Application of modified porous membranes in the combined process of isolation, enrichment and in situ culture of circulating tumor cells

Abstract

This invention belongs to the field of membrane separation and biomedical technology, and relates to the application of a modified porous membrane in the combined process of circulating tumor cell separation, enrichment, and in situ culture. First, a polydimethylsiloxane porous membrane is prepared using a vacuum filtration method. Then, carboxyl groups are introduced onto its surface through a dehydration condensation reaction, thereby improving the membrane's biocompatibility through carboxyl group modification. The porous separation membrane prepared by this invention combines the separation and culture of circulating tumor cells using membrane separation technology, achieving efficient and non-destructive separation and enrichment of circulating tumor cells from whole blood samples. This invention pre-removes red blood cells using a lysis buffer, and white blood cells of similar size can be distinguished by staining, thus improving the accuracy of detection. The separation and culture membrane has good biocompatibility and can be directly transferred to culture medium for observation, avoiding cell loss or inactivation during complex processing.

Description

Technical Field

[0001] This invention belongs to the field of membrane separation and biomedical technology, and relates to the application of a modified porous membrane in the combined process of separation, enrichment and in situ culture of circulating tumor cells. Background Technology

[0002] Cancer is one of the world's leading causes of death. Circulating tumor cells (CTCs), as a novel tumor biomarker, are of great significance for monitoring the disease, assessing prognosis, and predicting recurrence in cancer patients. They can also reflect the tumor burden level in real time, helping clinicians monitor disease progression and treatment. Furthermore, isolating and enriching CTCs from peripheral blood and culturing them in vitro to analyze their epigenetic characteristics can provide more information for clinical practice and research. This has significant clinical implications for personalized treatment and medication guidance, tumor diagnosis and staging, early screening, drug development, and drug resistance research. The isolation, enrichment, and observation of CTCs are of great importance for clinical medical research.

[0003] Currently, there are two main methods for the isolation and enrichment of circulating tumor cells (CTCs): First, physical separation methods based on cell characteristics such as size, density, and charge, including electrophoresis, gradient centrifugation, and microfluidics; second, biomarker methods based on detectable antibodies, including antigen-antibody labeling, immunomagnetic beads, and chromatography. These techniques suffer from low separation efficiency, inability to process large quantities of samples, and difficulty in releasing captured cells, leading to easy inactivation during subsequent culture. Membrane separation technology can process large quantities of samples efficiently and has wide applications in cell culture. However, there are currently no literature reports on the combined process of circulating tumor cell isolation, enrichment, and in situ culture, mainly because the influence of membrane properties on the viability of circulating tumor cells has not been effectively addressed. Summary of the Invention

[0004] This invention addresses the problems existing in traditional circulating tumor cell isolation, enrichment, and in situ culture by proposing a novel modified porous membrane for the combined process of circulating tumor cell isolation, enrichment, and in situ culture.

[0005] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0006] This invention proposes the application of modified porous membranes in the combined process of isolating, enriching, and culturing circulating tumor cells.

[0007] Preferably, the modified porous membrane is rich in carboxyl groups on its surface, and the modified porous membrane is a modified PDMS porous membrane with an average pore size of no more than 10.5 μm.

[0008] The above-mentioned method for preparing modified porous PDMS membranes involves first preparing polydimethylsiloxane (PDMS) porous membranes using a vacuum filtration method, and then introducing carboxyl groups onto the surface via a dehydration condensation reaction. Carboxyl group modification is then applied to improve the membrane's biocompatibility. The specific steps are as follows:

[0009] (1) Preparation of casting solution

[0010] The amino-terminated PDMS monomer (commercially available) was dissolved in n-heptane and stirred until completely dissolved. Then, a crosslinking agent and a catalyst were added, and the mixture was stirred until homogeneous before proceeding with the reaction to obtain the casting solution.

[0011] (2) Filter membrane formation

[0012] Using a large-pore membrane as a substrate, a casting solution is passed through the large-pore membrane substrate by vacuum filtration to obtain a porous PDMS membrane, which is then dried.

