CN108546681B - Cell resurfacing surface and uses thereof - Google Patents
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Abstract
The invention provides a cell re-etching surface and application thereof, and the cell re-etching surface is a micro-nano topological structure surface which can be used for high-purity capture of rare cells in blood and is complementary with a template cell structure, and a rare cell specific recognition antibody can be modified on the surface of the micro-nano topological structure surface, so that efficient specific capture of the rare cells can be realized under the synergistic effect of the rare cell specific recognition antibody and the template cell structure. The cell re-etching surface has the advantages of simple manufacturing process, low preparation cost, high capture efficiency on rare cells, excellent specific performance and broad spectrum capture of different rare cell types. Is particularly suitable for capturing and enriching rare cells in a patient body, particularly circulating tumor cells, and has important guiding functions on cancer prognosis, early diagnosis, curative effect evaluation, recurrence prediction, individualized treatment and the like.
Description
Technical Field
The invention belongs to the technical field of biological functional materials and analysis and detection, and particularly relates to a technology for forming a cell re-etching surface on the surface of a material and capturing rare cells in blood, in particular circulating tumor cells.
Background
The content of some cells in blood is very small but important for understanding diseases such as stem cells, circulating endothelial cells, residual diseased cells and circulating tumor cells. Accurate detection and analysis of these rare cells is critical to understanding the disease process and pathogenesis, especially circulating tumor cells are closely related to early diagnosis of cancer. Studies have shown that 90% of cancer deaths are caused by cancer metastases, and Circulating Tumor Cells (CTCs) refer to tumor cells that have been shed from solid tumor foci, undergo epithelial-to-mesenchymal transition (EMT), and then have flow properties into the peripheral blood. CTCs are closely related to cancer metastasis, so that the direct capture of CTCs from blood is a minimally invasive detection means, can deeply understand the effect of chemotherapy and can realize early diagnosis of cancer. In recent years, the CTCs capture technology is largely classified into a physical method and a biochemical method. The physical method is mainly to separate cancer cells by using the difference in size, density and the like between the cancer cells and normal blood cells. The biochemical method is based on the principle of immune recognition separation, and at present, mainly takes an epithelial cell adhesion molecule antibody (Anti-EpCAM) or aptamer as a recognition molecule, and magnetic nanoparticles or a microfluidic chip as a carrier to realize the capture of CTCs. Although these methods all have a certain CTCs capturing effect, they have various disadvantages. Documents Small 2015,11,3850, angelw. chem. int.ed.2016,55,1252, chem. soc.rev.,2017,46,4245, have reviewed methods and techniques for CTCs capture in recent years, and have also raised some existing problems. The separation purity is low by a physical method; the magnetic bead method is complex to operate and has no broad spectrum; the microfluidic chip has high preparation cost, long cell enrichment time and the like. In addition, the CTCs capture techniques reported in chinese patents CN201610760765.4, CN201380075303.3, and CN201580022705.6 are also difficult to overcome the above problems.
The micro-nano structure has high specific surface area and biocompatibility, and the cell surface morphology is taken as a multi-scale, complex and natural micro-nano structure in different micro-nano topological structures, and the micro-nano structure has nanometer-scale filopodia and micrometer-scale surface microprotrusions. Therefore, we propose to select a high molecular material, prepare a cell re-etching surface with a micro-nano topological structure complementary with the template cell by a soft etching method, and apply the cell re-etching surface to capture rare cells in blood. At present, no report exists on the preparation method and the application of the cell replica surface.
Disclosure of Invention
The invention aims to provide a cell re-engraving surface which is simple, convenient and quick to operate, low in cost, high in capture efficiency and capture purity. The cell re-etching surface is provided with a micro-nano topological structure surface which is complementary with a template cell structure, and the template cell is a tissue cell with a pseudopodous structure.
For the cell replication surface in the above technical solution, preferably, the cell replication surface further comprises a rare cell specific recognition antibody for modifying the surface in addition to the micro-nano topological structure surface, wherein the rare cell specific recognition antibody is a specific recognition antibody for a rare cell to be captured, and the rare cell to be captured is selected from stem cells, circulating endothelial cells, residual diseased cells and circulating tumor cells.
The surface of the micro-nano topological structure is a surface of the micro-nano topological structure which is complementary with a template cell structure, wherein the template cell is a tissue cell with a pseudopodous structure. The cell re-etching surface has a micro-nano structure with abundant template cell filamentous pseudo-feet, an adhesion enhancing effect is generated through the topological structure of the micro-nano structure and the template cells, meanwhile, a rare cell specific recognition antibody is modified on the surface, the antibody specific recognition improves the capture rate of cells to be captured and reduces the adhesion of blood cells.
For the cell replication surface in the above technical solution, preferably, the rare cell specific recognition antibody to be captured is modified on the surface of the micro-nano topology structure by chemical coupling or biological affinity. The chemical coupling method or the biological affinity method is a conventional technology in the field of biotechnology.
