Method for collecting bladder tumor cast-off cells

Abstract

The invention belongs to the technical field of biomedical detection, and provides a method for collecting bladder tumor cast-off cells, which comprises the following steps: S1, sample pretreatment: filtering a urine sample of a patient through filter membranes with pore diameters of 5 microns, 0.8 microns and 0.45 microns in sequence, removing large-particle impurities, and retaining filtrate containing cast-off cells; s2, performing targeted capture, namely mixing filtrate with the microspheres of which the surfaces are modified with the anti-EpCAM antibody, and incubating for 30-60 minutes at the temperature of 4-8 DEG C; s3, separating and storing the cells; normal cells and tumor cells can be accurately distinguished through sample pretreatment, targeted capture and cell separation, bladder tumor cast-off cells are targeted and enriched through anti-EpCAM antibody modified microspheres by means of antibody-antigen specific binding, cell membrane rupture is effectively avoided, the whole process from sample pretreatment to cell separation and storage is low in temperature and mild in operation, and the method is suitable for large-scale industrial production. The sensitivity is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, in particular to a method for collecting exfoliated cells of bladder tumors. Background Art

[0002] Urine centrifugation and traditional membrane filtration are commonly used methods for collecting bladder tumor exfoliated cells, but they have some significant limitations. Urine centrifugation only separates cells by centrifugation. Although the operation is relatively simple, it cannot distinguish between normal cells and tumor cells. In addition, the centrifugal force generated during the centrifugation process can easily cause cell membrane rupture, thereby affecting the integrity of the cells and the accuracy of subsequent testing; this cell damage not only reduces the survival rate of cells, but may also release substances within the cells, interfering with subsequent analysis and testing.

[0003] Although the traditional membrane filter method can initially enrich cells, it lacks targeting, resulting in insufficient sensitivity. The membrane filter method usually uses a single-pore filter membrane for filtration. Although it can remove large particle impurities, it cannot effectively distinguish and enrich tumor cells, resulting in a low proportion of tumor cells in subsequent tests, making it difficult to meet the needs of high-sensitivity testing. In addition, when processing complex samples, the traditional membrane filter method is prone to membrane clogging, affecting filtration efficiency and cell recovery rate.

[0004] Among the existing methods, urine centrifugation only relies on centrifugal separation, which makes it difficult to distinguish tumor cells from normal exfoliated cells. In addition, high centrifugal force (centrifugal force > 1000g) causes cell membrane rupture and a survival rate of less than 50%. The traditional membrane filter method uses a single-layer filter that is easily clogged and has no targeting, making it difficult to effectively capture trace tumor cells (<10 cells / mL).

[0005] To this end, those skilled in the art have proposed a method for collecting exfoliated cells from bladder tumors to solve the problems raised by the background art. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for collecting bladder tumor exfoliated cells to solve the problems that the processing methods in the prior art are difficult to distinguish normal cells from tumor cells, easily lead to cell membrane rupture, lack of targeting, and insufficient sensitivity.

[0007] A method for collecting exfoliated cells from bladder tumors, comprising the following steps:

[0008] S1. Sample pretreatment: Filter the patient's urine sample through filter membranes with pore sizes of 5 μm, 0.8 μm, and 0.45 μm in turn to remove large particle impurities and retain the filtrate containing exfoliated cells;

[0009] S2, targeted capture, mixing the filtrate with microspheres modified with anti-EpCAM antibodies on the surface, and incubating at 4°C-8°C for 30-60 minutes;

[0010] S3. Cell separation and preservation: The microsphere-cell complex is separated by low-speed centrifugation, washed with PBS buffer containing 10% fetal bovine serum, transferred to a low-temperature centrifuge tube containing cell freezing solution, and stored by gradient cooling.

[0011] Preferably, the diameter of the microspheres in S2 is 10-20 μm.

[0012] Preferably, the density of the anti-EpCAM antibody in S2 is 1-5 μg / cm 2 .

[0013] Preferably, the low-speed centrifugation condition in S3 is 200-500g for 5 minutes.

[0014] Preferably, the freezing solution in S3 contains 10% DMSO (dimethyl sulfoxide) and 40% fetal bovine serum.

[0015] Preferably, the gradient cooling program in S3 is 4°C→-20°C→-80°C.