[0013] (3) Modification of porous PDMS membrane

[0014] A carboxyl-containing compound was dissolved in a mixed EDC / NHS solution in a commercially available MES buffer solution (Sigma-Aldrich) to obtain an impregnation solution. The porous PDMS membrane was then immersed in this solution and repeatedly rinsed several times with a PBS buffer solution to obtain the modified porous PDMS membrane. A typical PBS buffer solution concentration is 10 mM, and its preparation method is as follows: Weigh 8.5 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄, dissolve them in 800 ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to a final volume of 1 L.

[0015] Preferably, in step (1) the casting solution, the mass fraction of amino-terminated PDMS is 5-20%, the mass fraction of crosslinking agent is 0.5-2%, the catalyst is dilauryl dibutyltin, and the mass fraction of the catalyst is 0.05-0.2%; the crosslinking agent is any one of tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxysilane.

[0016] Preferably, the reaction time in step (1) is 0.5-5h and the reaction temperature is 25-60°C.

[0017] Preferably, in step (2), the macroporous membrane is sheet-like with a circular cross-section, and is made of at least one of nylon, polysulfone, polyethersulfone, polycarbonate, polyester, and polyethyleneimine; the macroporous membrane substrate has a diameter of 50 mm, a thickness of 250 μm, a pore size range of 20-40 micrometers, and the pores are square woven holes or circular holes, and good light transmittance can be observed with the naked eye; the vacuum degree of vacuum filtration is 0.09 MPa, and the volume of filtrate added at one time is 25 ml.

[0018] Preferably, in step (3), the carboxyl-containing compound is any one of carboxymethyl cellulose, sodium alginate, graphene oxide, and polyacrylamide, and the mass fraction of the carboxyl-containing compound in the impregnation solution is 0.5-2%; the concentration of EDC is 0.5-2 mg / ml; the concentration of NHS is 0.5-2 mg / ml; the impregnation time is 8-12 h, and deionized water and PBS buffer solution are used for alternating rinsing, each rinsing 3 times.

[0019] The specific operating steps are as follows: Place the prepared modified porous PDMS membrane in a separation device, ensuring the membrane divides the device into upper and lower layers. Add whole blood sample and erythrocyte lysis buffer (the volume is the same as usual, just enough to lyse the erythrocytes) to the upper layer and mix thoroughly. Then, apply negative pressure to the lower layer, allowing the whole blood sample to pass through the separation membrane. Circulating tumor cells are trapped on the membrane. While maintaining negative pressure, collect the residual waste liquid in the upper layer using a blood collection tube. The membrane containing the enriched circulating tumor cells is then directly removed and placed in culture medium for further in vitro culture; or it can be used directly for fluorescent chromosome statistical analysis. A typical erythrocyte lysis buffer composition is as follows: 8g ammonium chloride, 1g potassium carbonate, 3.7g disodium EDTA, 10ml HCl, and bring the volume to 100ml in a volumetric flask. The culture environment can be DMEM medium or RPMI-1640 medium containing 5-10% bovine serum, with a CO2 atmosphere at 37°C.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0021] 1. The porous separation membrane prepared by this invention combines the separation and culture process of circulating tumor cells through membrane separation technology, thereby achieving efficient and non-destructive separation and enrichment of circulating tumor cells in whole blood samples.

[0022] 2. In this invention, red blood cells are removed beforehand using a lysis buffer, and white blood cells of similar size can be distinguished by staining, thereby improving the accuracy of detection.

[0023] 3. This invention uses a separation membrane for separation, which allows cells to be released and restored spontaneously through gas-liquid interfacial tension after sample filtration, avoiding the need for additional reagents to damage cell activity.

[0024] 4. The separation and culture membrane prepared by this invention has good optical transparency. When a strip of paper with writing is placed under the membrane, the writing can be clearly seen. It can be directly used for optical observation, which improves the efficiency of analysis and detection and avoids sample contamination.

[0025] 5. The separation culture membrane prepared by this invention has good biocompatibility and can be directly transferred to the culture medium for culture and observation, avoiding cell loss or inactivation during complex processing. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the principle of carboxyl modification of PDMS separation membranes. Detailed Implementation

[0027] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0028] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0029] Example 1

[0030] This embodiment provides a method for preparing a PDMS separation membrane.

