Magnetic genetically engineered cell as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a magnetic genetically engineered cell as well as a preparation method and application thereof. According to the invention, a magnetic nano material loaded with plasmids is efficiently transferred into an HEK293 cell or a derivative cell thereof through channel extrusion force and fluid acting force generated by a microfluidic technology, so that the magnetic genetically engineered cell is prepared, and the magnetic genetically engineered cell has strong magnetism and can express a specific antibody; and target cells (such as circulating tumor cells) can be specifically recognized, and high-purity separation can be realized. Compared with immunomagnetic nanoparticles, the immunomagnetic nanoparticles have lower nonspecificity, and when targeted target cells are circulating tumor cells, interference of a large number of white blood cells in peripheral blood can be effectively reduced, the purity of separated circulating tumor cells is improved, and good conditions are provided for downstream analysis of liquid biopsy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a magnetic genetically engineered cell and a preparation method and application thereof. Background Art

[0002] Cancer is a major public health problem, and metastasis of primary tumors accounts for nearly 90% of the high cancer-related mortality. Currently, the gold standard for cancer diagnosis is tissue biopsy, which has the disadvantages of difficult sampling, great pain for patients, and unsuitability for repeated sampling. Liquid biopsy is a non-invasive diagnostic technology that determines the progression of the disease by analyzing biomarkers in body fluids. During the progression of cancer, tumor cells can be separated from the extracellular matrix of the primary tumor and enter the circulatory system to become circulating tumor cells. Therefore, circulating tumor cells can be used as biomarkers for liquid biopsy, thereby diagnosing cancer in patients at an early stage and monitoring the efficiency of treatment in real time.

[0003] At present, the main technologies for separating circulating tumor cells from whole blood include density gradient centrifugation, cell filtration technology, cell adhesion technology, immunomagnetic bead separation technology, and microfluidic chip technology. Among them, immunomagnetic bead separation technology has attracted widespread attention due to its easy operation, high capture performance and rapid separation. However, there are only a small number of circulating tumor cells in the blood, but there are one billion white blood cells. The nonspecific adsorption of immunomagnetic beads to proteins, lipids and other substances will cause other biomarkers, blood cells, etc. to be adsorbed during the detection process, reducing the detection sensitivity and causing false negative results, which is not conducive to downstream analysis. For example, although it is a Johnson & Johnson approved by the U.S. Food and Drug Administration For CTC detection systems, the leukocyte background is still high, resulting in a purity of less than 0.5%, and the presence of leukocytes interferes with downstream analysis.

[0004] Therefore, there is an urgent need to develop new methods that can specifically bind to target cells (such as circulating tumor cells) while reducing nonspecific binding to other non-target cells (leukocytes). Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the immunomagnetic nanomaterials in the prior art, such as high non-specificity, low sensitivity of target cell (such as circulating tumor cell) detection results, high non-target cell (such as leukocyte) background and false negatives, and to provide a method for preparing magnetic genetically engineered cells.

[0006] The purpose of the present invention is to provide magnetic genetically engineered cells obtained by the preparation method.

[0007] Another object of the present invention is to provide applications of the magnetic genetically engineered cells.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing magnetic genetically engineered cells, comprising the following steps:

[0010] S1. Preparation of magnetic nanomaterials loaded with plasmids;

[0011] S2. Introduce HEK293 cells or cells derived therefrom and the magnetic nanomaterial loaded with plasmid obtained in step S1 into the microfluidic chip to prepare cells that have taken up the magnetic nanomaterial loaded with plasmid, and the obtained cells are magnetic genetically engineered cells.

