Method for freezing sample containing peripheral circulating tumor cells, and use thereof

Abstract

The present invention realizes: a method for freezing a sample containing peripheral circulating tumor cells, with which the number of false positives is reduced and good reproducibility is obtained; and use of the same. The present invention uses a method for freezing a sample containing peripheral circulating tumor cells, the method comprising a step for freezing a sample containing peripheral circulating tumor cells in a cryoprotective solution containing a complex lipid (excluding a cryoprotective solution containing a plasma component).

Description

Method for Freezing a Sample Containing Circulating Tumor Cells and Use Thereof

[0001] The present invention relates to a method for freezing a sample containing circulating tumor cells and use thereof.

[0002] Liquid biopsy is a technique for collecting tumor-derived substances from body fluids such as blood and analyzing the tumor-derived substances. Liquid biopsy has the advantage of being able to perform repeated examinations because it places less burden on patients. In particular, circulating tumor cells (CTCs) contained in blood are cells that can be obtained from blood and can provide various biological information (e.g., information related to tumors, etc.), and thus have attracted attention (see, for example, Patent Documents 1 and 2).

[0003] However, since CTCs are cells that cannot be stored for a long time, when performing examinations for diseases, etc. based on CTCs, it is necessary to analyze CTCs within 3 days from blood collection. For this reason, there are problems such as the need for pretreatment and analysis for each occurrence of the specimen, resulting in high costs, and the inability to select the specimen at a later date, etc. when performing examinations for diseases, etc. based on CTCs (see, for example, Non-Patent Documents 1 to 5).

[0004] Under such circumstances, the present inventor has started developing a technique for cryopreserving CTCs.

[0005] Patent No. 6198717, Patent No. 5943521

[0006] Peixuan Zhu et. al., "Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis" J.Transl.Med., (2016)14:198Marta Vismara et. al., "Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma" Clinical Chemistry, (2022)68:5, 691-701Sarah Nejlund et. al., "Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization" Biopreservation and Biobanking, (2016) Vol.14, No.4, 330-337Heming Li et. al., "Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells" Cancer Lett., (2017)403:216-223Daniel Brungs et. al., "Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer" World Journal of Gastroenterology, (2018), 24(7):810-818

[0007] In the process of developing cryopreservation technology for CTCs, the inventors encountered unique challenges when cryopreserving CTCs using dimethyl sulfoxide (DMSO) or the like, which have been conventionally used for cell cryopreservation. These challenges included a high number of false positives (for example, the number of cells in the thawed sample obtained by thawing the frozen sample that are mistakenly identified as peripheral blood circulating tumor cells) and poor reproducibility (for example, the reproducibility of the recovery rate of peripheral blood circulating tumor cells in the thawed sample obtained by thawing the frozen sample).

[0008] One aspect of the present invention aims to provide a method for freezing samples containing peripheral blood circulating tumor cells that reduces the number of false positives and exhibits good reproducibility, and to enable the use of the same.

[0009] In order to solve the above-mentioned problems, the inventors have conducted extensive research and have found that (i) by using a combination of a sample containing peripheral blood circulating tumor cells and a cryoprotective solution containing complex lipids (excluding a cryoprotective solution containing plasma components), the number of false positives can be reduced and reproducibility can be improved, and (ii) as will be clear from the examples described later, when a cryoprotective solution containing plasma components containing various substances is used, the number of false positives cannot be reduced and reproducibility cannot be improved, thus completing the present invention. One embodiment of the present invention encompasses the following invention.

[0010] [1] A method for freezing a sample containing peripheral blood circulating tumor cells, comprising the step of freezing the sample containing peripheral blood circulating tumor cells in a cryoprotective solution containing complex lipids (excluding a cryoprotective solution containing plasma components).

[0011] [2] The method for freezing a sample containing peripheral blood circulating tumor cells as described in [1], wherein the sample is obtained by hemolyzing a component of whole blood that includes peripheral blood circulating tumor cells, which is at least a part of the whole blood.

[0012] [3] The method for freezing a sample containing peripheral blood circulating tumor cells as described in [2], wherein the sample obtained by the hemolysis treatment has not undergone at least one treatment selected from the group consisting of fixation, permeabilization, and staining.

[0013] [4] The method for freezing a sample containing peripheral blood circulating tumor cells as described in [1], wherein the sample is obtained by density gradient centrifugation of a component of whole blood that includes peripheral blood circulating tumor cells, which is at least a portion of the whole blood.

[0014] A method for separating peripheral blood circulating tumor cells, comprising the steps of: thawing a frozen sample obtained by a method for freezing a sample containing peripheral blood circulating tumor cells described in any one of [5] [1] to [4] to obtain a thawed sample; and separating the peripheral blood circulating tumor cells from the thawed sample.