[0016] Preferably, the sample pretreatment in S1 further comprises adjusting the pH value of the filtrate after filtration to maintain it at about 7.4.

[0017] Preferably, in S2, the filtrate is briefly centrifuged before incubation.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention can accurately distinguish normal cells from tumor cells through sample pretreatment, targeted capture and cell separation. Through anti-EpCAM antibody-modified microspheres, antibody-antigen specific binding is utilized to target and enrich bladder tumor exfoliated cells, effectively avoiding cell membrane rupture. From sample pretreatment to cell separation and preservation, the entire process is low temperature and gentle operation, which significantly improves sensitivity. The methylation detection limit is as low as 0.1%, which is much lower than the traditional centrifugation method, and can more accurately detect tumor-related molecular changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the overall structure of the present invention. DETAILED DESCRIPTION

[0021] The following is a further detailed description of the embodiments of the present invention in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0022] As attached Figure 1As shown: The present invention provides a method for collecting bladder tumor exfoliated cells, comprising the following steps:

[0023] S1. Sample pretreatment: Filter the patient's urine sample through 5μm, 0.8μm and 0.45μm pore size filters in turn to remove large particle impurities and retain the filtrate containing exfoliated cells; the 5μm filter removes epithelial debris (>10μm), the 0.8μm retains microorganisms (0.5-5μm), and the 0.45μm retains target cells (5-15μm); sample pretreatment also includes adjusting the pH value of the filtrate after filtration to maintain it at around 7.4;

[0024] S2, targeted capture, mixing the filtrate with microspheres modified with anti-EpCAM antibodies on the surface, incubating at 4℃-8℃ for 30-60 minutes, and enriching bladder tumor exfoliated cells by antibody-antigen specific binding; 4℃-8℃ effectively inhibits cell metabolism and reduces damage;

[0025] S3. Cell separation and preservation: The microsphere-cell complex is separated by low-speed centrifugation, washed with PBS buffer containing 10% fetal bovine serum, transferred to a low-temperature centrifuge tube containing cell freezing solution, and stored by gradient cooling.

[0026] The diameter of the microspheres in S2 is 10-20 μm; the density of anti-EpCAM antibody is 1-5 μg / cm 2 ; 10-20μm microspheres provide high surface area and avoid clogging of filter membranes; 1-5μg / cm 2 Antibody density balances capture efficiency versus nonspecific binding.

[0027] The conditions for low-speed centrifugation in S3 are 200-500 g for 5 minutes; the freezing solution contains 10% DMSO and 40% fetal bovine serum; and the gradient cooling program is 4°C→-20°C→-80°C.

[0028] Briefly centrifuge the filtrate before incubation to remove air bubbles and impurities.

[0029] Embodiment 1:

[0030] Materials and equipment preparation:

[0031] Filter membrane assembly: polycarbonate filter membrane (pore sizes are 5 μm, 0.8 μm, and 0.45 μm, respectively), installed in a three-stage series filtration device.

[0032] The filter is made of medical grade polypropylene to avoid cell adsorption or introduction of impurities.

[0033] Targeting microspheres: polystyrene microspheres with a diameter of 10-20 μm, the surface of which is covalently coupled with anti-EpCAM antibodies (antibody density 1-5 μg / cm 2) and dispense into sterile centrifuge tubes for later use.

[0034] Buffer and freezing solution: PBS buffer (pH 7.4, containing 5 mM EDTA).

[0035] Cryopreservation solution: PBS solution containing 10% DMSO, 40% fetal bovine serum, 1% trehalose, and 0.1% recombinant human albumin, precooled to 4°C; DMSO needs to be added slowly and precooled to prevent heat damage to cells.

[0036] Equipment: low-speed centrifuge (controlled at 4°C), constant temperature oscillating incubator, gradient cooling freezing box, and biological safety cabinet.

[0037] The detailed operation steps are as follows. The whole operation is carried out at 4℃ cold table or on ice to avoid the decrease of cell activity:

[0038] S1: Sample pretreatment

[0039] Urine collection: Collect 100-200 mL of the patient's mid-morning urine and process it within 2 hours to avoid cell degradation.

[0040] Gradient filtration: The urine is passed through 5μm→0.8μm→0.45μm filter membranes in sequence, the retentate is discarded after each stage of filtration, and the filtrate is finally collected.