[0012] The present invention adopts microfluidic technology, and the squeezing force of the microchannel and the microfluidic force temporarily destroy the cell membrane to allow a large amount of magnetic nanomaterials loaded with plasmids to enter, so that the cells have strong magnetism that can carry themselves and other cells for magnetic migration. The magnetic genetically engineered cells prepared by the above method can specifically identify the target cells and drag them to separate, reducing the interference of background cells. Specifically, the surface of the magnetic genetically engineered cells loaded with exogenous plasmids can express specific antibodies, and the target protein has a high affinity, so that the interaction force between cells during the magnetic migration process is greater than the pulling force generated by the magnetic field, and the magnetic genetically engineered cells can be firmly combined with the target cells; in addition, the microfluidic technology reduces the magnetic nanomaterials on the surface of the cell membrane, and most of the materials are wrapped in a complete natural biofilm. The complete natural biofilm can reduce the nonspecific adsorption with other substances in the sample and reduce the interference of background substances.

[0013] Furthermore, the HEK293 (human embryonic kidney cell 293) or its derivative cells include one or more of HEK-293, HEK-293T, HEK-293A, and HEK-293FT.

[0014] Preferably, the HEK293 or its derivative cells are HEK-293T.

[0015] Furthermore, the HEK-293T cell is a derivative of HEK293 cells, obtained by integrating the SV40 large T antigen gene into the genome of HEK293 cells.

[0016] Furthermore, the method for preparing magnetic genetically engineered cells using a microfluidic chip comprises the following steps: introducing a single cell suspension into the microfluidic chip, and simultaneously introducing a magnetic nanomaterial loaded with a plasmid, collecting cells flowing out of the microfluidic chip, and culturing until the plasmid is expressed, thereby obtaining magnetic genetically engineered cells;

[0017] The speed of introducing the single cell suspension is greater than the speed of introducing the magnetic nanomaterial loaded with plasmids, in order to maximize the amount of magnetic nanomaterials internalized by the cells.

[0018] Preferably, the speed of passing the single cell suspension is 8-12 μL / min.

[0019] Preferably, the speed of introducing the magnetic nanomaterial loaded with plasmids is 2.5-7.5 μL / min.

[0020] Furthermore, the culturing until the plasmid is expressed also includes post-processing, and the post-processing includes washing, incubation and resuspension. The subsequent sufficient washing further reduces the magnetic nanomaterials on the cell membrane surface, and most of the materials are wrapped in the complete natural biofilm, which can reduce non-specific adsorption with other substances in the sample.

[0021] Specifically, the post-treatment includes fully washing the cells after the culture is completed, adding a digestion solution for incubation, and resuspending the incubated cells to obtain magnetic genetically engineered cells.

[0022] Furthermore, the sufficient flushing is to use an appropriate amount of phosphate buffer to fully wash on a shaker for 3 to 5 times to fully remove the magnetic nanomaterials that have not been internalized.

[0023] Furthermore, the digestive juice is a digestive juice containing digestive enzymes, and the digestive enzymes are preferably pancreatic enzymes.

[0024] Furthermore, the incubation condition is 35-38° C. for 3-10 minutes.

[0025] Furthermore, a single cell suspension refers to cells that are individually dispersed in the suspension rather than aggregated into agglomerates or cell clusters.

[0026] Furthermore, the single cell suspension is formed as follows: the digested cells are added to a buffer solution and resuspended.

[0027] Furthermore, the digestion is to use digestive enzymes (preferably pancreatic enzymes) to decompose or weaken the connection between the cells and the culture substrate (such as the surface of a culture flask or a culture dish), so as to separate the cells from the culture substrate and form a single cell suspension.

[0028] Furthermore, the culture conditions are 35-38° C. and 3-7% CO 2 for 24-36 hours.

[0029] Furthermore, the cell density of the single cell suspension is (0.1-10)×10 5 cells / mL.

[0030] Specifically, the method for preparing magnetic genetically engineered cells using a microfluidic chip comprises the following steps: resuspending the digested cells in phosphate buffer to a density of 1×10 5 A single cell suspension of 10 cells is introduced into a microfluidic chip at a flow rate of 8 to 12 μL / min, and a magnetic nanomaterial loaded with a plasmid is introduced into the microfluidic chip at a flow rate of 2.5 to 7.5 μL / min. The treated cells are collected in a culture dish filled with culture medium and cultured at 37°C and 5% CO2 for 24 to 36 hours. The obtained cells are magnetic genetically engineered cells.

