CN111575239A - A method and device for enriching circulating tumor cells - Google Patents

Abstract

The invention relates to the field of molecular biology, in particular to a method and a device for enriching circulating tumor cells, wherein the method comprises the following steps: mixing and incubating the sample and immunomagnetic beads capable of being combined with white blood cells; adding erythrocyte lysate into the sample for incubation; diluting the incubated sample, and then sequentially carrying out magnetic separation and membrane filtration; the method combines the negative enrichment technology with the membrane filtration technology, integrates the advantages of negative enrichment and membrane filtration, ensures the high recovery rate of CTCs, ensures the purity and activity of CTCs, avoids the reduction of the recovery rate of CTCs and the reduction of cell damage activity caused by the steps of centrifugation, liquid transfer and the like, simultaneously uses the membrane filtration to intercept circulating tumor cells to remove other impurities and cells, retains the circulating tumor cells as much as possible, simplifies the process, can complete the separation of 5ml of whole blood within 2h, and realizes the capture of more than 90 percent of CTCs and the removal of more than 95 percent of white blood cells.

Description

A method and device for enriching circulating tumor cells

technical field

The invention relates to the field of molecular biology, in particular to a method for enriching circulating tumor cells and a device thereof.

Background technique

Circulating tumor cells were first discovered by Thomas Ashworth in 1869. With the development of repetitive detection technology, circulating tumor cells have received more and more attention. Circulating tumor cells (CTCs), a collective term for various types of tumor cells in the peripheral blood, are shed from the primary tumor tissue and released into the blood, and develop into new tumors in distant places to achieve metastasis. Therefore, CTCs are considered to be a necessary prerequisite for tumor metastasis, and can then be used as the core research objects for tumor disease diagnosis, prognosis evaluation, and personalized monoclonal antibody drug screening, and become a "liquid biopsy" of metastatic tumors. However, due to their extreme rarity and high heterogeneity, the isolation and detection of CTCs from whole blood presents great challenges. Therefore, the development of high-efficiency, high-recovery and high-purity CTC separation technology is the future direction of development.

Currently, the developed effective separation and detection techniques are mainly based on specific tumor markers and physical properties. In the context of CTCs capture, these methods are divided into "affinity-based strategies" and "label-free strategies" according to whether the separation is affinity-based or not. . Affinity-based capture depends on immunochemical interactions between antigen-antibodies, such as EpCAM and CD45 antigens expressed on the cell surface and their corresponding antibodies immobilized on magnetic beads or patterned structures, such as using positive (forward) selection by Target binding to tumor-associated biomarkers (EpCAM, CEA, EphB4, EGFR, PSA, etc.) to capture CTCs, or use negative (reverse) selection to deplete blood cells by targeting binding to CD45 not expressed on CTCs. The negative depletion method will remove "normal" cells, including red blood cells, white blood cells and platelets, and retain "abnormal cells" such as CTCs for further analysis, such as a circulating tumor cell negative enrichment method disclosed in Chinese patent document CN106635995B, using lymphocytes The density gradient centrifugation based on the cell separation tube pre-processes the blood sample, which simplifies the operation steps of leukocyte separation and shortens the enrichment time of circulating tumor cells. The whole operation can be completed within 2.5 hours. The protocol described above involves a typical negative enrichment method, which involves chemical lysis of red blood cells or gradient centrifugation to remove white blood cells, followed by immunohistochemical or molecular analysis to identify "abnormal cells". However, the aforementioned negative enrichment techniques involve multiple steps, such as RBC lysis and centrifugation, which will increase the risk of losing CTCs and adversely affect target cells.

So far, CellSearch with affinity-based strategies is the only FDA-approved platform for CTC detection in breast, prostate, and colorectal cancers, and has become the gold standard in the field of CTCs. In the CellSearch system, magnetic beads coated with anti-EpCAM positively select rare CTCs. However, recent studies have shown that EpCAM-based methods may significantly underestimate the number of CTCs, as CTCs undergoing epithelial-mesenchymal transition may downregulate EpCAM expression.

In contrast, label-free strategies, including physical methods such as centrifugal deflection, dielectrophoretic separation, and size-based filtration, enable separation of epithelial and mesenchymal phenotypes and are more suitable for analyzing tumor heterogeneity. However, the above methods still suffer from technical limitations, namely, it is often difficult to make trade-offs between capture efficiency, purity and cell viability, which are important criteria for basic and clinical research. For example, a major problem with cell size-based filtration techniques is that if the pore size is small enough to achieve high tumor cell capture efficiency, there is usually a high retention rate of non-tumor cells, ie, low purity.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is to overcome the defects of high recovery rate, high purity and high viability in the enrichment method of circulating tumor cells in the prior art, so as to provide a method for enriching circulating tumor cells and a method for enriching circulating tumor cells. The device can obtain circulating tumor cells with high recovery rate, high purity and high viability at the same time.