[0031] (1) Preparation of porous PDMS membrane: After cleaning and drying a 150ml conical flask, add 5g of amino-terminated PDMS and 50ml of n-heptane solution. Place the flask in a rotor, cover it to isolate it from air, and stir at 400r / min for 15min to disperse it evenly. Add 0.5g of tetraethyl orthosilicate crosslinking agent and 0.05g of dilauryl dibutyltin catalyst, and finally add 50ml of n-heptane solvent. Cover the flask again and stir for 0.5h under air-isolated conditions until it is evenly dispersed. Stop stirring and let the sealed conical flask stand at room temperature (25°C) for 5h for crosslinking reaction. Use the casting solution immediately. Measure 25ml of the casting solution and use a nylon membrane with an average pore size of 20μm as the substrate to prepare a porous PDMS membrane by vacuum filtration at 0.09MPa atmospheric pressure. Dry the prepared PDMS membrane in a 60°C oven for 3h.

[0032] (2) Hydrophilic modification of porous PDMS membrane: Take 50 ml of MES buffer solution, add 0.25 g of carboxymethyl cellulose, 0.025 g of EDC and 0.025 g of NHS, sonicate for 5 min and stir for 20 min to obtain a mixture, so that the mass fraction of carboxymethyl cellulose in the mixture is 0.5%, the concentration of EDC is 0.5 mg / ml and the concentration of NHS is 0.5 mg / ml. At room temperature, put the membrane prepared and dried in (1) into the mixture and react for 8 h. Rinse with 10-30 mL of deionized water and PBS buffer solution 3 times each to obtain a porous PDMS membrane with carboxyl groups on the surface. The PBS buffer solution concentration is 10mM. The specific preparation method is as follows: Weigh 8.5g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to make up to 1L.

[0033] Example 2

[0034] This embodiment provides a method for separating PDMS membranes.

[0035] (1) Preparation of porous PDMS membrane: After cleaning and drying a 150ml conical flask, add 10g of amino-terminated PDMS and 50ml of n-heptane solution. Place the flask in a rotor and cover it to isolate it from air. Stir at 400r / min for 15min to disperse it evenly. Add 0.5g of 3-aminopropyltrimethoxysilane crosslinking agent and 0.1g of dilauryl dibutyltin catalyst. Finally, add 50ml of n-heptane solvent. Cover the flask and stir for 0.5h under air-isolated conditions until it is evenly dispersed. Stop stirring and let the sealed conical flask stand at 40°C for 3h for crosslinking reaction. Use the casting solution immediately. Take 25ml of the casting solution and prepare a porous PDMS membrane by vacuum filtration at 0.09MPa atmospheric pressure using a polycarbonate membrane with an average pore size of 30μm as the substrate. Place the prepared PDMS membrane in a 60°C oven for drying for 3h.

[0036] (2) Hydrophilic modification of porous PDMS membrane: Take 50 ml of MES buffer solution, add 0.5 g of graphene oxide, 0.05 g of EDC and 0.05 g of NHS, sonicate for 5 min and stir for 20 min to obtain a mixture, so that the mass fraction of carboxymethyl cellulose in the mixture is 0.5%, the concentration of EDC is 1 mg / ml and the concentration of NHS is 1 mg / ml. At room temperature, put the membrane prepared and dried in (1) into the mixture and react for 10 h. Rinse with deionized water and PBS buffer solution 3 times each to obtain a porous PDMS membrane with carboxyl groups on the surface.

[0037] Example 3

[0038] This embodiment provides a method for separating PDMS membranes.

[0039] (1) Preparation of porous PDMS membrane: After cleaning and drying a 150ml conical flask, add 15g of amino-terminated PDMS and 50ml of n-heptane solution. Place the flask in a rotor and cover it to isolate it from air. Stir at 400r / min for 15min to disperse it evenly. Add 1.5g of 3-aminopropyltriethoxysilane crosslinking agent and 0.15g of dilauryl dibutyltin catalyst. Finally, add 50ml of n-heptane solvent. Cover the flask and stir for 0.5h under air-isolated conditions until it is evenly dispersed. Stop stirring and let the sealed conical flask stand at 50°C for 1.5h for crosslinking reaction. Use the casting solution immediately. Measure 25ml of the casting solution and prepare a porous PDMS membrane by vacuum filtration at 0.09MPa atmospheric pressure using a polyester membrane with an average pore size of 35μm as the substrate. Place the prepared PDMS membrane in a 60°C oven for drying for 3h.