[0031] Furthermore, the microfluidic chip is a PDMS microfluidic chip.

[0032] As an optional embodiment, the PDMS microfluidic chip is prepared by the following steps: first, PDMS and a cross-linking agent are mixed evenly in proportion, and then poured into a microfluidic chip mold, and the microfluidic chip mold filled with PDMS is placed under vacuum or heating conditions for curing; after the curing is completed, the PDMS substrate is peeled off from the mold, and necessary injection holes and output holes are formed on it or on another substrate that cooperates with it; finally, sealing is achieved by plasma treatment and hot pressing bonding (so that the contact surface of the PDMS substrate and the other substrate is reversibly or irreversibly bonded), thereby forming a PDMS microfluidic chip.

[0033] The prepared PDMS microfluidic chip has a microchannel structure.

[0034] Furthermore, the mass ratio of the PDMS to the cross-linking agent is (8-12):1.

[0035] Furthermore, after the PDMS and the cross-linking agent are uniformly mixed in proportion, a bubble removal treatment may be performed, wherein the bubble removal method is to remove the bubbles by vacuuming at room temperature.

[0036] Furthermore, the heating condition is 80-120° C., 20-40 min.

[0037] Furthermore, the other substrate is preferably a glass sheet.

[0038] Furthermore, the conditions of the thermal compression bonding are 80-100° C. (preferably 80° C.) and 30-120 min (preferably 60 min).

[0039] Specifically, the PDMS microfluidic chip is prepared by the following steps: 20g of PDMS and 2g of cross-linking agent are mixed evenly, placed in a vacuum drying oven, and vacuumed for 30 minutes at room temperature to remove bubbles. 1mL of PDMS is poured into a microfluidic chip mold, and the PDMS chip is demolded after heating on a 100°C heating plate for 30 minutes. The obtained PDMS chip and a clean glass sheet are placed in a plasma surface treatment instrument for 1 minute, pressed and sealed, and the sealed chip is placed in an oven and heated at 80°C for 1 hour to complete the sealing, thereby obtaining a microfluidic chip with a microchannel structure.

[0040] Furthermore, the magnetic nanomaterial loaded with plasmids is prepared by the following steps: the magnetic nanomaterial and the plasmids are co-incubated to obtain the magnetic nanomaterial loaded with plasmids.

[0041] Furthermore, the mass ratio of the magnetic nanomaterial to the plasmid is (2-10):1.

[0042] Preferably, the mass ratio of the magnetic nanomaterial to the plasmid is (5-10):1, more preferably (6-10):1.

[0043] Further, the magnetic nanomaterial is ferroferric oxide nanoparticles modified by a positively charged polymer. Further, the positively charged polymer includes one or more of polyethyleneimine, polydimethylaminoethyl methacrylate, chitosan, and polypyridine polymers.

[0044] Preferably, the positively charged polymer is polyethyleneimine.

[0045] Furthermore, the positively charged polymer modified ferroferric oxide nanoparticles are prepared by the following steps: subjecting a solution containing at least ferrous ions to a coprecipitation reaction under alkaline conditions, the reaction being complete, adding a positively charged polymer solution, mixing, and then subjecting the solution to a solvothermal reaction at 130-140° C., and post-treating to obtain positively charged polymer modified ferroferric oxide nanoparticles.

[0046] Furthermore, the alkaline condition is provided by an alkaline reagent, and the alkaline reagent is selected from one or more of ammonia water, sodium hydroxide, and potassium hydroxide.

[0047] Furthermore, the ferrous ion is selected from ferrous chloride, ferrous sulfate, ferrous nitrate, ferrous acetate or hydrates of the above ferrous salts.

[0048] Furthermore, the product expressed by the plasmid specifically binds to the target cell through antibody-antigen interaction.