[0015] A method for obtaining data for diagnosing a disease, comprising the step of analyzing peripheral blood circulating tumor cells isolated by the method for isolating peripheral blood circulating tumor cells described in [6] and [5].

[0016] [7] The above disease is cancer. A method for obtaining data to diagnose the disease described in [6].

[0017] According to one aspect of the present invention, a method for freezing a sample containing peripheral blood circulating tumor cells, which reduces the number of false positives and exhibits good reproducibility, and its use are realized.

[0018] This graph shows the test results for the number of false positives and the CTCs recovery rate of various frozen samples in the embodiments of the present invention.

[0019] The following describes in detail some examples of embodiments of the present invention, but the present invention is not limited to these. The present invention can be modified in various ways within the scope of the claims. Embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents mentioned herein are incorporated herein by reference. Unless otherwise specified herein, "X to Y" representing a numerical range means "X or more and Y or less".

[0020] [1. Method for Freezing Samples Containing Peripheral Blood Circulating Tumor Cells] A method for freezing samples containing peripheral blood circulating tumor cells according to one embodiment of the present invention comprises the step of freezing a sample containing peripheral blood circulating tumor cells in a cryoprotective solution containing complex lipids (excluding a cryoprotective solution containing plasma components). Here, a cryoprotective solution containing plasma components refers to, for example, the supernatant obtained by centrifuging blood or a mixture of the supernatant and DMSO. With this configuration, the number of false positives (for example, the number of cells in a thawed sample obtained by thawing a frozen sample that are mistakenly identified as peripheral blood circulating tumor cells) can be reduced, and reproducibility (for example, the reproducibility of the recovery rate of peripheral blood circulating tumor cells contained in a thawed sample obtained by thawing a frozen sample) can be improved.

[0021] The sample containing peripheral blood circulating tumor cells mentioned above is acceptable as long as it contains peripheral blood circulating tumor cells, and its specific composition is not limited. Examples of such samples include blood (e.g., whole blood) and blood (e.g., whole blood) that has undergone a predetermined treatment and contains peripheral blood circulating tumor cells. From the viewpoint of obtaining a higher desired effect, it is preferable that the sample containing peripheral blood circulating tumor cells is blood (e.g., whole blood) that has undergone a predetermined treatment and contains peripheral blood circulating tumor cells. The predetermined treatment is not limited, and examples include treatments for separating or concentrating peripheral blood circulating tumor cells from blood (e.g., whole blood).

[0022] Examples of processes for separating or concentrating peripheral blood circulating tumor cells from the above-mentioned blood (e.g., whole blood) include one or more processes selected from the group consisting of hemolysis, fixation, permeabilization, staining, and separation. More specifically, examples of processes for separating or concentrating peripheral blood circulating tumor cells from the above-mentioned blood (e.g., whole blood) include (i) hemolysis, (ii) hemolysis and fixation in that order, (iii) hemolysis, fixation and permeabilization in that order, (iv) hemolysis, fixation, permeabilization and staining in that order, and (v) hemolysis, fixation, permeabilization, staining and separation in that order.

[0023] Preferably, the sample described above is obtained by hemolyzing a component of whole blood that includes peripheral blood circulating tumor cells, which is at least a portion of the whole blood. This configuration reduces the number of false positives and improves reproducibility. Furthermore, this configuration is excellent when analyzing the DNA of peripheral blood circulating tumor cells contained in a thawed sample obtained by thawing a frozen sample.

[0024] Preferably, the above-mentioned sample is one that has not undergone at least one treatment selected from the group consisting of fixation, permeabilization, and staining, after being obtained by the above-mentioned hemolysis treatment. This configuration is superior in that it results in a small number of false positives, a high CTC recovery rate, and does not exceed 100%. Furthermore, this configuration is superior when analyzing DNA from peripheral blood circulating tumor cells contained in a thawed sample obtained by thawing a frozen sample.

[0025] The above hemolysis treatment can be any treatment that can remove red blood cells, and its specific composition is not limited. The above hemolysis treatment may, for example, be (i) treating at least a portion of the components of whole blood with a hemolytic agent containing ammonium chloride or saponin, etc. A commercially available hemolytic agent can be used as the above hemolytic agent.

[0026] The above fixation treatment can be any treatment that can fix the desired cells, and its specific configuration is not limited. The above fixation treatment may, for example, be (i) treating the desired cells obtained after hemolysis with a fixative containing formalin, alcohol, glutaraldehyde, chloroform, or acetone. A commercially available fixative can be used as the above fixative.

[0027] The above permeabilization treatment can be any treatment that can impart permeability to the cell membrane of the desired cells, and its specific configuration is not limited. The above permeabilization treatment may, for example, be (i) treating the desired cells obtained after fixation with a permeabilizing agent containing a surfactant, methanol, acetone, or proteinase K. Commercially available permeabilizing agents can be used as the above permeabilizing agent.