[0040] (2) Hydrophilic modification of porous PDMS membrane: Take 50 ml of MES buffer solution, add 0.75 g sodium alginate, 0.075 g EDC and 0.075 g NHS, sonicate for 5 min and stir for 20 min to obtain a mixture, so that the mass fraction of carboxymethyl cellulose in the mixture is 1.5%, the concentration of EDC is 1.5 mg / ml and the concentration of NHS is 1.5 mg / ml. At room temperature, put the membrane prepared and dried in (1) into the mixture and react for 9 h. Rinse with deionized water and PBS buffer solution 3 times each to obtain a porous PDMS membrane with carboxyl groups on the surface.

[0041] Example 4

[0042] This embodiment provides a method for separating PDMS membranes.

[0043] (1) Preparation of porous PDMS membrane: After cleaning and drying a 150ml conical flask, add 20g of amino-terminated PDMS and 50ml of n-heptane solution. Place the flask in a rotor and cover it to isolate it from air. Stir at 400r / min for 15min to disperse it evenly. Add 2g of 3-aminopropyltriethoxysilane crosslinking agent and 0.2g of dilauryl dibutyltin catalyst. Finally, add 50ml of n-heptane solvent. Cover the flask and stir for 0.5h under air-isolated conditions until it is evenly dispersed. Stop stirring and let the sealed conical flask stand at 60°C for 0.5h for crosslinking reaction. Use the casting solution immediately. Take 25ml of the casting solution and prepare a porous PDMS membrane by vacuum filtration at 0.09MPa atmospheric pressure using a polycarbonate membrane with an average pore size of 40μm as the substrate. Place the prepared PDMS membrane in a 60°C oven and dry it for 3h.

[0044] (2) Hydrophilic modification of porous PDMS membrane: Take 50 ml of MES buffer solution, add 1 g of sodium alginate, 0.1 g of EDC and 0.1 g of NHS, sonicate for 5 min and stir for 20 min to obtain a mixture, so that the mass fraction of carboxymethyl cellulose in the mixture is 2%, the concentration of EDC is 2 mg / ml and the concentration of NHS is 2 mg / ml. At room temperature, put the membrane prepared and dried in (1) into the mixture and react for 12 h. Rinse with deionized water and PBS buffer solution 3 times each to obtain a porous PDMS membrane with carboxyl groups on the surface.

[0045] Unless otherwise specified in Examples 1-4, all operations are routine. Unless otherwise specified in Examples 2-4, they are consistent with Example 1.

[0046] Testing showed that the porous PDMS membranes with carboxyl-rich surfaces prepared in Examples 1-4 could effectively retain circulating tumor cells, with retention efficiencies all exceeding 96%, and samples could be filtered within 1.5 minutes. Furthermore, the tumor cell-enriched membrane sheets could be directly transferred to culture medium for in situ cell culture. In the culture medium, circulating tumor cells spontaneously recovered through gas-liquid interfacial tension release, with no significant change in cell viability, indicating that the modified porous PDMS membranes prepared in Examples 1-4 possess excellent circulating tumor cell separation capabilities and biocompatibility. The following tests, using the separation membrane obtained in Example 4 as an example, will conduct membrane performance verification and circulating tumor cell separation experiments. Unless otherwise specified, the procedures described in Example 1 or conventional chemical experimental operations will be followed in the following tests.

[0047] A. Contact angle and hydrophilicity test

[0048] The hydrophilicity of the PDMS separation membrane before and after carboxyl grafting was tested using a contact angle meter. The tests showed that the contact angle of the PDMS separation membrane after carboxyl grafting decreased from 143° to 62.8°, and the electronegativity changed from -13.2 eV to -18.7 eV. This indicates that the hydrophilicity and electronegativity of the modified membrane increased. The increased hydrophilicity facilitates the formation of a hydrated layer on the membrane surface, while the increased electronegativity helps the membrane repel negatively charged proteins, indicating that the modification treatment improved the biocompatibility of the separation membrane.