For this reason, the invention provides the following technical solutions:

The present invention provides a method for enriching circulating tumor cells, comprising:

Incubate the sample with immunomagnetic beads that can bind to leukocytes;

adding red blood cell lysate to the sample for incubation;

The incubated samples were diluted, followed by magnetic separation and membrane filtration in sequence.

Preferably, the sample is subjected to magnetic separation and membrane filtration in sequence at a flow rate of 1/12ml/min-1/4ml/min.

Preferably, the sample is diluted to 5-10 times the original volume.

Further, it also includes the step of washing the filter membrane with a buffer solution, and the washing volume is 10-15ml.

Further, after the membrane filtration, the step of staining and marking the enriched circulating tumor cells in the filter membrane is also included.

In the step of staining and marking, before staining and marking, a blocking solution is added to the filter membrane enriched in circulating tumor cells for blocking, and then the liquid is washed and drained.

In the step of staining and marking, during staining and marking, adding cell staining solution to the filter membrane enriched with circulating tumor cells, incubating in the dark, then washing and draining the liquid; the ratio of the volume of cell staining solution to the diameter of the filter membrane was 100-150:13 (μl:mm).

In the dyeing and labeling step, the dyeing and labeling are performed twice. For the first dyeing and labeling, cell staining solution A is used, and the cells are incubated at 4°C for 30-40 minutes in the dark; Incubate at 4°C for 10 minutes in the dark;

Preferably, the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33%-5.00%, preferably 3.85%; the volume percentage of EpCAM-PE is 0.67%-1.00% , preferably 0.77%;

Preferably, the cell staining solution B contains 1/15ug/ml DAPI.

Further, the filter membrane in the membrane filtration step is selected from polycarbonate membrane (PC membrane) or PVDF membrane (polyvinylidene fluoride membrane); the pore size of the filter membrane is 0.65 μm-8 μm. Preferably, it is 8 μm.

The erythrocyte lysate contains fixative components.

CD45 antibody is coupled to the immunomagnetic beads.

The present invention provides an enrichment device for circulating tumor cells, including:

The sample collector has a sample inlet at the top and a sample outlet at the bottom;

a separation column, located below the sample collector, comprising a top opening and a bottom opening, and the top opening can be sealedly connected with the sample outlet;

The filter is located below the separation column; the filter has a cavity, the top of the cavity is provided with an inlet, which can be sealedly connected with the bottom end of the separation column, and the bottom end of the cavity is provided with an outlet; A filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity.

Further, the filter membrane is selected from polycarbonate membrane or PVDF membrane; the pore size of the filter membrane is 0.65 μm-8 μm. Preferably, it is 8 μm.

Further, the diameter of the filter membrane is 13mm-25mm.

Further, the filter membrane divides the cavity into an upper chamber and a lower chamber, and the volume of the upper chamber is less than or equal to 300 μl.

Further, a magnetic device is provided outside the separation column. Preferably, the interior of the separation column is filled with micro-iron beads. Further preferably, the diameter of the micro-iron beads is 100 μm. Further preferably, the gap between two adjacent micro-iron beads is 200 μm. Further preferably, the micro-iron beads are micro-iron beads treated with a hydrophilic coating layer to ensure that they can be dissolved in the solution.

Further, the outlet at the bottom end of the cavity can be tightly connected with the pump.

The technical scheme of the present invention has the following advantages:

1. A method for enriching circulating tumor cells provided by the present invention, comprising: mixing and incubating a sample with immunomagnetic beads that can bind to leukocytes; adding erythrocyte lysate to the sample for incubation; The sample is diluted, followed by magnetic separation and membrane filtration in sequence; the above method combines negative enrichment technology with membrane filtration technology, and combines the advantages of negative enrichment and membrane filtration to ensure high recovery rate of CTCs while ensuring the purity of CTCs and viability, avoiding the reduction of CTCs recovery rate and cell damage and viability caused by steps such as centrifugation and liquid transfer. At the same time, membrane filtration is used to retain circulating tumor cells to remove other impurities and cells, and to retain circulating tumor cells as much as possible. The separation of 5ml of whole blood is completed within 2h, and the capture of more than 90% of CTCs and the removal of more than 95% of leukocytes can be achieved.

