a non-cyan substitution type electroless Au plating, for 20 minutes at 80 ° C. to perform substitution gold plating.

After washing with water, it was immersed in HGS-5400 (trade name, manufactured by Hitachi Chemical Co., Ltd.), which is non-cyan reduced electroless Au plating, at 65 ° C. for 10 minutes, plated with gold, washed with water and dried.

(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 a sample (blood sample or the like) to be a test solution using 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 that is put into the processing liquid storage container 5 include a staining liquid for staining rare cells, a fixative, 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 being sucked 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.

The above units are 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.

(Filter coating with MPC polymer)
The filter was immersed in an ethanol solution of 0.5% MPC polymer (LIPIDURE (registered trademark) -CM5206; manufactured by NOF Corporation). The filter pulled up from the solution was naturally dried to obtain a filter coated with MPC polymer.

(Filtration of blood sample)
As shown in FIG. 5, 3 mL of a blood sample collected by adding 7 mL of PBS solution (hereinafter referred to as “buffer solution”) containing 0.5% BSA and 2 mM EDTA in the reservoir and collected with an anticoagulant-containing blood collection tube was added. Gently apply to the lower layer of the buffer solution with a micropipette, and introduce the blood and buffer solution in the reservoir into the filter at a flow rate of 600 μL / min. Then, cells in the blood were captured on the filter. Further, a buffer solution was introduced into the filter to wash away blood components remaining on the filter.

(Blood staining)
In order to detect that the cells captured on the filter are leukocytes, CD45 antigen, which is a surface antigen of leukocytes, and nuclei were stained. 1.25 mL of R-Phycoerythrin (hereinafter referred to as PE) modified anti-human CD45 mouse monoclonal antibody clone: 2D1 (manufactured by Thermo Fisher Scientific Co., Ltd.) and Hoechst 33342 mixed into a filter at a flow rate of 400 μL / min This was introduced and reacted at room temperature for 30 minutes. 3 mL of buffer solution was introduced into the filter at a flow rate of 400 μL / min, and the reaction solution in the filter was discharged. The filter was then removed from the CTC capture device.

(White blood cell count)
The filter was placed on a fluorescence microscope. A fluorescent mirror unit was used to excite the fluorescent dyes (PE and Hoechst 33342) on the cells, respectively. The fluorescence emitted from each fluorescent dye was photographed, and the resulting images were synthesized. From the synthesized image, cells showing leukocytes were extracted visually or using image analysis software, and the number of each cell was determined. Here, the cells showing leukocytes are PE-positive and Hoechst33342-positive cells.

(Test Example 1)
Fill the filter not coated with MPC polymer with buffer in advance, set it in the CTC capture device, add 7 mL of buffer in the reservoir, and then add 3 mL of blood sample collected from a healthy person to the lower layer of the buffer The mixture (Comparative Example 1) and the filter not coated with MPC polymer were previously filled with a buffer containing 0.5% MPC polymer (LIPIDURE (registered trademark) -BL203; manufactured by NOF Corporation) and set in the CTC capture device. Then, 7 mL of a buffer solution containing 0.5% MPC polymer (LIPIDURE (registered trademark) -BL203; manufactured by NOF Corporation) is added to the reservoir, and 3 mL of a blood sample collected from a healthy person is added to the lower layer of the buffer solution. The two-layered mixture (Example 1) was applied to a CTC capture device. Blood samples were filtered and blood cells were stained with a CTC capture device, and white blood cells were counted with a fluorescence microscope.
The results are shown in FIG. When the residual white blood cell count of Comparative Example 1 was 100, the residual white blood cell count of Example 1 was 86. The number of cancer cells captured in Comparative Example 1 and Example 1 was the same. From the above results, it was confirmed that by filtering a blood sample containing MPC polymer, the leukocyte removal rate was improved as compared with the case where MPC polymer was not included.

(Test Example 2)
The filter coated with MPC polymer was previously filled with 0.5% MPC polymer (LIPIDURE (registered trademark) -BL203; manufactured by NOF Corporation), set in a CTC capture device, and 0.5% MPC polymer (LIPIDURE (LIPIDURE ( (Registered trademark) -BL203; manufactured by NOF Co., Ltd.) 7 mL of a mixture was added, and a blood sample collected from a healthy person was mixed in 2 layers so that a blood sample 3 mL was a lower layer of the buffer (Example 2), a filter coated with MPC polymer Is filled with 0.5% MPC polymer (LIPIDURE (registered trademark) -BL405; manufactured by NOF Corporation) and set in a CTC capture device, and 0.5% MPC polymer (LIPIDURE (registered trademark) -BL405; (Made by NOF Corporation) 7mL is added, and 3mL blood sample collected from healthy people is buffered Subjected mixture was two layers so that the the lower layer (Example 3) to CTC capture device were captured cancer cells. In addition, cancer cells were captured under the same conditions as in Comparative Example 1 (Comparative Example 2). Blood samples were filtered and blood cells were stained with a CTC capture device, and white blood cells were counted with a fluorescence microscope. The results are shown in FIG. When the residual white blood cell count of Comparative Example 2 was 100, the residual white blood cell count of Example 2 was 32, and the residual white blood cell count of Example 3 was 30. The numbers of cancer cells captured in Comparative Example 2, Example 2 and Example 3 were the same. From the above results, it was confirmed that the leukocyte removal rate was further improved by filtering a blood sample containing MPC polymer using a filter coated with MPC polymer.

The front and back surfaces of the filter after filtration and the bottom of the cartridge were observed with a scanning electron microscope (SEM). In Example 2 and Example 3, the number of contaminating cells attached to the front and back surfaces of the filter and the bottom of the cartridge was smaller than that of Comparative Example 2.

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 DESCRIPTION OF SYMBOLS ... Waste liquid tank, 48 ... Control part, 49 ... Selection valve driver, 50 ... Valve driver, 51 ... Pump driver, 100 ... CTC capture device, 110 ... Through-hole, 120 ... Thin film, 200 ... Filter, 300 ... Buffer solution, 310 ... blood samples.

Claims

A method for capturing rare cells in a blood sample, comprising:
Filtering the blood sample with a filter in the presence of a polymer selected from the group consisting of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine and a copolymer of 2-methacryloyloxyethyl phosphorylcholine;
Including methods.
The method according to claim 1, wherein the filter is a filter coated with a polymer selected from the group consisting of a homopolymer of 2-methacryloyloxyethyl phosphorylcholine and a copolymer of 2-methacryloyloxyethyl phosphorylcholine.
The method according to claim 1 or 2, wherein the rare cells are circulating cancer cells in the blood.