[0049] Furthermore, the product expressed by the plasmid is an anti-epithelial cell adhesion molecule single-chain antibody. When the target cell is a circulating tumor cell, since the plasmid can express the anti-epithelial cell adhesion molecule single-chain antibody, the resulting magnetic genetically engineered cell can successfully identify and bind to the circulating tumor cell expressing the epithelial cell adhesion molecule, and the target cell can be separated by dragging under the action of the magnetic field.

[0050] The present invention also protects the magnetic genetically engineered cells prepared by the preparation method.

[0051] The present invention also protects the use of the magnetic genetically engineered cells in the preparation of detection and / or sorting target cell products.

[0052] Furthermore, the target cells include circulating tumor cells.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention constructs a magnetic genetically engineered cell that can express specific antibodies and wrap magnetic nanomaterials. The channel squeezing force and fluid force generated by microfluidic technology increase the cell's internalization of magnetic nanomaterials while maintaining high cell activity, so that the cell has sufficient magnetism to drag the migration of target cells; the cell expresses antibodies through genetic engineering technology, retaining the high affinity of the antibody to the target cell; the magnetic nanomaterial is covered with a complete natural biomembrane to effectively reduce the high non-specific adsorption of the magnetic nanomaterial. Compared with the traditional immunomagnetic bead separation method, the magnetic genetically engineered cells of the present invention show better specific results and lower non-target cell background in target cell detection or sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a statistical graph of the particle size distribution of polyethyleneimine modified ferroferric oxide nanoparticles.

[0056] Figure 2 The figure shows the binding of magnetic nanoparticles and plasmids and the effect of magnetic separation.

[0057] Figure 3 This is a statistical chart of magnetic genetically engineered cell activity data.

[0058] Figure 4 Statistical diagram of data verifying the entry of magnetic nanomaterials into cells using Prussian blue staining.

[0059] Figure 5 Statistical graph of the data of magnetic genetically engineered cells expressing green fluorescent protein.

[0060] Figure 6Statistical graph showing the binding of wild-type cells (left) and magnetic genetically engineered cells (right) to epithelial cell adhesion molecules labeled with phycoerythrin.

[0061] Figure 7 This is a statistical diagram of the data for magnetic genetically engineered cell recognition and dragging of target cells for separation, where the left figure is a fluorescence microscopy image of the supernatant, and the right figure is a fluorescence microscopy image of the magnetic separation product.

[0062] Figure 8 This is a statistical graph showing the capture of trace tumor cells by magnetic genetically engineered cells.

[0063] Fig. 9 Fluorescence images of magnetically engineered cells (left) and cells captured by immunomagnetic nanoparticles (right).

[0064] Fig.10 Statistical graphs showing flow cytometry quantitative analysis of human breast cancer cells captured by magnetic genetically engineered cells (left) and immunomagnetic nanoparticles (right).

[0065] Fig.11 The data statistics of the magnetic separation rate (Figure A) and the transfection efficiency (Figure B) of the engineered cells obtained by microfluidic technology and conventional mixed incubation are shown. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0067] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0068] PDMS (polydimethylsiloxane) and the cross-linking agent are derived from Dow Corning PDMS curing agent, that is, Dow Corning PDMS curing agent contains PDMS and a cross-linking agent and is a two-component product.

[0069] The 293T cells used in the examples are HEK-293T cells.

[0070] The plasmid was constructed by Youbao Biotechnology and can express single-chain antibodies against epithelial cell adhesion molecules.

[0071] Example 1 Preparation of magnetic genetically engineered cells

[0072] (1) Preparation of magnetic nanomaterials loaded with plasmids

[0073] Dissolve 0.25g of ferrous chloride tetrahydrate in 12.75mL of water, add 1.25mL of ammonia water, and stir in the air for 10 minutes. Add 5mL of 0.01g / mL polyethyleneimine, mix well and pour into the reactor, and react for 3h at 134℃ and 2bar. After the reaction is completed, cool to room temperature, and the black precipitate collected by magnetic separation is polyethyleneimine-modified ferroferric oxide nanoparticles. Take 2-20μg of polyethyleneimine-modified ferroferric oxide nanoparticles (hereinafter referred to as magnetic nanoparticles) and 2μg of plasmid (capable of expressing anti-epithelial cell adhesion molecule single-chain antibody) and incubate at room temperature for 30min to obtain magnetic nanomaterials loaded with plasmids.