[0028] The above staining treatment can be any treatment that can visualize the desired substance present in the desired cells, and its specific configuration is not limited. The above staining treatment may, for example, be (i) contacting the desired cells obtained after permeabilization with a primary antibody that recognizes the desired substance present in the cells, and a secondary antibody that recognizes the primary antibody and is labeled with a fluorescent substance or the like, or (ii) physically staining the desired cells obtained after permeabilization with a staining agent that physically binds to the cells. Commercially available antibodies can be used as the primary and secondary antibodies.

[0029] The above separation process can be any process capable of separating or concentrating peripheral blood circulating tumor cells, and its specific configuration is not limited. The above separation process may, for example, involve subjecting the desired cells obtained after staining to a cell separation device (e.g., a cell sorter, centrifuge, bead purification device, filter device, or microchip device) to separate or concentrate peripheral blood circulating tumor cells. Commercially available devices can be used as the cell separation device.

[0030] The above sample is preferably a component of whole blood, specifically a component containing peripheral blood circulating tumor cells, obtained by density gradient centrifugation. This configuration reduces the number of false positives and improves reproducibility. Furthermore, this configuration is excellent when analyzing RNA from peripheral blood circulating tumor cells contained in a thawed sample obtained by thawing a frozen sample.

[0031] The density gradient centrifugation process described above can be any process that can separate or concentrate peripheral blood circulating tumor cells, for example, a density gradient centrifugation method and a commercially available kit may be used.

[0032] The above-mentioned complex lipids are not limited to those mentioned above, and examples include glycolipids and phospholipids.

[0033] The above glycolipids are not limited to, but include, for example, sophorose lipid, rhamnolipid, n-dodecyl-β-D-maltoside, trelipid, cellobiopid, n-octyl-β-glucoside, galactosyldiacylglycerol, cerebroside, and alkyl polyglucoside.

[0034] The phospholipids mentioned above are not limited to those listed above, but include, for example, sphingosine-1-phosphate, phosphatidylserine, phosphatidylcholine, and lecithin.

[0035] The complex lipid contained in the above cryoprotective solution may be, for example, at least one selected from the group consisting of sophorose lipid, rhamnolipid, n-dodecyl-β-D-maltoside, and sphingosine-1-phosphate.

[0036] The sophorose lipids mentioned above are a biosurfactant discovered in the culture medium of Starmerella (Candida) bombicola. Commercially available sophorose lipids and cryoprotective solutions containing them can be used. Alternatively, the sophorose lipids can be prepared according to the description in Japanese Patent Publication No. 2016-160244.

[0037] The above-mentioned rhamnolipid is one of the biosurfactants produced by Pseudomonas aeruginosa. Commercially available rhamnolipid and cryoprotective solutions containing rhamnolipid can be used. Alternatively, the above-mentioned rhamnolipid can be prepared from Pseudomonas aeruginosa cultures.

[0038] The amount of complex lipids contained in the above cryoprotective solution is not limited. For example, the above cryoprotective solution may contain 0.001 to 100% by weight, 0.001 to 90% by weight, 0.001 to 80% by weight, 0.001 to 70% by weight, 0.001 to 60% by weight, 0.001 to 50% by weight, 0.001 to 40% by weight, 0.001 to 30% by weight, 0.001 to 20% by weight, 0.001 to 10% by weight, 0.001 to 5% by weight, or 0.001 to 1% by weight of complex lipids per 100% by weight of the above cryoprotective solution. The lower limit of each numerical range indicating the complex lipid content is not limited to 0.001% by weight, but may be, for example, 0.005% by weight, 0.01% by weight, 0.1% by weight, or 1% by weight.

[0039] The freezing rate in the above freezing process is not limited. The freezing rate may be rapid freezing (freezing rate of -4.0°C / min or higher) which freezes the sample in a short time, but slow freezing which freezes the sample over a long period of time is preferred.

[0040] Examples of freezing rates in the slow freezing described above include rates of less than -4.0°C / min, -3.0°C / min or lower, -2.0°C or lower, or -1.0°C / min or lower. Freezing the sample for a longer period of time can reduce the number of false positives and improve reproducibility.

[0041] [2. Method for separating peripheral blood circulating tumor cells, and method for obtaining data for diagnosing a disease] A method for separating peripheral blood circulating tumor cells according to one embodiment of the present invention comprises: step A, which involves thawing a frozen sample obtained by a method for freezing a sample containing peripheral blood circulating tumor cells according to one embodiment of the present invention to obtain a thawed sample; and step B, which involves separating the peripheral blood circulating tumor cells in the thawed sample.

[0042] The thawed sample obtained by step A above has a low number of cells that are judged as false positives in the subsequent step B (for example, the number of cells that are mistakenly identified as peripheral blood circulating tumor cells). By separating peripheral blood circulating tumor cells from this thawed sample in step B, the number of false positives can be reduced and peripheral blood circulating tumor cells can be separated with good reproducibility.