[0049] B. Circulating tumor cell isolation, enrichment, staining, and counting assay

[0050] The specific steps are as follows:

[0051] I. Isolation and enrichment of circulating tumor cells

[0052] Load the separation membrane into the separator. The separator itself is not particularly critical, as long as the upper and lower layers separated by the membrane are sealed. Add 5 ml of sample (1 ml peripheral blood sample and 4 ml PBS buffer solution containing circulating tumor cells) and 5 ml red blood cell lysis buffer to the upper layer of the separation membrane. Mix thoroughly using a pipette. Pass the sample through the separation membrane under vacuum. Circulating tumor cells will accumulate on the membrane. Use a blood collection tube to collect and remove the waste liquid. After separation, remove the separation membrane for further counting and analysis.

[0053] II. Circulating tumor cell counting analysis

[0054] Because leukocyte size and circulating tumor cell size may partially overlap, to improve counting accuracy, CD45-FITC staining agent was first added to the separation membrane and incubated at 4°C for 30 min, followed by incubation with Triton X-100 reagent for 10 min, and excess staining agent was removed with PBS; then DAPI staining agent was added and incubated at 37°C for 30 min, and cells were fixed with paraformaldehyde. The separation membrane was then transferred to a fluorescence microscope for observation. (CD45-FITC was used to label leukocytes, and DAPI was used to label circulating tumor cells).

[0055] The isolated cells were observed, counted, and their retention efficiency was calculated. The summary table is as follows.

[0056] Table 1. Statistical analysis of cell counts obtained from isolation

[0057]

[0058] As shown in Table 1, the present invention has a good enrichment effect on circulating tumor cells, with a retention efficiency of over 96%, and the sample can be filtered within 1.5 minutes, which greatly saves analysis time.

[0059] C. Experiment on the effect of membrane pore size on separation and enrichment efficiency

[0060] The cell line selected was Miapaca-2, and the number of cells was 100.

[0061] (1) Adjust the mass percentage of the casting solution to 5%, crosslink at room temperature for 5 hours, take 25 ml of the casting solution, filter, and carboxylate to prepare a porous PDMS membrane #1; (2) Adjust the mass percentage of the casting solution to 10%, crosslink at room temperature for 3.5 hours, take 25 ml of the casting solution, filter, and carboxylate under the same conditions to prepare a porous PDMS membrane #2; (3) Adjust the mass percentage of the casting solution to 15%, crosslink at room temperature for 3 hours, take 25 ml of the casting solution, filter, and carboxylate under the same conditions to prepare a porous PDMS membrane #3; (4) Adjust the mass percentage of the casting solution to 20%, crosslink at room temperature for 3 hours, take 25 ml of the casting solution, filter, and carboxylate under the same conditions to prepare a porous PDMS membrane #4; Test its pore size distribution and circulating tumor cell retention efficiency, and summarize in Table 2 below:

[0062] Table 2. Results of pore size distribution and circulating tumor cell retention efficiency test

[0063]

[0064] As shown in Table 2 above, when the membrane pore size is greater than about 10.5 μm, the retention efficiency is poor. When the membrane pore size is less than about 10.5 μm, the retention efficiency is not much different and can meet the enrichment requirements.

[0065] D. Experiments on the effect of membrane properties on cell viability

[0066] The prepared membranes have a pore size of 6.5 μm and different surface properties.

[0067] The uncarboxylated PDMS membrane was designated #1; during carboxylation treatment, 0.5 wt% carboxymethyl cellulose, 0.5 mg / ml EDC solution, and 0.5 mg / ml NHS solution were added, designated #2; during carboxylation treatment, 1.5 wt% carboxymethyl cellulose, 1.5 mg / ml EDC solution, and 1.5 mg / ml NHS solution were added, designated #3; the separation membranes prepared above were used for the separation and enrichment of circulating tumor cells, and the membranes containing circulating tumor cells were cultured in a 37°C carbon dioxide (5%) incubator for 12 h.

[0068] Cell suspension was seeded into 96-well plates (200 μL / well), and 50 μL of WST-1 reagent was added to each well for 2 h of further incubation. The absorbance change was measured at 450 nm using a microplate reader, and cell viability was calculated. The results are summarized in Table 3 below.

[0069] Table 3 Cell viability statistics

[0070]

[0071] According to the test results in Table 3, although the PDMS membrane has a certain degree of biocompatibility, its surface compatibility increases after carboxylation treatment, which is more conducive to cell culture.