[0074] The particle size of the obtained magnetic nanomaterial was measured, and the structure can be found in Figure 1 The particle size of the obtained magnetic nanomaterial is 14.06±2.5nm. Figure 2 It can be seen that when the mass ratio of magnetic nanomaterials: plasmids is ≥5:1, the plasmid loading rate reaches 94% or above.

[0075] (2) Preparation of microfluidic chips

[0076] Take 20g PDMS (polydimethylsiloxane) and 2g cross-linking agent, mix them evenly, put them in a vacuum drying oven, and evacuate them at room temperature for 30 minutes to remove bubbles. Take 1mL of cross-linked PDMS and pour it into the microfluidic chip mold. Heat it on a 100℃ heating plate for 30 minutes and then demold it. Put the prepared PDMS chip and a clean glass sheet into a plasma surface treatment instrument for 1 minute and press to seal. Put the sealed chip into an oven and heat it at 80℃ for 1 hour to complete the sealing and obtain a microfluidic chip with a microchannel structure.

[0077] (3) Preparation of magnetic genetically engineered cells

[0078] 293T cells were cultured at 37°C and 5% CO2 in DMEM medium containing 10% serum and 1% double antibody. The cultured 293T cells were digested with trypsin and resuspended in phosphate buffer to a density of 1×10 5cells / ml to obtain a single cell suspension. Use a micro-control pump to pass the obtained single cell suspension into the microfluidic chip at a flow rate of 10μL / min, and at the same time, pass the magnetic nanomaterial loaded with plasmids into the microfluidic chip at a flow rate of 7.5μL / min. Each treatment lasts ten minutes. The treated cells flowing out of the microfluidic chip are collected in a culture dish filled with culture medium and cultured for 24-36 hours at 37°C and 5% CO2. After the culture is completed, add an appropriate amount of phosphate buffer and wash thoroughly on a shaker for 3-5 times to fully remove the magnetic nanomaterials that have not been internalized. After washing, add digestive fluid (containing trypsin), incubate at 37°C for 5 minutes, then blow off the cells with a pipette, resuspend them in phosphate buffer, and obtain magnetic genetically engineered cells.

[0079] The activity of the obtained magnetic genetically engineered cells was measured, and the results were as follows Figure 3 As shown, the obtained magnetic genetically engineered cells maintained a high activity of more than 93% within 48 hours.

[0080] Example 2 Functional verification of magnetic genetically engineered cells

[0081] (1) Magnetic verification of magnetic genetically engineered cells

[0082] By adding the Prussian blue staining kit to stain the magnetic genetically engineered cells, the iron-containing magnetic nanomaterials can be stained blue. Figure 4 It can be seen that there are blue magnetic nanomaterials distributed in the magnetic genetically engineered cells, indicating that the magnetic nanomaterials have been successfully introduced into the 293T cells.

[0083] Digest the magnetic genetically engineered cells, resuspend them in 1 mL of phosphate buffer, and count the total number. Place the cell suspension on a magnetic separation rack and let it stand for 2 minutes, remove the supernatant, add 1 mL of phosphate buffer to resuspend the magnetic separation product, and count. The cell magnetic separation efficiency is calculated to be 87.8% by cell magnetic separation efficiency = number of magnetically separated cells / total number of cells, which means that most of the magnetic genetically engineered cells are successfully separated, further indicating that the magnetic genetically engineered cells are successfully prepared, and the obtained magnetic genetically engineered cells have good magnetic response ability.