For the cell re-etching surface in the above technical solution, preferably, the method for preparing the micro-nano topological structure surface includes the following steps: inoculating the template cells on a cell culture surface for proliferation culture (wherein the cell culture surface is a plane formed by a cell culture medium in general); fixing the template cells on a cell culture surface through paraformaldehyde, acetone, methanol or glutaraldehyde, dehydrating and shrinking the template cells, pouring a high-molecular prepolymer on the surface of the template cells, and stripping the high-molecular prepolymer after thermosetting, crosslinking and curing to obtain a micro-nano topological structure surface; the micro-nano topological structure surface is connected with an antibody specifically recognized by rare cells through a chemical coupling method or a biological affinity method to obtain a final product, namely a cell re-etching surface, and the micro-nano topological structure surface can be widely used for efficiently capturing target rare cells from blood.
For the cell replication surface described in the above technical scheme, preferably, the preparation method dehydrates and shrinks the template cell by dehydration of anhydrous ethanol, anhydrous crystalline salt, polyhydroxy compound and the like, so as to obtain a harder and rougher surface structure.
For the cell re-etching surface in the above technical scheme, preferably, the micro-nano topological structure surface is a micro-nano topological structure complementary to the template cell structure obtained by a soft-etching method or a cell imprinting method. The soft etching method comprises the steps of pouring a high-molecular prepolymer on the surface of a template cell, and stripping after thermosetting crosslinking curing to obtain a cell re-etching surface.
The template material in the conventional soft lithography technology is usually selected from a lithographically prepared artificial patterned template, and the reason for rarely using a natural biological surface (e.g. mammalian cells, microorganisms, etc.) is that: natural biological surfaces are not sufficiently hard and surfaces with rich water content do not easily acquire a fine structure complementary to the template material during the soft lithography process. In order to overcome the technical problems, the invention innovatively provides that the template cells are fixed on a cell culture dish or other similar cell culture surfaces through paraformaldehyde, acetone, methanol or glutaraldehyde, and are dehydrated through anhydrous ethanol, anhydrous crystal salt, polyhydroxy compounds and the like to shrink the cells, so that the hard and rough template cell surfaces are obtained, and thus the template cell re-etching surfaces can be prepared through soft etching of high molecular materials.
For the cell re-etching surface in the above technical solution, preferably, the material of the micro-nano topological structure surface is a polymer material, and is generally selected from polydimethylsilane, polypyrrole peroxide, and polyurethane.
For the above-described cell replica surface of the technical solution, preferably, the template cell is selected from the group consisting of cancer cell, tissue cell, leukocyte and phagocyte.
For the cell re-etching surface in the above technical scheme, preferably, the area occupied by the micro-nano topological structure complementary to the template cell is not less than 65% of the total area.
Another aspect of the present invention is the use of the protective cell replica surface described above for the efficient capture of rare cells in blood. The invention selects high molecular materials, prepares cell re-etching surfaces with different substrate appearances by a soft etching technology, and connects the antibody specifically recognized by the surface of rare cells to be captured (mainly cancer cells in the embodiment). The efficient specific capture of rare cells is realized under the coordination of the cell replication surface topology effect and the antibody specific recognition effect. And higher capture rate and capture purity are achieved in whole blood, so that the possibility is provided for later clinical application.
The experimental result shows that the cell repeated etching surface of the invention can realize higher capture rate for rare cells to be captured, especially, in the embodiment, taking cancer cells as an example, the capture rate can reach 73%, after modifying specific antibodies, the capture rate for antibody positive cancer cells can reach 90%, the capture rate for antibody negative cancer cells can reach 72%, and the invention has the cancer cell broad-spectrum capture capability. The adhesion and the specificity of the antibody modified cell re-etching surface to cancer cells are excellent, the capture efficiency of the trace CTCs artificial blood sample can reach 85%, and the capture purity can reach more than 90%.
Has the advantages that: the invention discloses a cell re-etching surface for capturing rare cells in blood, which has the advantages of simple preparation method and low preparation cost, shows high capture rate of the rare cells in a specific application example, has excellent specific performance, and also shows high capture rate and capture purity of the rare cells in whole blood. The invention is particularly suitable for capturing and enriching rare cells in a patient body, particularly circulating tumor cells, and has important guiding functions on cancer prognosis, early diagnosis, curative effect evaluation, recurrence prediction, individualized treatment and the like.
Drawings
FIG. 1 is a scanning electron micrograph of a cell replica surface prepared in example 1 of the present invention, with a scale bar of 1 μm.
FIG. 2 is a confocal microscope photograph of the replica surface and the planar surface of the cell of example 1. Wherein A is a three-dimensional topography picture of a planar surface; b is a three-dimensional appearance picture of the cell re-engraved surface.
FIG. 3 is a bar graph of the capture efficiency of the replica surface and the planar surface of the cells of example 1 of the present invention for cancer cells.
FIG. 4 is a bar graph of the capture efficiency of the replica surface of cells modified with the antibody of example 2 of the present invention against different cancer cell lines.