[0043] A method for obtaining data for diagnosing a disease according to one embodiment of the present invention includes step C of analyzing peripheral blood circulating tumor cells separated by a method for separating peripheral blood circulating tumor cells according to one embodiment of the present invention. More specifically, a method for obtaining data for diagnosing a disease according to one embodiment of the present invention includes step A of thawing a frozen sample obtained by a method for freezing a sample containing peripheral blood circulating tumor cells according to one embodiment of the present invention to obtain a thawed sample, step B of separating the peripheral blood circulating tumor cells in the thawed sample, and step C of analyzing the peripheral blood circulating tumor cells separated by step B.

[0044] The number of cells determined to be false positives (for example, the number of cells erroneously determined to be circulating tumor cells in peripheral blood) in the subsequent step B is small in the thawed sample obtained by the above step A. If circulating tumor cells in peripheral blood are separated from the thawed sample in step B, the number of false positives can be reduced, and circulating tumor cells in peripheral blood can be separated with good reproducibility. Then, in the above step C, if the circulating tumor cells in peripheral blood obtained with a small number of false positives and good reproducibility are analyzed, the disease can be accurately diagnosed.

[0045] Regarding the above step A, since the frozen sample obtained by the freezing method of the sample containing circulating tumor cells in peripheral blood has been described in [1. Freezing method of the sample containing circulating tumor cells in peripheral blood] described above, the description thereof will be omitted here.

[0046] Regarding the above step A, the method of thawing the frozen sample to obtain a thawed sample is not limited. For example, the frozen sample can be thawed by placing the frozen sample in an environment of 25°C to 40°C, 30°C to 40°C, or 35°C to 37°C (for example, a water bath) to obtain a thawed sample.

[0047] Regarding the above step B, the method of separating circulating tumor cells in peripheral blood from the thawed sample is not limited. Examples of such methods include methods of separating circulating tumor cells in peripheral blood from the thawed sample using a cell sorter, a centrifuge, a bead purification device, a filter device, or a microchip device.

[0048] Regarding the above step C, the viewpoints and methods for analyzing peripheral blood circulating tumor cells are not limited. In the above step C, for example, (i) measuring the number of peripheral blood circulating tumor cells using a flow cytometer or the like, (ii) analyzing peripheral blood circulating tumor cells using a fluorescence microscope or the like, (iii) analyzing proteins (for example, vimentin, cytokeratin) of peripheral blood circulating tumor cells, (iv) analyzing DNA of peripheral blood circulating tumor cells (for example, DNA mutation analysis (KRAS and BRAF)), and / or (v) analyzing RNA (for example, PGR and / or SCGB2A1, PIP) of peripheral blood circulating tumor cells (see, for example, Cancer Discov. 2018(10):1286-1299) may be performed. Since these analysis methods are well-known, detailed descriptions thereof are omitted.

[0049] Regarding the method for obtaining data for diagnosing a disease according to an embodiment of the present invention, the above disease is not limited. Examples of the above disease include diseases related to peripheral blood circulating tumor cells (for example, diseases in which the number of peripheral blood circulating tumor cells changes according to the onset or cure of the disease), or diseases that may be related to peripheral blood circulating tumor cells (for example, diseases in which the number of peripheral blood circulating tumor cells may change according to the onset or cure of the disease).

[0050] The above disease may be cancer, and examples of the cancer include gastric cancer, lung cancer, breast cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, and colorectal cancer. According to this configuration, these diseases can be accurately diagnosed.

[0051] An embodiment of the present invention may contribute to the achievement of, for example, Goal 3, "Good health and well-being for all", of the Sustainable Development Goals (SDGs) proposed by the United Nations.

[0052] <Test 1: Examination of the components of the cryoprotectant> <1-1. Preparation of blood samples> After culturing PC9 cells derived from lung adenocarcinoma, the number of the PC9 cells was counted under microscopic observation. A known number of PC9 cells were added to 4 mL of blood. In subsequent tests, the blood containing the PC9 cells was used as a blood sample.

[0053] <1-2. Hemolysis Treatment> 36 mL of ultrapure water (Merck) and 4 mL of RBC Lysis Buffer (10x) (Biolegend) were added to a 50 mL centrifuge tube (IWAKI) and mixed.

[0054] The blood sample prepared in <1-1. Preparation of Blood Sample> was added to the centrifuge tube described above, and after inverting the tube to mix, it was left to stand for 20 minutes (at room temperature).

[0055] The above centrifuge tubes were subjected to centrifugation (conditions: 500 x g, 30 minutes, room temperature), and the supernatant was removed. 10 mL of 0.5% BSA / T-Buffer was added to the centrifuge tubes after the supernatant was removed, and the cell pellet was suspended. The centrifuge tubes were then subjected to centrifugation (conditions: 500 x g, 5 minutes, room temperature), and the supernatant was removed.