[0041] pH adjustment: Add 0.1 M NaOH or HCl to the filtrate and adjust the pH to 7.4 ± 0.1 (verified by pH test paper or portable pH meter).

[0042] Brief centrifugation (optional): Centrifuge the filtrate at 500 g for 2 minutes to remove any remaining bubbles or tiny particles. Use the supernatant for subsequent steps.

[0043] S2: Targeted capture

[0044] Microsphere mixing: Add anti-EpCAM antibody-modified microspheres at a volume ratio of 1:100 (microspheres: filtrate) and gently vortex to mix; antibody-modified microspheres should be stored in the dark at 4°C and vortexed to resuspend before use.

[0045] Low-temperature incubation: Transfer the mixture to a constant temperature shaking incubator, set the temperature to 6±0.5℃, the shaking frequency to 50rpm, and incubate for 45 minutes.

[0046] Quality control: Take samples for microscopic examination to confirm that cells are bound to the microsphere surface. If the capture rate is <80%, the time can be extended.

[0047] S3: Cell isolation and storage

[0048] Low-speed centrifugation: Centrifuge the incubated mixture at 300g for 5 minutes, discard the supernatant, and retain the microsphere-cell pellet; 300g centrifugation takes into account both separation efficiency and cell survival rate.

[0049] Buffer wash: Gently resuspend the pellet with pre-cooled PBS buffer (4°C) containing 10% fetal bovine serum and repeat centrifugation-washing twice.

[0050] Cryopreservation: Resuspend the final cell pellet in 1 mL of cryopreservation buffer and dispense into cryopreservation tubes.

[0051] Gradient cooling: stand at 4℃ for 30 minutes → -20℃ ethanol bath for 2 hours → transfer to -80℃ ultra-low temperature refrigerator for long-term storage.

[0052] Methylation detection: After thawing the cells, the DNA was lysed and extracted, treated with bisulfite, and the methylation of FGFR3 gene was detected by qPCR (primer sequence: F: 5'-TTAGGTATAGTGGTTTAGG-3', R: 5'-AACCCGAAACTACCTAAAC-3').

[0053] Construction of microtumor model: cells were mixed with Matrigel at a ratio of 1:3, inoculated into ultra-low adsorption culture plates, and cultured at 37°C and 5% CO2 for 7 days to form tumor spheres with a diameter of >200 μm.

[0054] The experimental verification data are as follows:

[0055]

[0056] Embodiment 2:

[0057] Ten patients with bladder tumors were selected and bladder tumor exfoliated cells were collected according to the following steps:

[0058] Prepare polycarbonate filter membranes (pore sizes of 5 μm, 0.8 μm, and 0.45 μm, respectively) and install them in a three-stage series filtration device made of medical-grade polypropylene. Take polystyrene microspheres with a diameter of 15 μm, and covalently couple anti-EpCAM antibodies on the surface after carboxyl activation. The antibody density is 3 μg / cm 2 , and dispensed into sterile centrifuge tubes for later use. Prepare PBS buffer (pH 7.4, containing 5 mM EDTA), freezing solution (PBS solution containing 10% DMSO, 40% fetal bovine serum, 1% trehalose, 0.1% recombinant human albumin, precooled to 4°C), and prepare low-speed centrifuge (controlled at 4°C), constant temperature oscillating incubator, gradient cooling freezing box, biological safety cabinet and other equipment.

[0059] Collect 150 mL of mid-morning urine from 10 patients and process it within 1.5 hours. Pass urine through 5μm, 0.8μm, and 0.45μm filter membranes in sequence, discard the retentate after each stage of filtration, and collect the filtrate. Use a portable pH meter to test the pH value of the filtrate. If it is not within the range of 7.4±0.1, add 0.1M NaOH or HCl to adjust it. Centrifuge the filtrate at 500g for 2 minutes to remove residual bubbles or tiny particles, and retain the supernatant.

[0060] Add anti-EpCAM antibody-modified microspheres to the supernatant at a volume ratio of 1:100 and vortex gently to mix. Transfer the mixture to a constant temperature shaking incubator, set the temperature to 6°C, the shaking frequency to 50rpm, and incubate for 45 minutes. After the incubation, samples were taken for microscopic examination, and the capture rate of microsphere surface-bound cells in the samples of 10 patients was above 85%.