[0084] (2) Verification of exogenous gene expression in magnetic genetically engineered cells

[0085] The plasmid carrying the green fluorescent protein reporter gene was transferred into the cells, and the fluorescence results were as follows Figure 5 As shown, magnetic genetically engineered cells expressing green fluorescent protein were prepared, indicating that the exogenous plasmid can be successfully expressed in the obtained magnetic genetically engineered cells.

[0086] (3) Magnetic genetically engineered cell recognition and dragging of target cells for separation

[0087] Take 1×10 5 Magnetic genetically engineered cells and wild-type cells expressing single-chain antibodies against epithelial cell adhesion molecules were plated in confocal dishes and cultured for 16-24 hours. After the culture, rinsed three times with phosphate buffer, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and rinsed three times with phosphate buffer. The fixed cells were blocked at 37°C for 30 minutes with phosphate buffer containing 1% serum, and the blocking solution was discarded after the incubation. According to the instructions, epithelial cell adhesion molecules labeled with phycoerythrin were added to the magnetic genetically engineered cells and wild-type cells and incubated at 4°C for 16-18 hours, and rinsed three times with phosphate buffer after the incubation. Add 0.5mL 10μg / mL Hoechst 33342, stain at 37°C for 5 minutes, and rinsed three times with phosphate buffer after the staining. Finally, add 1mL phosphate buffer to the confocal dish to keep the cells moist. Observe the cells under a confocal microscope.

[0088] The results are as follows Figure 6 There is no red fluorescence on the surface of wild-type cells (left picture), indicating that they do not bind to epithelial cell adhesion molecules; there is red fluorescence on the surface of magnetic genetically engineered cells expressing anti-epithelial cell adhesion molecule single-chain antibodies (right picture), indicating that they bind to epithelial cell adhesion molecules and anti-epithelial cell adhesion molecule single-chain antibodies are successfully expressed.

[0089] Add 1mL 10μg / mL Hoechst 33342 to the magnetic genetically engineered cells in a 6cm dish and stain at 37℃ for 20min. After staining, rinse three times with phosphate buffer, digest the magnetic genetically engineered cells, and resuspend in normal saline. Add 0.5mL 2nM / mL red cell membrane dye to human breast cancer cells MCF-7 (target cells) in a 3cm dish and stain at 37℃ for 20min. After staining, rinse three times with phosphate buffer, digest the cultured human breast cancer cells MCF-7 (target cells), and resuspend in normal saline.

[0090] Add 1×10 5 magnetic genetically engineered cells and 5×10 3 Target cells were added, mixed thoroughly, and incubated at 37°C for 30 minutes. After the incubation, the centrifuge tube containing the cell mixture was placed on a magnetic separation rack and allowed to stand for 2 minutes. The supernatant was placed in a 12-well plate. 1 mL of phosphate buffer was added to the centrifuge tube to resuspend the magnetic separation product, and the resuspended solution was placed in a 12-well plate.

[0091] The results of fluorescence microscopy observations were as follows Figure 7As shown, no red fluorescence was observed in the supernatant (left), while significantly more red fluorescence was observed in the magnetic separation product (right). The red fluorescence is human breast cancer cells labeled with red cell membrane dyes. The presence of red fluorescence in the magnetic separation product in the right figure proves that the human breast cancer cells were successfully dragged and separated; the absence of red fluorescence in the supernatant in the left figure indicates that all human breast cancer cells were magnetically separated. This shows that the magnetic genetically engineered cells successfully recognized and bound to human breast cancer cells expressing epithelial cell adhesion molecules, and achieved the separation of target cells by dragging under the action of a magnetic field. After the anti-epithelial cell adhesion molecule single-chain antibody binds to the epithelial cell adhesion molecule on the cell surface, a red light label is generated, thereby verifying the effectiveness of the magnetic separation of magnetic genetically engineered cells.