[0056] The above 0.5% BSA / T-Buffer was prepared by mixing 95 mL of T-Buffer (manufactured by OnChip Biotechnology) with 5 mL of 10% BSA. The above 10% BSA was prepared by weighing 10 g of BSA (bovine serum albumin) (manufactured by Sigma-Aldrich) into a 100 mL tube, and then adding 100 mL of ultrapure water to the tube and mixing.

[0057] To the centrifuge tube described above, 10 mL of 0.5% BSA / T-Buffer was added again to suspend the cell pellet. The centrifuge tube was subjected to centrifugation (conditions: 500 × g, 5 minutes, room temperature), and the supernatant was removed.

[0058] A cell suspension was obtained by adding 1 mL of various cryoprotective solutions to the obtained cell pellet and resuspending the cells by pipetting. When CP-1 was used as the cryoprotective solution, a cell suspension was obtained by adding 0.5 mL of 0.5% BSA / T-Buffer and 0.5 mL of CP-1 preparation solution to the obtained cell pellet and resuspending the cells by pipetting.

[0059] The following cryoprotective solutions were used: (1) SOFORO Cryo (manufactured by SARAYA, containing sophorose lipid), (2) CellBanker 1 plus (manufactured by Xenogen Pharma, containing 10% DMSO), (3) Autologous plasma (the supernatant obtained by centrifuging blood at 2000 x g for 5 minutes at 4°C was used as autologous plasma), (4) 10% DMSO-containing autologous plasma (9 mL of autologous plasma and 1 mL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to make 10% DMSO-containing autologous plasma), (5) CP-5E (manufactured by Kyokuto Pharmaceutical Industries, Ltd., containing 5% DMSO, 6% hydroxyethyl starch, and 5% EG (ethylene glycol)), (6) CP-1 preparation solution (CP-1 High (7) A mixture of 6.8 mL of Grade (manufactured by Kyokuto Pharmaceutical Co., Ltd., containing 10% DMSO and hydroxyethyl starch) and 3.2 mL of autologous plasma on ice was used as the CP-1 preparation solution. (8) A mixture of Rhamnolipids (manufactured by Sigma-Aldrich, Grade 95%) with a final concentration of 0.1% (w / w) and glycerol with a final concentration of 30% (w / w). (9) A mixture of n-dodecyl-β-D-maltoside (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Grade 98%) with a final concentration of 0.1% (w / w) and glycerol with a final concentration of 30% (w / w). (9) A mixture of phosphate-1-phosphorate (Sigma-Aldrich, Grade 95%) with a final concentration of 0.1% (w / w) and glycerol with a final concentration of 30% (w / w).

[0060] The obtained cell suspension was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). This cryopreservation tube was then placed in a Coolcell container (manufactured by Biocision Co., Ltd.), and the Coolcell container was placed in a -80°C freezer. This allowed the cell suspension to freeze for more than 4 hours, and a frozen sample was prepared. The freezing speed at this time was approximately -1°C / min (slow freezing).

[0061] <1-3. Density Gradient Centrifugation Treatment> The blood sample prepared in <1-1. Preparation of Blood Sample> was added to a 15 mL centrifuge tube (manufactured by Sumitomo Bakelite Co., Ltd.), and 2% FBS-containing PBS (manufactured by STEMCELL Technologies) was added to the blood sample to dilute it.

[0062] Four mL of Lymphoprep (STEMCELL Technologies) was added to SepMate-15 (STEMCELL Technologies). The diluted blood sample was gently layered on top of the Lymphoprep in the SepMate-15. The inside of the 15 mL centrifuge tube that contained the blood sample was washed with 1 mL of 0.5% BSA / T-Buffer, and this 0.5% BSA / T-Buffer was also layered on top of the Lymphoprep.

[0063] After centrifuging the above SepMate-15 (conditions: 500 x g, 20 minutes, room temperature), it was confirmed that the red blood cells and buffy coat had separated. The buffy coat was collected using a pipette and added to a new 50 mL centrifuge tube. 10 mL of 0.5% BSA / T-Buffer was added to the centrifuge tube and mixed. The centrifuge tube was then centrifuged (conditions: 500 x g, 10 minutes, room temperature), and the supernatant was removed. 5 mL of 0.5% BSA / T-Buffer was added to the centrifuge tube after the supernatant was removed to suspend the cell pellet. The centrifuge tube was then centrifuged (conditions: 500 x g, 5 minutes, room temperature), and the supernatant was removed.

[0064] A cell suspension was obtained by adding 1 mL of various cryoprotective solutions to the obtained cell pellet and resuspending the cells by pipetting. When CP-1 was used as the cryoprotective solution, a cell suspension was obtained by adding 0.5 mL of 0.5% BSA / T-Buffer and 0.5 mL of CP-1 preparation solution to the obtained cell pellet and resuspending the cells by pipetting. The same cryoprotective solutions as those described in <1-2. Hemolysis Treatment> were used.