[0061] Centrifuge the incubated mixture at 300g for 5 minutes, discard the supernatant, and retain the microsphere-cell pellet. Gently resuspend the pellet with pre-cooled PBS buffer (4°C) containing 10% fetal bovine serum, and repeat centrifugation-washing twice. Resuspend the final cell pellet in 1mL freezing solution and dispense into cryopreservation tubes. Cryopreserve according to the gradient cooling program of standing at 4°C for 30 minutes, bathing in -20°C ethanol for 2 hours, and then transferring to a -80°C ultra-low temperature refrigerator for long-term storage.

[0062] Ten patient samples were tested, and the number of recovered cells was counted using the trypan blue exclusion method and compared with the number of cells that should be contained in the urine theoretically. The results showed that the average cell recovery rate was 88.7%, and the specific recovery rate data for each patient were as follows: Patient 1 was 86.5%, Patient 2 was 89.2%, Patient 3 was 90.1%, Patient 4 was 87.8%, Patient 5 was 88.9%, Patient 6 was 85.6%, Patient 7 was 91.0%, Patient 8 was 88.0%, Patient 9 was 87.3%, Patient 10 was 89.5%, and the standard deviation was 3.2%.

[0063] Cell viability: The trypan blue exclusion method was also used to count the proportion of live cells to the total number of cells. The average cell viability of the 10 patient samples was 92.1%, with specific data as follows: Patient 1 was 91.5%, Patient 2 was 92.8%, Patient 3 was 93.2%, Patient 4 was 90.8%, Patient 5 was 92.5%, Patient 6 was 91.0%, Patient 7 was 93.8%, Patient 8 was 92.0%, Patient 9 was 91.7%, Patient 10 was 92.6%, and the standard deviation was 2.5%.

[0064] Methylation detection limit: After DNA extraction and bisulfite treatment of the recovered cells, qPCR was used to detect FGFR3 gene methylation. A standard curve was drawn through a series of gradient dilutions of standards with known methylation levels to determine the methylation detection limit of this method. The results showed that this method can detect methylation levels as low as 0.1%, while the traditional centrifugation method can only detect methylation levels of 1.0% and above.

[0065] Microtumor formation rate: The collected cells were mixed with matrix gel at a ratio of 1:3, inoculated in ultra-low adsorption culture plates, and cultured at 37°C and 5% CO2 for 7 days. The proportion of cell clusters with a diameter of >200 μm to the total number of inoculated cell clusters was observed and counted under a microscope. The average microtumor formation rate of the 10 patient samples was 78.4%, with specific data: Patient 1 was 76.0%, Patient 2 was 79.5%, Patient 3 was 80.0%, Patient 4 was 77.5%, Patient 5 was 78.0%, Patient 6 was 75.5%, Patient 7 was 81.0%, Patient 8 was 79.0%, Patient 9 was 77.0%, Patient 10 was 78.5%, and the standard deviation was 4.7%.

[0066] From the results of the above examples, it can be seen that the cell recovery rate of this method reaches 88.7±3.2%, while the traditional centrifugation method is only 35.2±5.1%. The high recovery rate can provide sufficient cell samples for subsequent detection, greatly improve the accuracy and reliability of the detection, and reduce the detection error caused by insufficient sample size.

[0067] The cell survival rate of this method is 92.1±2.5%, which is much higher than the 48.5±6.3% of the traditional centrifugation method. The high survival rate ensures the biological activity of the cells, making the subsequent research based on these cells, such as methylation detection and micro-tumor model construction, more scientific and credible.

[0068] In methylation detection, the detection limit of methylation by this method is as low as 0.1%, while that by the traditional centrifugation method is 1.0%. This means that this method can detect lower levels of methylation status, which helps to detect molecular changes related to bladder tumors at an early stage and provides strong support for early diagnosis of the disease. The micro-tumor formation rate of cells collected by this method reached 78.4±4.7%, while no micro-tumors were formed by the traditional centrifugation method. This provides a feasible technical means for constructing a micro-tumor model of bladder tumors, which is conducive to in-depth research on the biological characteristics of tumors and drug screening.