[0072] E. Effect of membrane separation method on cell release efficiency (experiment)

[0073] The prepared membrane had a pore size of 6.5 μm. The sample solution to be separated was passed through the membrane until the membrane surface was completely dry under negative pressure. The membrane was then transferred to DMEM culture medium and allowed to stand for 5 min. The membrane was then removed and rinsed at least three times with PBS buffer. The membrane was treated with DAPI staining and CD45-FITC staining (as in Experiment B) and observed under a fluorescence microscope. The fluorescence results showed no fluorescent cells on the membrane surface, indicating that the cells on the membrane surface had been completely released into the culture medium.

[0074] The principle of complete release using membrane separation is as follows: During the separation and enrichment process, the cells are always immersed in the solution environment, and the downward negative pressure binds them to the membrane surface; when the liquid sample on the membrane surface is filtered out and the surface is dry, the cells remain on the membrane surface, but the binding force is weakened; when there is no negative pressure and the cells are transferred to the liquid culture medium, the cells are transferred from the membrane surface to the liquid culture medium under the action of the gas-liquid interface.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. Application of modified porous membranes in the combined process of isolating, enriching, and culturing circulating tumor cells; wherein the modified porous membrane is rich in carboxyl groups on its surface, and the modified porous membrane is a modified porous PDMS membrane with an average pore size not exceeding 10.5 μm. The preparation steps of the modified porous PDMS membrane are as follows: (1) Preparation of casting solution The amino-terminated PDMS monomer was dissolved in n-heptane, and after stirring to dissolve, a crosslinking agent and a catalyst were added. After stirring evenly, the reaction was carried out to obtain the casting solution. (2) Filter membrane formation Using a large-pore membrane as a substrate, a casting solution is passed through the large-pore membrane substrate by vacuum filtration to obtain a porous PDMS membrane, which is then dried. (3) Modification of porous PDMS membrane A carboxyl-containing compound was dissolved in a mixed EDC / NHS solution in a MES buffer solution to obtain an impregnation solution. A porous PDMS membrane was then placed in the impregnation solution for reaction. The membrane was then repeatedly rinsed several times with deionized water and PBS buffer solution to obtain a modified porous PDMS membrane. In step (1), the mass fraction of amino-terminated PDMS monomer in the casting solution is 5-20%, the mass fraction of crosslinking agent is 0.5-2%, the catalyst is dilauryl dibutyltin, and the mass fraction of the catalyst is 0.05-0.2%; the crosslinking agent is any one of tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxysilane; the reaction time in step (1) is 0.5-5h, and the reaction temperature is 25-60℃; In step (2), the macroporous membrane material is at least one of nylon, polysulfone, polyethersulfone, polycarbonate, polyester, and polyethyleneimine; the diameter of the macroporous membrane is 50 mm, and the pore size range is 20-40 micrometers. In step (3), the carboxyl-containing compound is any one of carboxymethyl cellulose, sodium alginate, graphene oxide, and polyacrylamide. The mass fraction of the carboxyl-containing compound in the impregnation solution is 0.5-2%; the concentration of EDC is 0.5-2 mg / ml; the concentration of NHS is 0.5-2 mg / ml; and the impregnation time is 8-12 h.

2. The application according to claim 1, characterized in that, Step (2) The vacuum degree of vacuum filtration is 0.09 MPa, and the volume of filtrate added in a single vacuum filtration is 25 ml.

3. The application according to claim 1, characterized in that, A modified porous PDMS membrane was placed in a separation device. Whole blood samples and erythrocyte lysis buffer were added to the upper part of the modified porous PDMS membrane and mixed thoroughly. Then, negative pressure was applied to the lower layer of the modified porous PDMS membrane for separation, allowing the whole blood sample to pass through the separation membrane while circulating tumor cells were retained on the membrane. After separation, the residual waste liquid on the upper layer was removed, and the modified porous PDMS membrane containing the enriched circulating tumor cells was removed and placed in culture medium for in situ in vitro culture. The erythrocyte lysis buffer consisted of 8g ammonium chloride, 1g potassium carbonate, 3.7g disodium ethylenediaminetetraacetate, 10ml HCl, and deionized water was added to bring the volume to 100ml. The culture medium used was DMEM medium or RPMI-1640 medium containing 5-10% bovine serum, and the culture environment was a CO2 atmosphere at 37°C.

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