[0065] The obtained cell suspension was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). This cryopreservation tube was then placed in a Coolcell container (manufactured by Biocision Co., Ltd.), and the Coolcell container was placed in a -80°C freezer. This allowed the cell suspension to freeze for more than 4 hours, and a frozen sample was prepared. The freezing speed at this time was approximately -1°C / min (slow freezing).

[0066] <1-4. Thawing Treatment> The frozen samples obtained in <1-2. Hemolysis Treatment> and <1-3. Density Gradient Centrifugation Treatment> described above were thawed. Specifically, the cell suspension inside the cryopreservation tubes was thawed by immersing the cryopreservation tubes, which had been removed from a -80°C freezer, in a 37°C water bath.

[0067] The thawed cell suspension was added to 5 mL of 0.5% BSA / T-Buffer, and the mixture was subjected to centrifugation (conditions: 500 × g, 5 minutes, room temperature), after which the supernatant was removed.

[0068] The obtained cell pellet (thawed sample) was subjected to the procedure described later in <1-5. Measurement of CTCs>.

[0069] <1-5. Measurement of peripheral blood circulating tumor cells> True Nuclear 4× Fix Concentrate (manufactured by Biolegend) was added to the cell pellet obtained from the frozen sample in <1-2. Hemolysis> as described in <1-4. Thawing> above, and the cells were fixed.

[0070] After the above fixation treatment, True Nuclear 10× Perm (manufactured by Biolegend) was added and mixed, and the cells were subjected to permeabilization treatment.

[0071] The cells after the above permeabilization treatment were washed with 0.5% BSA / T-Buffer. Human TruStain FcX (Biolegend) was added to the washed cells and mixed, and the cells were subjected to blocking treatment. Anti-Cytokeratin (CK3-6H5)-FITC, human (Miltenyi Biotec) was added to the blocked cells and mixed, and the cells were subjected to fluorescent antibody staining treatment. The cells after the fluorescent antibody staining treatment were washed with 0.5% BSA / T-Buffer. The cells obtained after washing are called cell A.

[0072] On the other hand, in the cell pellet obtained from the frozen sample in the above-mentioned <1-3. Density gradient centrifugation treatment> in the <1-4. Thawing treatment> described above, 1×DNase I Reaction Buffer was added and mixed. The 1×DNase I Reaction Buffer was prepared by mixing 900 μL of ultrapure water with 100 μL of DNase I Reaction Buffer (manufactured by New England Biolabs).

[0073] To the resulting mixture, DNase I (RNase-free) (New England Biolabs) was added and mixed, and the cells were subjected to DNA degradation treatment. The cells after degradation treatment were washed with 0.5% BSA / T-Buffer.

[0074] The cells, after washing as described above, were mixed with CD326 (EpCAM) Antibody, anti-human, REAfinity (trademark) (manufactured by Milltenyi Biotec), and the cells were subjected to fluorescent antibody staining. The cells, after the fluorescent antibody staining treatment, were washed with 0.5% BSA / T-Buffer. The cells obtained after washing are referred to as cell B.

[0075] After the fluorescent antibody staining treatment described above, cells A and B were detected and collected using On-chip Sort (manufactured by On-chip Biotechnologies), and the number of peripheral blood circulating tumor cells contained in each cell was counted.

[0076] Regarding the identification of peripheral blood circulating tumor cells, first, a large number of cells that had been previously confirmed to be peripheral blood circulating tumor cells were subjected to On-chip Sorting, and the region containing the measurement data corresponding to these peripheral blood circulating tumor cells was set as a gate for identifying peripheral blood circulating tumor cells. Next, the sample to be measured was subjected to On-chip Sorting, and cells whose measurement data was contained within the range of the gate were identified as peripheral blood circulating tumor cells.

[0077] <1-6. Test Results> The test results are shown below. The table below shows the test results for seven blood samples (blood samples 1-7) collected on different days. In the table below, "spike count" refers to the total number of cells subjected to analysis by On-chip Sort, "detection count" refers to the number of cells determined to be peripheral blood circulating tumor cells in the analysis by On-chip Sort, "recovery rate" refers to the percentage shown by "(detection count / spike count) × 100", and "N.A." indicates that peripheral blood circulating tumor cells could not be isolated by the normal isolation method due to a large number of false positives.

[0078] As is clear from Tables 1 to 3, the combination of SOFORO Cryo with hemolysis treatment, and the combination of SOFORO Cryo with density gradient centrifugation treatment, consistently showed high recovery rates that never exceeded 100% in any of the multiple tests. This indicates that these combinations can reduce the number of false positives (the number of cells mistakenly identified as peripheral blood circulating tumor cells) and improve reproducibility (the reproducibility of the results of the identification of peripheral blood circulating tumor cells).