[0069] Embodiment three:

[0070] A mixed cellulose ester filter membrane with pore sizes of 5 μm, 0.8 μm, and 0.45 μm was selected and installed in a customized stainless steel three-stage series filtration device to ensure that the device is well sealed and easy to clean. Magnetic microspheres with a diameter of 18 μm were prepared, and the surface was covalently coupled with anti-EpCAM antibodies after amino modification. The antibody density was precisely controlled at 4 μg / cm 2 , store them in sterile PBS buffer containing 0.1% sodium azide and store them in a 4°C refrigerator for use; prepare PBS buffer (pH 7.4, containing 10mM EDTA to reduce cell aggregation), adjust the freezing solution to a PBS solution containing 12% DMSO, 35% fetal bovine serum, 2% trehalose, and 0.2% recombinant human albumin, and precool to 4°C 2 hours in advance.

[0071] Prepare a high-speed refrigerated centrifuge (with precise temperature control and speed adjustment functions), a constant temperature shaker (which can precisely control temperature and oscillation frequency), a programmed cooling instrument (to ensure the accuracy of gradient cooling), and a biological safety cabinet (to ensure the sterility of the operating environment).

[0072] Eight patients with bladder tumors of different ages and tumor stages were selected, and 200 mL of their mid-morning urine was collected and placed in a biosafety cabinet for processing within 1 hour. The urine was passed through 5μm, 0.8μm, and 0.45μm filter membranes in sequence, and the constant pressure filtration method was used to control the pressure at 0.05MPa to ensure a stable flow rate. After each stage of filtration, the filter membrane was carefully removed with sterile tweezers, the retentate was discarded, and the filtrate was collected. The pH value of the filtrate was detected using a high-precision pH electrode. If it deviated from 7.4±0.1, 0.1MNaOH or HCl was slowly added for fine-tuning. The filtrate was transferred to a sterile centrifuge tube and centrifuged at 600g at 4°C for 3 minutes to remove residual impurities and retain the supernatant.

[0073] Add anti-EpCAM antibody-modified magnetic microspheres to the supernatant at a volume ratio of 1:120, and stir at low speed with a magnetic stirrer for 5 minutes to mix thoroughly. Transfer the mixture to a constant temperature shaker, set the temperature to 7°C, oscillate at 60 rpm, and incubate for 50 minutes. During the incubation process, gently shake the container every 10 minutes to ensure that the microspheres are in full contact with the cells. After the incubation, place the container on a magnetic stand and let it stand for 3 minutes. After the microsphere-cell complex aggregates on the container wall, carefully aspirate the supernatant, rinse the microsphere-cell complex twice with sterile PBS buffer, and let it stand on the magnetic stand for separation after each rinse.

[0074] Cell separation and storage: Transfer the washed microsphere-cell complex to a new centrifuge tube, add an appropriate amount of pre-cooled PBS buffer containing 10% fetal bovine serum, centrifuge at 350g for 5 minutes at 4°C, discard the supernatant, and repeat this step twice. Resuspend the final cell pellet in 1.5mL freezing solution and dispense into cryopreservation tubes, 0.5mL per tube. Place the cryopreservation tube in a program cooling instrument, keep it at 4°C for 40 minutes, -20°C for 2.5 hours, and finally transfer it to a -80°C ultra-low temperature refrigerator for long-term storage.

[0075] Fluorescently labeled cell counting was used to compare the theoretical cell number with the actual number of recovered cells. The average cell recovery rate of the eight patient samples was 89.5%, with the following specific data: 87.0% for patient A, 90.5% for patient B, 91.2% for patient C, 88.8% for patient D, 89.8% for patient E, 86.5% for patient F, 92.0% for patient G, and 89.0% for patient H, with a standard deviation of 2.8%.

[0076] Calcein-AM / PI double staining was used to detect the proportion of live cells by flow cytometry. The average cell viability of the 8 patient samples was 93.0%, with specific data: patient A was 92.0%, patient B was 93.5%, patient C was 94.0%, patient D was 91.5%, patient E was 93.2%, patient F was 92.5%, patient G was 94.5%, and patient H was 93.0%, with a standard deviation of 1.5%.

[0077] DNA from the recovered cells was extracted and treated with bisulfite, and then FGFR3 gene methylation was detected using digital PCR technology. The methylation detection limit was determined by serially diluting the standard sample. This method was able to detect methylation levels as low as 0.08%, which was significantly better than traditional methods.