2. A method for enriching circulating tumor cells provided by the present invention, the sample is subjected to magnetic separation and membrane filtration at a flow rate of 1/12ml/min-1/4ml/min in turn. The flow rate of filtration is an important factor affecting the capture efficiency (ie, the recovery rate) of circulating tumor cells, and within the above-mentioned range, a higher capture efficiency can be obtained.

3. A method for enriching circulating tumor cells provided by the present invention, the sample is diluted to 5-10 times of the original volume, and it is found in the study that the dilution of the sample affects the capture efficiency (ie recovery rate) of circulating tumor cells. An important factor is that the dilution factor within the above range can obtain higher capture efficiency.

4. A method for enriching circulating tumor cells provided by the present invention further includes the step of washing the filter membrane with a buffer solution, and the washing volume is 10-15ml. In the study, it was found that when the sample flow rate was 0.25ml/min, the washing volume was controlled. At 10-15ml, higher capture efficiency can be obtained.

5. A method for enriching circulating tumor cells provided by the present invention, after membrane filtration, further includes the step of dyeing and labeling the circulating tumor cells enriched in the filter membrane, since the aforementioned steps are carried out directly on the filter membrane. Dyeing, high degree of automation, no need to transfer liquid and centrifugation, save steps and high efficiency.

6. In a method for enriching circulating tumor cells provided by the present invention, when staining and labeling, a cell staining solution is added to the filter membrane for enriching circulating tumor cells, incubated in the dark, and then washed and drained; the cell staining solution The ratio of volume to filter membrane diameter is 100-150:13 (μl:mm); by controlling the ratio of cell staining solution volume to filter membrane diameter, the resulting staining pattern has the advantages of low background interference and high specificity.

7. A method for enriching circulating tumor cells provided by the present invention, staining and marking twice, the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33%-5.00% , the volume percentage of EpCAM-PE is 0.67% to 1.00%; the cell staining solution B contains 1/15ug/ml DAPI; by selecting the above cell staining solutions A and B, the obtained staining map further has low background interference and high specificity advantage.

8. This embodiment provides a device for enriching circulating tumor cells, including: a sample collector, with a sample inlet at the top and a sample outlet at the bottom; a separation column, located below the sample collector, including an opening at the top and the bottom opening, the top opening can be sealed with the sample outlet; the filter is located below the separation column; the filter has a cavity, and the top of the cavity is provided with an inlet to be open to the bottom end of the separation column The bottom end of the cavity is provided with an outlet; a filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity. In the above-mentioned enrichment device for circulating tumor cells, the outlet is tightly connected to the peristaltic pump during use. After the sample collector collects the sample, the peristaltic pump is turned on to perform suction filtration. The sample flows through the separation column at a constant flow rate, and the separation column is placed in the magnetic device. The immunomagnetic beads in the sample are adsorbed on the inner side wall of the separation column to achieve the purpose of removing the cells bound to the immunomagnetic beads in the sample. The separated sample enters the filter at a constant flow rate and passes through the filter membrane , the circulating tumor cells in the sample are trapped on the filter, and the remaining foreign cells or impurities are removed. The above device combines the negative enrichment strategy with membrane filtration, and combines the advantages of negative enrichment and membrane filtration, taking into account the recovery rate, purity and cell viability, and obtains the effects of high recovery rate, high purity and high cell viability. The device can realize red blood cell lysis and magnetic separation to remove leukocytes directly in the device, realize the removal of non-circulating tumor cells in the sample, and avoid the defects of losing CTCs or cell damage during centrifugation and sample transfer. Membrane filtration retains circulating tumor cells to remove other impurities and cells, and retains circulating tumor cells as much as possible. The process is simplified, and the separation of 5ml of whole blood can be completed within 2 hours, achieving the capture of more than 90% of CTCs and the removal of more than 95% of white blood cells.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

FIG. 1 is a schematic structural diagram of an enrichment device for circulating tumor cells in Example 1 of the present invention;

2 is a schematic diagram of the principle of enriching cells by the enrichment device for circulating tumor cells in Example 1 of the present invention;

Fig. 3 is the fluorescence imaging results of different samples to be tested in the experimental example of the present invention (scale bar: all are 50 μm);

Fig. 4 is the influence result of different dilution times on capture efficiency in the experimental example of the present invention;