[0079] In combinations of CellBanker 1 plus with hemolysis, and with density gradient centrifugation, many tests showed excessively high recovery rates, resulting in a large number of false positives. This indicates that these combinations have problems in terms of reducing false positives (the number of cells mistakenly identified as peripheral blood circulating tumor cells) and reproducibility.

[0080] In combinations of CP-1 preparation solution and hemolysis treatment, the recovery rate exceeded 100% in all multiple tests, or peripheral blood circulating tumor cells could not be isolated due to a large number of false positives. This indicates that this combination is problematic, mainly in terms of the number of false positives.

[0081] The combination of CP-5E and hemolysis treatment resulted in high recovery rates, sometimes exceeding 100%, but also in cases where peripheral blood circulating tumor cells could not be isolated due to a large number of false positives. This indicates that this combination has problems in terms of reducing the number of false positives (the number of cells mistakenly identified as peripheral blood circulating tumor cells) and in terms of reproducibility.

[0082] The combination of autologous plasma and hemolysis resulted in a recovery rate exceeding 100%. This primarily indicates a problem in terms of the number of false positives.

[0083] The combination of autologous plasma containing 10% DMSO and hemolysis treatment resulted in a recovery rate exceeding 100%. This primarily indicates a problem in terms of the number of false positives.

[0084] <Test 2: Investigation of Freezing Timing> <2-1. Preparation of Blood Samples> After culturing lung adenocarcinoma-derived PC9 cells, the number of PC9 cells was counted under microscopic observation. The known number of PC9 cells was added to 4 mL of blood. In subsequent tests, the blood containing these PC9 cells was used as the blood sample.

[0085] <2-2. Hemolysis Treatment> 36 mL of ultrapure water (Merck) and 4 mL of RBC Lysis Buffer (10x) (Biolegend) were added to a 50 mL centrifuge tube (IWAKI) and mixed.

[0086] The blood sample prepared in <2-1. Preparation of Blood Sample> was added to the centrifuge tube described above, and after inverting the tube to mix, it was left to stand for 20 minutes (at room temperature).

[0087] The above centrifuge tubes were subjected to centrifugation (conditions: 500 x g, 30 minutes, room temperature), and the supernatant was removed. 10 mL of 0.5% BSA / T-Buffer was added to the centrifuge tubes after the supernatant was removed, and the cell pellet was suspended. The centrifuge tubes were then subjected to centrifugation (conditions: 500 x g, 5 minutes, room temperature), and the supernatant was removed.

[0088] The above 0.5% BSA / T-Buffer was prepared by mixing 95 mL of T-Buffer (manufactured by OnChip Biotechnology) with 5 mL of 10% BSA. The above 10% BSA was prepared by weighing 10 g of BSA (bovine serum albumin) (manufactured by Sigma-Aldrich) into a 100 mL tube, and then adding 100 mL of ultrapure water to the tube and mixing.

[0089] To the centrifuge tube described above, 10 mL of 0.5% BSA / T-Buffer was added again to suspend the cell pellet. The centrifuge tube was subjected to centrifugation (conditions: 500 × g, 5 minutes, room temperature), and the supernatant was removed.

[0090] A cell suspension was obtained by adding 1 mL of cryoprotection solution to the resulting cell pellet and resuspending the cells by pipetting.

[0091] The following cryoprotective solutions were used: (1) CellBanker 1 plus (manufactured by Xenogen Pharma, containing 10% DMSO); The results regarding the timing of freezing were not affected by the type of cryoprotective solution and are highly likely to also apply to SOFORO Cryo (manufactured by SARAYA, containing sophorolipid).

[0092] A portion of the obtained cell suspension was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). This cryopreservation tube was then placed inside a Coolcell container (manufactured by Biocision Co., Ltd.), and the Coolcell container was placed in a -80°C freezer. This froze the cell suspension over a period of more than 4 hours, preparing frozen sample A. The freezing speed at this time was approximately -1°C / min (slow freezing).

[0093] Separately from the slow freezing test described above, a portion of the cell suspension after hemolysis was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). This cryopreservation tube was placed in a -80°C freezer as is. This froze the cell suspension in a short time, and frozen sample B was prepared.

[0094] Specifically, after 10 minutes had elapsed since transferring the above-mentioned cryopreservation tube from room temperature (25°C) to a -80°C freezer, the cell suspension inside the tube was in a state where liquid and solid matter were mixed. After 15 minutes had elapsed since transferring the above-mentioned cryopreservation tube from room temperature (25°C) to a -80°C freezer, the cell suspension inside the tube was completely frozen. The freezing speed at this time was estimated to be "-4.0°C / min" using the formula "(-15°C (freezing point) - 25°C) / 10 min" (rapid freezing).

[0095] A portion of the resulting cell suspension was subjected to the fixation process described later in section 2-3.