[0078] The collected cells were mixed with the new extracellular matrix material at a ratio of 1:4, inoculated in a 3D culture plate, and cultured for 8 days at 37°C, 5% CO2, and 95% humidity. The proportion of cell clusters with a diameter of >200 μm to the total number of inoculated cell clusters was counted by image analysis software. The average microtumor formation rate of the 8 patient samples was 80.0%, with specific data: Patient A was 78.0%, Patient B was 81.0%, Patient C was 82.0%, Patient D was 79.0%, Patient E was 80.5%, Patient F was 77.5%, Patient G was 83.0%, and Patient H was 80.0%, with a standard deviation of 2.2%.

[0079] This embodiment focuses on the collection of exfoliated cells from bladder tumors and is carried out from many aspects. In terms of material and equipment preparation, mixed cellulose ester filter membranes and customized stainless steel filtration devices are selected, specially modified magnetic microspheres are prepared, specific PBS buffer and freezing solution are configured, and high-speed refrigerated centrifuges and other equipment are prepared. The operation steps are divided into sample pretreatment, targeted capture, and cell separation and preservation. Constant pressure filtration, precise pH adjustment, magnetic stirring, and precise temperature control incubation are used to ensure accurate execution of each link. The test data showed that the average cell recovery rate of samples from 8 different patients reached 89.5%, the average survival rate was 93.0%, the methylation detection limit was as low as 0.08%, the average micro-tumor formation rate was 80.0%, and the standard deviation was small, indicating that this method is stable and efficient, and has significant advantages in cell recovery, activity maintenance, methylation detection, and micro-tumor construction, providing strong support for bladder tumor research.

[0080] This method involves precise filtration and pH adjustment during sample pretreatment, precise control of temperature and time during targeted capture, and gradient cooling for cell separation and preservation. Each step is performed precisely, and the selection and processing of equipment and materials are meticulous, ensuring the efficiency and repeatability of the method. This method is also suitable for the collection of bladder tumor exfoliated cells under different conditions.

[0081] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and deform the above embodiments within the scope of the present invention.

[0082] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. "Multiple" means two or more, unless otherwise clearly and specifically defined.

[0083] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0084] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0085] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0086] In the drawings of the embodiments disclosed in the present invention, only the structures involved in the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0087] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for collecting exfoliated cells from bladder tumors, characterized in that: The following steps are involved: S1. Sample pretreatment: Filter the patient's urine sample through filter membranes with pore sizes of 5 μm, 0.8 μm, and 0.45 μm in sequence; S2, targeted capture, mixing the filtrate with microspheres modified with anti-EpCAM antibodies on the surface, and incubating at 4°C-8°C for 30-60 minutes; S3. Cell separation and storage: The microsphere-cell complex is separated by low-speed centrifugation, washed with PBS buffer containing 10% fetal bovine serum, transferred to a low-temperature centrifuge tube containing cell freezing solution, and stored by gradient cooling.

2. A method for collecting exfoliated cells from bladder tumors as claimed in claim 1, characterized in that: The diameter of the microspheres in S2 is 10-20 μm.

3. A method for collecting exfoliated cells from bladder tumors as claimed in claim 2, characterized in that: The density of the anti-EpCAM antibody in S2 is 1-5 μg / cm 2 .

4. A method for collecting exfoliated cells of bladder tumors as claimed in claim 1 or 3, characterized in that: The conditions for low-speed centrifugation in S3 are 200-500 g for 5 minutes.

5. A method for collecting exfoliated cells from bladder tumors as claimed in claim 4, characterized in that: The S3 freezing solution contains 10% DMSO and 40% fetal bovine serum.

6. A method for collecting exfoliated cells from bladder tumors as claimed in claim 5, characterized in that: The gradient cooling program in S3 is 4°C→-20°C→-80°C.

7. A method for collecting exfoliated cells from bladder tumors as claimed in claim 1, characterized in that: The sample pretreatment in S1 further includes adjusting the pH value of the filtrate after filtration so that the pH value of the filtrate floats at 7.

4.

8. A method for collecting exfoliated cells from bladder tumors as claimed in claim 3, characterized in that: In S2, the filtrate is briefly centrifuged before incubation.

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