Fig. 5 is the influence result of different flow velocity on capture efficiency in the experimental example of the present invention;

Fig. 6 is the influence result of different washing volumes on the capture efficiency and the number of leukocytes in the experimental example of the present invention;

Fig. 7 is the number of A549 cells recovered after enrichment under optimal conditions for blood samples containing different numbers of A549 cells in the experimental example of the present invention;

Fig. 8 shows the results of fluorescence imaging without magnetic separation and with magnetic separation in the experimental example of the present invention (scale bar: Figures (a) to (b) are all 100 μm);

Fig. 9 is the quantitative evaluation result of the leukocyte depletion effect of using different filters in the experimental example of the present invention;

FIG. 10 is a comprehensive evaluation result of the capture performance of circulating tumor cells under optimal conditions in the experimental example of the present invention.

Reference numerals: 1 - sample collector; 11 - sample inlet; 12 - sample outlet;

2-separation column; 21-top opening; 22-bottom opening; 23-micro iron beads;

3-filter; 31-cavity; 32-inlet; 33-outlet; 34-filter membrane; 35-rubber pad; 36-protrusion;

4-magnetic device; 5-peristaltic pump.

Detailed ways

The following examples are provided for a better understanding of the present invention, and are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by combining with the features of other prior art shall fall within the protection scope of the present invention.

If the specific experimental steps or conditions are not indicated in the examples, it can be carried out according to the operations or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.

The one-step lysis fixatives involved in the following examples were purchased from eBioscience ^™ 1-step Fix/LyseSolution (10X). JEG cells were purchased from Shanghai Guandao Bioengineering Co., Ltd.; CD45-labeled immunomagnetic beads (50nm in diameter) solution were selected from Miltenyi brand (Item No.: 130-045-801); Micro-iron beads treated with hydrophilic coating ( diameter 100 μm) was purchased from Miltenyi brand.

Example 1

This embodiment provides an enrichment device for circulating tumor cells, as shown in FIG. 1 , including:

The sample collector 1 is provided with a sample inlet 11 at the top and a sample outlet 12 at the bottom;

The separation column 2, located below the sample collector 1, includes a top opening 21 and a bottom opening 22, and the top opening 21 can be sealedly connected with the sample outlet 12;

The filter 3 is located below the separation column 2; the filter 3 has a cavity 31, the top of the cavity 31 is provided with an inlet 32 which can be sealed with the bottom opening 22 of the separation column 2, the cavity 31 The bottom end of the cavity is provided with an outlet 33 ; a filter membrane 34 is horizontally arranged in the cavity 31 , and the edge of the filter membrane 34 is tightly connected with the inner wall of the cavity 31 .

In the above-mentioned enrichment device for circulating tumor cells, as shown in FIG. 2 , the outlet 33 is tightly connected to the peristaltic pump during use. After the sample collector 1 collects the sample, the peristaltic pump is turned on to perform suction filtration, and the sample flows through at a constant flow rate. Separation column 2, the immunomagnetic beads in the sample are adsorbed on the inner side wall of the separation column 2 under the cooperation of the magnetic device to achieve the purpose of removing the cells in the sample bound to the immunomagnetic beads. The flow rate of 1000000000000000 enters the filter 3 and passes through the filter membrane 34, the circulating tumor cells in the sample are trapped on the filter membrane 34, and the remaining foreign cells or impurities are removed. The above device combines the negative enrichment strategy with membrane filtration, and combines the advantages of negative enrichment and membrane filtration, taking into account the recovery rate, purity and cell viability, and obtains the effects of high recovery rate, high purity and high cell viability. The device can realize red blood cell lysis and magnetic separation to remove leukocytes directly in the device, realize the removal of non-circulating tumor cells in the sample, and avoid the defects of losing CTCs or cell damage during centrifugation and sample transfer. Membrane filtration retains circulating tumor cells to remove other impurities and cells, and retains circulating tumor cells as much as possible. The process is simplified, and the separation of 5ml of whole blood can be completed within 2 hours, achieving the capture of more than 90% of CTCs and the removal of more than 95% of white blood cells.

Further, the filter membrane is selected from polycarbonate membrane or PVDF membrane; the pore size of the filter membrane is 0.65 μm-8 μm, and in this embodiment, a polycarbonate membrane (PC membrane) with a pore size of 5 μm is selected.

Further, the diameter of the filter membrane 34 is 13mm-25mm, and in this embodiment, the diameter of the filter membrane is 13mm.