[0096] <2-3. Fixation Treatment> 4× Fix Concentrate (manufactured by Biolegend) was added to the cell suspension obtained in <2-2. Hemolysis Treatment> described above, and the cells were fixed.

[0097] A portion of the cell suspension after fixation was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). The cryopreservation tube was then placed in a -80°C freezer. This allowed the cell suspension to freeze rapidly, preparing frozen sample C. The freezing speed at this time was estimated to be "-4.0°C / min" (rapid freezing), based on the same formula as described in <2-2. Hemolysis Treatment> above: "(-15°C (freezing point) - 25°C) / 10 min".

[0098] A portion of the cell suspension after fixation was subjected to the process described later in <2-4. Permeabilization>.

[0099] <2-4. Permeabilization Treatment> True Nuclear 10× Perm (manufactured by Biolegend) was added to the cell suspension after the fixation treatment described above and mixed, and permeabilization treatment was performed.

[0100] The cell suspension, after fixation and permeabilization, was injected into a 2.0 mL cryopreservation tube (manufactured by Sumitomo Bakelite Co., Ltd.). The cryopreservation tube was then placed in a -80°C freezer. This allowed the cell suspension to freeze rapidly, preparing frozen sample D. The freezing speed at this time was estimated to be "-4.0°C / min" (rapid freezing), based on the same formula as described in <2-2. Hemolysis Treatment> above: "(-15°C (freezing point) - 25°C) / 10 min".

[0101] <2-5. Thawing Process> Frozen samples A to D described above, and frozen sample E, which was rapidly frozen from the blood sample prepared in <2-1. Preparation of Blood Samples> described above, were stored in a -80°C freezer for 30 days and then thawed. Specifically, the cell suspension inside the cryopreservation tubes was thawed by immersing the cryopreservation tubes, which had been removed from the -80°C freezer, in a 37°C water bath.

[0102] The thawed cell suspension was added to 5 mL of 0.5% BSA / T-Buffer, and the mixture was subjected to centrifugation (conditions: 500 × g, 5 minutes, room temperature), after which the supernatant was removed.

[0103] The obtained cell pellet (thawed sample) was subjected to the measurement procedure described in <1-5. Measurement of peripheral blood circulating tumor cells> above. The explanation of this measurement procedure is omitted here.

[0104] <2-6. Test Results> The test results are shown in Figure 1. In Figure 1, "false positives" refers to the number of cells that were incorrectly identified as peripheral blood circulating tumor cells in the On-chip Sorting analysis, "CTC recovery rate" refers to the ratio shown by "(number of cells identified as peripheral blood circulating tumor cells in the On-chip Sorting analysis) / (total number of cells subjected to On-chip Sorting analysis) × 100", and "NA" indicates that the test results cannot be shown numerically due to a large number of false positives.

[0105] As is clear from Figure 1, frozen samples A to D all had fewer false positives and higher CTC recovery rates compared to frozen sample E, and none of them exceeded 100%.

[0106] Comparing frozen samples B through D, frozen sample B was considered the best in that it had a low number of false positives, a high CTC recovery rate, and never exceeded 100%.

[0107] Comparing frozen sample A and frozen sample B, it became clear that slow freezing is superior to rapid freezing in terms of reducing the number of false positives.

[0108] This invention can be widely used in the medical and research fields, and more specifically, it can be used for the diagnosis of diseases.

Claims

1. A method for freezing a sample containing peripheral blood circulating tumor cells, comprising the step of freezing the sample containing peripheral blood circulating tumor cells in a cryoprotective solution containing complex lipids (excluding a cryoprotective solution containing plasma components).

2. The method for freezing a sample containing peripheral blood circulating tumor cells according to claim 1, wherein the sample is obtained by hemolyzing a component of whole blood that includes peripheral blood circulating tumor cells, which is at least a portion of the whole blood.

3. The method for freezing a sample containing peripheral blood circulating tumor cells according to claim 2, wherein the sample obtained by the hemolysis treatment has not undergone at least one treatment selected from the group consisting of fixation, permeabilization, and staining.

4. The method for freezing a sample containing peripheral blood circulating tumor cells according to claim 1, wherein the sample is obtained by density gradient centrifugation of a component of whole blood that includes peripheral blood circulating tumor cells, which is at least a portion of the whole blood.

5. A method for separating peripheral blood circulating tumor cells, comprising the steps of: thawing a frozen sample obtained by a method for freezing a sample containing peripheral blood circulating tumor cells according to any one of claims 1 to 4 to obtain a thawed sample; and separating the peripheral blood circulating tumor cells in the thawed sample.

6. A method for obtaining data for diagnosing a disease, comprising the step of analyzing peripheral blood circulating tumor cells isolated by the method for isolating peripheral blood circulating tumor cells described in claim 5.

7. A method for obtaining data for diagnosing the disease described in claim 6, wherein the disease is cancer.

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