[0092] Example 3 Application verification of magnetic genetically engineered cells

[0093] Preparation of immunomagnetic nanoparticles: 500 μg of streptavidin magnetic beads (Beaver Biotech) and 10 μg of biotin-modified anti-epithelial cell adhesion molecule antibody (BD Biosciences) were added to 500 μL PBS and incubated at 4 °C.

[0094] The immunomagnetic nanoparticles were obtained by incubating for 16-18 hours.

[0095] To verify the ability of magnetic genetically engineered cells to specifically identify and drag trace tumor cells, 10-100 human breast cancer MCF-7 cells were added to 1 mL of normal saline to simulate trace circulating tumor cells in the blood, and 1×10 5 The cells were identified and captured by magnetic genetically engineered cells. Among them, human breast cancer cells were labeled with a red cell membrane dye. The cell mixture was incubated at 37°C for 30 minutes. After the incubation, the cell mixture was separated by magnetic adsorption for 2 minutes and washed three times with 1 mL of phosphate buffer. Finally, the supernatant and the magnetic separation product were resuspended in 100 μL of phosphate buffer and placed in a 96-well plate for observation. The number of tumor cells in the supernatant and the magnetic separation product was calculated under a fluorescence microscope, and the cell capture efficiency was calculated as follows = number of magnetically separated cells / (number of supernatant cells + number of magnetically separated cells) × 100%.

[0096] The results are as follows Figure 8 As shown, the magnetic genetically engineered cells have good capture performance for trace tumor cells, with an average capture efficiency of 84.7%.

[0097] To verify that magnetic genetically engineered cells can specifically identify tumor cells and separate them from leukocytes, and the separation purity is higher than that of immunomagnetic nanoparticles. 1×10 5 Jurkat T lymphocyte leukemia cells simulated white blood cells in the blood, and 5×10 31×10 MCF-7 breast cancer cells were added to simulate circulating tumor cells in the blood. 5 100 μg of the above-prepared immunomagnetic nanoparticles were used for identification and capture. Among them, human breast cancer cells were labeled with red cell membrane dye, human T lymphocyte leukemia cells were labeled with calcein, and magnetic genetically engineered cells were labeled with Hoechst33342. The cell mixture was incubated at 37°C for 30 minutes. After the incubation, the cell mixture was separated by magnetic adsorption for 2 minutes and washed three times with 1 mL of phosphate buffer. The supernatant and the magnetic separation product were placed in a 12-well plate and observed under a fluorescence microscope.

[0098] The results are as follows Fig. 9 As shown, compared with immunomagnetic nanoparticles (right), magnetic genetically engineered cells (left) have fewer green interfering cells in the tumor cells captured and have higher purity.

[0099] Similarly, the magnetic separation product was resuspended in 200 μL phosphate buffer and passed into a flow cytometer for flow cytometric analysis. Fig.10 As shown, the magnetic genetically engineered cells (left) have a higher tumor cell capture purity of 91.9%, while the immunomagnetic nanoparticles (right) have a capture purity of 61.2%.

[0100] Example 4 Comparison of engineered cells obtained by microfluidics and conventional mixed incubation

[0101] The digested 293T cells or HUVEC cells were resuspended in culture medium to a density of 2 × 10 per ml. 4 1 mL of cell suspension was added to a 6-well plate and cultured at 37°C and 5% CO2 for 12-18 hours to allow the cells to adhere. Jurkat cells were resuspended in culture medium to 2 × 10 4 cells. 20 μg / mL of magnetic nanomaterials loaded with plasmids were added to the cells. The mixed incubation group was directly placed in an incubator for culture. The magnetic transfer group placed the cells with added magnetic nanomaterials in a magnetic field for incubation for 20 minutes, and then placed in an incubator for culture. The preparation method of the microfluidic chip transfer group was the same as the preparation of magnetic engineered cells in "Example 1".