Further, the filter membrane 34 divides the cavity 31 into an upper chamber and a lower chamber. In this embodiment, the upper chamber and the lower chamber of the filter 3 are detachably connected, and a rubber pad 35 is provided on the inner side wall of the bottom of the upper chamber for pressing the filter membrane 34. The lower chamber A protrusion 36 is provided on the inner side wall of the top of the chamber for supporting the filter membrane, and the filter membrane 34 is tightly connected to the side wall of the filter 34 through the cooperation of the rubber pad 35 and the protrusion 36 .

Further, the volume of the upper chamber is less than or equal to 300 μl. In this embodiment, the volume of the upper chamber is 300 μl, which can perform immunofluorescence staining in a small volume.

Further, a magnetic device is provided on the outside of the separation column 2 . In this embodiment, N magnetic poles and S magnetic poles are respectively arranged on both sides of the outside of the separation column 2 .

Further, the interior of the separation column 2 is filled with micro-iron beads 23, the gap between two adjacent micro-iron beads 23 is 200 μm, the diameter of the micro-iron beads 23 is 100 μm, and the micro-iron beads 23 It is a micro-iron bead treated with a hydrophilic coating layer. The micro-iron bead 23 itself is non-magnetic, and the micro-iron bead 23 plays the role of expanding the magnetic field. , the different cells can be separated without too much immunomagnetic beads to bind leukocytes, and at the same time, CTCs can flow through for subsequent continuous operations while removing leukocytes with high efficiency.

Further, the outlet 33 at the bottom end of the cavity 31 is tightly connected with the peristaltic pump.

Example 2

This embodiment provides a method for enriching circulating tumor cells, comprising the following steps:

(1) Mix 5ml of fresh peripheral blood sample with 500μl of CD45-labeled immunomagnetic bead solution, and incubate at 4°C for 0.5h;

(2) adding 15ml of one-step lysis fixative to the peripheral blood sample incubated in step (1) and incubating for 15min;

(3) Add PBS buffer (pH 7.2) to the peripheral blood sample incubated in step (2) for dilution, dilute to 5 times the original volume (5ml) of the peripheral blood sample, and then load it into the sample of Example 1. The sample collector 1 in the enrichment device for circulating tumor cells, then the peristaltic pump is turned on to perform suction filtration, and the sample is subjected to magnetic separation and membrane filtration in sequence at a flow rate of 0.25 ml/min;

(4) When the sample liquid level in the device reaches the top of the separation column 2, add PBS buffer (pH7.2) as a cleaning solution, and the cleaning volume is 10-15ml, in this embodiment, 10ml is selected, and 0.25 The flow rate of ml/min flows through the filter membrane 34;

(5) After the liquid has been filtered, turn off the peristaltic pump, stain and label the circulating tumor cells enriched in the filter membrane, first remove the separation column 2, and add a blocking solution to the filter 3 to seal the For the circulating tumor cells enriched on the filter membrane 34, in this example, 300 μl of 1wt% BSA solution was selected as the blocking solution, the blocking time was 15 minutes, and then 1 ml of PBS solution was used to wash and drain the liquid;

(6) Turn off the peristaltic pump, add 100-150ul (130ul in this embodiment) of cell staining solution A to the filter membrane 34, and incubate at 4°C for 30-40 minutes in the dark (selected in this embodiment) 35 minutes), then wash with 1 ml of PBS solution and drain the liquid; the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33% to 5.00%, which is selected in this example. 3.85%, the volume percentage of EpCAM-PE is 0.67% to 1.00%, and 0.77% is selected in this embodiment;

(7) Turn off the peristaltic pump, add 100-150ul (130ul in this embodiment) of cell staining solution B to the filter membrane 34, incubate at 4°C for 10 minutes in the dark, then wash with 1ml of PBS solution and rinse Drain the liquid; the cell staining solution B contains 1/15ug/ml DAPI;

(8) Remove the filter membrane 34 for packaging, and then use a high-throughput multicolor fluorescence imaging system to perform fluorescence imaging and result analysis, and finally determine the number of circulating tumor cells.

Example 3

This embodiment provides a method for enriching circulating tumor cells, comprising the following steps:

(1) Mix 5ml of fresh peripheral blood sample with 500μl of CD45-labeled immunomagnetic bead solution, and incubate at 4°C for 0.5h;

(2) adding 15ml of one-step lysis fixative to the peripheral blood sample incubated in step (1) and incubating for 15min;

(3) Add PBS buffer (pH 7.2) to the peripheral blood sample incubated in step (2) for dilution, dilute to 10 times the original volume (5 ml) of the peripheral blood sample, and then load it into the sample of Example 1. The sample collector 1 in the enrichment device for circulating tumor cells, and then the peristaltic pump is turned on to perform suction filtration, and the sample is subjected to magnetic separation and membrane filtration in sequence at a flow rate of 1/12 ml/min;

(4) When the sample liquid level in the device reaches the top of the separation column 2, add PBS buffer (pH7.2) as a cleaning solution, and the cleaning volume is 10-15ml, in this embodiment, select 15ml, and continue to use 1 The flow rate of /12ml/min flows through the filter membrane 34;

(5) After the liquid has been filtered, turn off the peristaltic pump, stain and label the circulating tumor cells enriched in the filter membrane, first remove the separation column 2, and add a blocking solution to the filter 3 to seal the For the circulating tumor cells enriched on the filter membrane 34, in this example, 300 μl of 1wt% BSA solution was selected as the blocking solution, the blocking time was 15 minutes, and then 1 ml of PBS solution was used to wash and drain the liquid;

(6) Turn off the peristaltic pump, add 100-150 ul (100 ul in this embodiment) of cell staining solution A to the filter membrane 34, and incubate at 4°C in the dark for 30-40 minutes (in this embodiment, select 100 ul) 30 minutes), then wash with 1 ml of PBS solution and drain the liquid; the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33% to 5.00%, which is selected in this example. 3.33%, the volume percentage of EpCAM-PE is 0.67% to 1.00%, and 0.67% is selected in this embodiment;

(7) Turn off the peristaltic pump, add 100-150 ul (100 ul in this embodiment) of cell staining solution B to the filter membrane 34, incubate at 4° C. for 10 minutes in the dark, and then wash with 1 ml of PBS solution. Drain the liquid; the cell staining solution B contains 1/15ug/ml DAPI;

(8) Remove the filter membrane 34 for packaging, and then use a high-throughput multicolor fluorescence imaging system to perform fluorescence imaging and result analysis, and finally determine the number of circulating tumor cells.

Example 4

This embodiment provides a method for enriching circulating tumor cells, comprising the following steps:

(1) Mix 5ml of fresh peripheral blood sample with 500μl of CD45-labeled immunomagnetic bead solution, and incubate at 4°C for 0.5h;

(2) adding 15ml of one-step lysis fixative to the peripheral blood sample incubated in step (1) and incubating for 15min;

(3) Add PBS buffer (pH 7.2) to the peripheral blood sample incubated in step (2) for dilution, dilute to 8 times the original volume (5 ml) of the peripheral blood sample, and then load it into the sample of Example 1. The sample collector 1 in the enrichment device for circulating tumor cells, then the peristaltic pump is turned on to perform suction filtration, and the sample is subjected to magnetic separation and membrane filtration in sequence at a flow rate of 1/8ml/min;

(4) When the sample liquid level in the device reaches the top of the separation column 2, add PBS buffer (pH7.2) as a cleaning solution, and the cleaning volume is 10-15ml, in this embodiment, select 13ml, and continue to use 1 The flow rate of /8ml/min flows through the filter membrane 34;

(5) After the liquid has been filtered, turn off the peristaltic pump, stain and label the circulating tumor cells enriched in the filter membrane, first remove the separation column 2, and add a blocking solution to the filter 3 to seal the For the circulating tumor cells enriched on the filter membrane 34, in this example, 300 μl of 1wt% BSA solution was selected as the blocking solution, the blocking time was 15 minutes, and then 1 ml of PBS solution was used to wash and drain the liquid;

(6) Turn off the peristaltic pump, add 100-150ul (select 150ul in this embodiment) of cell staining solution A to the filter membrane 34, and incubate at 4°C for 30-40 minutes in the dark (selected in this embodiment) 40 minutes), then wash with 1 ml of PBS solution and drain the liquid; the cell staining solution A contains CD45-FITC and EpCAM-PE, and the volume percentage of CD45-FITC is 3.33% to 5.00%, which is selected in this example. 5%, the volume percentage of EpCAM-PE is 0.67% to 1.00%, and 1% is selected in this embodiment;

(7) Turn off the peristaltic pump, add 100-150ul (150ul in this embodiment) of cell staining solution B to the filter membrane 34, incubate at 4°C for 10 minutes in the dark, then wash with 1ml of PBS solution and rinse Drain the liquid; the cell staining solution B contains 1/15ug/ml DAPI;

(8) Remove the filter membrane 34 for packaging, and then use a high-throughput multicolor fluorescence imaging system to perform fluorescence imaging and result analysis, and finally determine the number of circulating tumor cells.