[0102] The results of magnetic nanomaterial transfer are as follows Fig.11As shown in Figure A, compared with the mixed incubation and magnetic mixed incubation methods, the microfluidic chip transfer method can increase the magnetic nanomaterials obtained by the three cells, thereby enhancing the efficiency of their magnetic separation. For 293T cells, the magnetic separation rate of microfluidic chip transfer can be increased to 87%, compared with 50% and 42% for mixed incubation and magnetic mixed incubation methods. The excellent magnetic separation rate means that 293T cells can be more effectively separated from the mixed cell population, reducing the interference of non-target cells and improving the purity of target cell capture.

[0103] The results of magnetic nanomaterial transfection efficiency are as follows Fig.11 As shown in Figure B, compared with the mixed incubation method, the microfluidic chip transfer method can improve the transfection efficiency of the three types of cells by increasing the magnetic nanomaterials obtained. For 293T cells, the transfection efficiency of the microfluidic chip transfer can be increased to 20%, while the mixed incubation transfection efficiency is 8%. Higher transfection efficiency means that cells can effectively take up plasmid DNA, which is conducive to the prepared magnetic genetically engineered cells to fully express specific antibodies and enhance the ability to specifically recognize target cells.

[0104] The above results show that the magnetic genetically engineered cells prepared based on 293T cells have both excellent magnetic separation rate and transfection efficiency, and can specifically identify target cells and achieve high-purity separation. However, the magnetic genetically engineered cells prepared based on non-HEK293 cells using microfluidic technology cannot have both excellent magnetic separation rate and transfection efficiency.

[0105] It can be seen that the present invention efficiently transfers the magnetic nanomaterial loaded with plasmids into HEK293 cells or their derivative cells through the channel extrusion force and fluid force generated by microfluidic technology to obtain magnetic genetically engineered cells, which contain strong magnetism and can express specific antibodies, and can specifically identify target cells (such as circulating tumor cells) and achieve high-purity separation. Compared with immunomagnetic nanoparticles, the present invention has lower non-specificity. When the target cells targeted are circulating tumor cells, it can effectively reduce the interference of a large number of white blood cells in peripheral blood, improve the purity of separated circulating tumor cells, and provide good conditions for downstream analysis of liquid biopsy.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing magnetic genetically engineered cells, characterized in that: The steps include: S1. Preparation of magnetic nanomaterials loaded with plasmids; S2. HEK293 cells or cells derived therefrom and the magnetic nanomaterial loaded with plasmids obtained in step S1 are introduced into the microfluidic chip to prepare cells that take up the magnetic nanomaterial loaded with plasmids, and the obtained cells are magnetic genetically engineered cells.

2. The preparation method according to claim 1, characterized in that: The HEK293 cells or cells derived therefrom include one or more of HEK-293, HEK-293T, HEK-293A, and HEK-293FT.

3. The preparation method according to claim 1, characterized in that: The method for preparing magnetic genetically engineered cells using a microfluidic chip specifically comprises the following steps: introducing a single cell suspension into the microfluidic chip, and simultaneously introducing a magnetic nanomaterial loaded with a plasmid, collecting cells flowing out of the microfluidic chip, and culturing until the plasmid is expressed, thereby obtaining magnetic genetically engineered cells; The speed of introducing the single cell suspension is greater than the speed of introducing the magnetic nanomaterial loaded with the plasmid.

4. The preparation method according to claim 1, characterized in that: The magnetic nanomaterial loaded with plasmids is prepared by the following steps: the magnetic nanomaterial and the plasmids are co-incubated to obtain the magnetic nanomaterial loaded with plasmids.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the magnetic nanomaterial to the plasmid is (2-10):

1.

6. The preparation method according to claim 1, characterized in that: The magnetic nano material is ferroferric oxide nanoparticles modified by a positively charged polymer.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The product expressed by the plasmid specifically binds to the target cell through antibody-antigen interaction.

8. The preparation method according to claim 7, characterized in that: The product expressed by the plasmid is an anti-epithelial cell adhesion molecule single-chain antibody.

9. The magnetic genetically engineered cell prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the magnetic genetically engineered cells according to claim 9 in preparing products for detecting and / or sorting target cells.

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