Experimental example

1. Fluorescence imaging and result analysis

Samples to be tested: A negative control: no cancer cells in peripheral blood; B: peripheral blood red mixed with A549 cells; C: peripheral blood red mixed with JEG cells.

The above-mentioned samples to be tested were respectively implemented according to Example 2. The results of fluorescence imaging are shown in Figure 3. CD45-FITC stains leukocytes, EpCAM-PE stains CTCs, and DAPI stains cell nuclei. From the above figure, the cycle of the present invention can be drawn. The tumor cell enrichment method is feasible, with clear staining and low background interference, and can accurately identify and count CTCs.

2. Different factors of capture efficiency and leukocyte contamination and sensitivity test for CTC under optimal conditions

2.1 Influence of dilution factor on capture efficiency

According to Example 2, in step (3), the dilution ratios were selected as 1, 3, 5, 7, and 10, respectively, and the rest of the steps were the same. The capture efficiency was investigated. Capture efficiency = the number of identified circulating tumor cells/in the original sample Total number of circulating tumor cells.

The results are shown in Fig. 4 (error bars represent the standard deviation of three independent experiments), with a dilution factor of 5-10 and a high capture efficiency.

2.2 Influence of flow rate on capture efficiency

According to Example 2, in step (3), the samples were subjected to magnetic separation and membrane filtration at the flow rates of 1/12, 1/8, 1/4, 1/2, and 1 ml/min, respectively, to investigate the capture efficiency. .

The results are shown in Figure 5 (error bars represent the standard deviation of three independent experiments), and the capture efficiency was high when the flow rate was between 1/12-1/4.

2.3 Influence of cleaning volume on capture efficiency

According to Example 2, in step (4), the washing volume was selected as 1, 5, 10, 15, and 20 ml, and the rest of the steps were the same, and the capture efficiency and the number of leukocytes were investigated.

The results are shown in Figure 6 (error bars represent the standard deviation of three independent experiments), when the wash volume was 10 ml, the capture efficiency was high and the number of leukocytes was low.

2.4 Sensitivity test to CTC under optimal conditions

1, 10, 100, and 200 A549 cells were mixed into 1ml of blood respectively, and the above blood samples were carried out according to Example 2. In step (3), the dilution factor was 5 times, and the samples were sequentially performed at a flow rate of 0.25ml/min. Perform magnetic separation and membrane filtration. In step (4), the washing volume is selected to be 10 ml, and the remaining steps are the same to determine the number of A549 cells.

The results are shown in Figure 7 (three parallel experiments were performed for each sample), and the sensitivity of the CTCs enrichment method of the present invention to detect CTCs can reach 1 cell/ml.

3. Qualitative and quantitative evaluation of leukocyte depletion effect and comprehensive evaluation of cell capture performance

3.1 Qualitative evaluation of leukocyte depletion effect

According to Example 2, magnetic separation is performed and magnetic separation is not performed, respectively, the filter membrane is a PVDF membrane of 0.65 μm, and the rest of the steps are the same.

The results are shown in Figure 8. In Figure 8, (a) is without magnetic separation of leukocytes, and (b) is magnetic separation of leukocytes. A large number of leukocytes are shown in (a), and only a small amount of leukocytes are shown in (b). , indicating that the method for enriching circulating tumor cells of the present invention can remove leukocytes in the sample, and it can be seen from the figure that the leukocyte removal rate reaches more than 95%.

3.2 Quantitative assessment of leukocyte depletion effect

According to Example 2, 0.65 μm PVDF film, 5 μm PVDF film, 5 μm PC film and 8 μm PC film were selected for the filter membrane, respectively, without magnetic separation of leukocytes and magnetic separation of leukocytes, and the remaining steps were the same.

The results are shown in Fig. 9. It is obtained that on the same type and size of membrane, the removal rate of leukocytes with magnetic separation of leukocytes is higher than that without magnetic separation of leukocytes.

3.3 Comprehensive evaluation of the capture performance of circulating tumor cells

Implemented according to Example 2, wherein in step (3), the dilution factor was 5 times, and the sample was subjected to magnetic separation and membrane filtration at a flow rate of 0.25ml/min in turn, and in step (4), the cleaning volume was selected 10ml; 5μm PVDF film, 5μm PC film and 8μm PC film were selected for the membrane respectively; the rest of the steps were the same.

The results are shown in Fig. 10. After magnetic separation and membrane filtration, the CTC capture rate of the 5 μm PVDF membrane can reach more than 90%.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (18)

1. A method for enriching circulating tumor cells, comprising: Incubate the sample with immunomagnetic beads that can bind to leukocytes; adding red blood cell lysate to the sample for incubation; The incubated samples were diluted, followed by magnetic separation and membrane filtration in sequence. 2. The enrichment method for circulating tumor cells according to claim 1, wherein, The samples were sequentially subjected to magnetic separation and membrane filtration at flow rates of 1/12ml/min-1/4ml/min. 3. The enrichment method for circulating tumor cells according to claim 1 or 2, wherein the sample is diluted to 5-10 times the original volume. 4 . The method for enriching circulating tumor cells according to claim 1 , further comprising the step of washing the filter membrane with a buffer solution, and the washing volume is 10-15 ml. 5 . 5 . The method for enriching circulating tumor cells according to claim 1 , wherein after the membrane filtration, the method further comprises the step of staining and labeling the circulating tumor cells enriched in the filter membrane. 6 . The method for enriching circulating tumor cells according to claim 5, characterized in that, in the step of staining and marking, before staining and marking, a blocking solution is added to the filter membrane for enriching circulating tumor cells for blocking, Then wash and drain the liquid. 7. The method for enriching circulating tumor cells according to claim 5 or 6, characterized in that, during staining and labeling, a cell staining solution is added to the filter membrane for enriching circulating tumor cells, incubated in the dark, and then washed and cleaned. Drain the liquid; the ratio of the volume of the cell staining solution to the diameter of the filter membrane is 100-150:13, and the proportional relationship is μl/mm. 8 . The enrichment method for circulating tumor cells according to claim 7 , wherein the method for enriching circulating tumor cells according to claim 7 is characterized in that the staining and marking are carried out twice, and cell staining solution A is used for the first staining and marking, and incubated at 4° C. for 30-40 minutes in the dark; Use cell staining solution B for secondary staining and incubate at 4°C for 10 minutes in the dark; Preferably, the cell staining solution A contains CD45-FITC and EpCAM-PE, the volume percentage of CD45-FITC is 3.33%-5.00%, and the volume percentage of EpCAM-PE is 0.67%-1.00%; Preferably, the cell staining solution B contains 1/15ug/ml DAPI. 9. The enrichment method for circulating tumor cells according to any one of claims 1-8, wherein, The filter membrane in the membrane filtration step is selected from polycarbonate membrane or PVDF membrane; the pore size of the filter membrane is 0.65 μm-8 μm. 10 . The method for enriching circulating tumor cells according to claim 1 , wherein the red blood cell lysate contains a fixative component. 11 . 11. The method for enriching circulating tumor cells according to any one of claims 1-10, wherein the immunomagnetic beads are coupled with CD45 antibody. 12. A device for enriching circulating tumor cells, comprising: The sample collector has a sample inlet at the top and a sample outlet at the bottom; a separation column, located below the sample collector, comprising a top opening and a bottom opening, and the top opening can be sealedly connected with the sample outlet; The filter is located below the separation column; the filter has a cavity, the top of the cavity is provided with an inlet, which can be sealedly connected with the bottom end of the separation column, and the bottom end of the cavity is provided with an outlet; A filter membrane is horizontally arranged in the cavity, and the edge of the filter membrane is tightly connected with the inner wall of the cavity. 13 . The enrichment device according to claim 12 , wherein the filter membrane is selected from polycarbonate membrane or PVDF membrane; the pore size of the filter membrane is 0.65 μm-8 μm. 14 . 14. The enrichment device according to claim 13, wherein the diameter of the filter membrane is 13mm-25mm. 15 . The enrichment device according to claim 12 , wherein the filter membrane divides the cavity into an upper chamber and a lower chamber, and the volume of the upper chamber is less than or equal to 300 μl. 16 . 16. The enrichment device according to any one of claims 12-14, wherein a magnetic device is provided outside the separation column. 17 . The enrichment device according to claim 12 , wherein the separation column is filled with micro-iron beads, and the gap between two adjacent micro-iron beads is 200 μm. 18 . , the diameter of the micro-iron beads is 100 μm. 18. The enrichment device according to any one of claims 12-14, wherein the outlet at the bottom end of the cavity can be tightly connected with a pump.

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