FIG. 5 is a plot of the capture efficiency and capture purity of artificial blood samples of different numbers of CTCs on the surface of cells after antibody modification in example 3 of the present invention. Wherein A is a dot plot of capture efficiency; b is a dot plot of capture purity.
FIG. 6 is a scanning electron micrograph of cancer cells and leukocytes captured on the surface of the modified cell of example 3, with a scale bar of 5 μm. Wherein A is a captured SEM of the cancer cell; b is the scanning electron micrograph of the white blood cells which are captured.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical companies.
Example 1
The template cell selected in this example was human breast cancer MCF-7 cell (guangzhou seiku biotechnology limited), and the soft lithography material was poly-dimethylsilane (PDMS); by taking the capture of cancer cells by the cell replica PDMS surface and the planar surface as an example, the capture system of the invention is further explained and verified, which comprises the following steps:
1) preparation of cell replica PDMS surface:
template cell MCF-7 cells were plated at 25000cells/cm2Is inoculated in culture at a density ofIn a dish, and placed in CO at 37 ℃2In the cell incubator, after 24 hours, fixing with a 4% paraformaldehyde solution for 20 minutes, dehydrating the fixed cells in absolute ethanol for 30 minutes, and drying to obtain a template cell surface. Then pouring the PDMS prepolymer on the surface of the template cell, thermally curing and crosslinking for 6 hours at 80 ℃, stripping to obtain a cell re-etched PDMS surface, and observing through a scanning electron microscope (figure 1), wherein the coverage rate of the cell on the re-etched PDMS surface is 90%.
In addition, the cell replica PDMS surface and the plane PDMS surface are respectively observed and photographed by a white light confocal microscope, the roughness of the cell replica PDMS surface is observed to be 12.3 μm from the three-dimensional morphology surface, and the roughness of the plane PDMS surface is only 0.04 μm.
2) Preparing a circulating tumor cell sample to be tested:
human breast cancer cells (MCF-7 cells) were cultured in DMEM cell culture medium. Taking 100 mu L of cell suspension, counting by using a cell counter and calculating the concentration of the cell suspension; aspirating a certain amount of the above cell suspension, diluting to 1X 10 with DMEM medium5Individual cells/mL.
3) Capturing circulating tumor cells in a sample to be tested:
placing the cell re-etched PDMS surface obtained in the step 1) into a cell culture plate, then dropwise adding 0.5mL of the uniformly mixed cell suspension obtained in the step 2) onto the PDMS surface, and then placing the PDMS surface in CO at 37 DEG C2In the cell incubator, the capture time was 60 minutes.
4) Evaluation of capturing effect:
after 60 minutes, the captured circulating tumor cells were fixed with 4% by mass aqueous paraformaldehyde solution for 20 minutes and washed three times with Phosphate Buffered Saline (PBS); then staining the cells with 10 mu g/mL fluorescent dye DAPI solution for 30 minutes; and finally, respectively photographing under 10 times of an Olympus inverted fluorescence microscope, randomly photographing 20 fluorescence photographs under different visual fields, counting the captured circulating tumor cells by using ImageJ software, and calculating the capturing efficiency.
The experimental result shows that the cell re-etching PDMS surface prepared by using the soft etching technology has low price and simple operation, and the micro-nano scale cell re-etching surface can be obtained (figure 1). Compared with a planar PDMS surface (figure 2A), the cell re-etching PDMS surface has a rougher structure (figure 2B), and the capture rate of cancer cells is improved from 15% to 73% of the planar surface (figure 3), which indicates that the cell re-etching PDMS surface prepared by the method has a rough micro-nano topological structure and can be used for capturing circulating tumor cells.
Example 2
In the embodiment, a PDMS surface is re-etched by selecting cells, and an anti-EpCAM specific antibody is connected to a substrate material by utilizing the interaction of biotin/biotin avidin; the capture system of the invention is further elaborated and verified by investigating the capture efficiency of the cell replica PDMS surface to different kinds of cancer cells, and comprises the following steps:
1) preparation of cell replica PDMS surface was the same as in example 1:
2) the bioaffinity method links anti-EpCAM specific antibodies:
the cell surface was re-engraved with uv ozone for 10 minutes to hydrophilize it, and then placed in a cell culture plate, and a 4% by mass n-octadecyl triethoxysilane solution (C18, diluted with ethanol) was added thereto, and after 45 minutes of reaction at room temperature, washed once with ethanol and water, and 3 times with Phosphate Buffered Saline (PBS). 0.5mg/mL biotinylated albumin (biotin-BSA) solution (PBS dilution) was added, and after soaking at 37 ℃ for 2 hours, PBS was washed 3 times. After adding 10. mu.g/mL avidin solution (PBS dilution) and soaking at 37 ℃ for 30 minutes, PBS was washed 3 times. Finally, biotinylated anti-EpCAM (PBS) containing 10 mu g/mL is added for dilution, the mixture is reacted at room temperature for 30 minutes and then washed for 3 times by PBS, and the cell replica PDMS surface modified by the anti-EpCAM specific antibody is obtained.
3) Preparing a circulating tumor cell sample to be tested:
human breast cancer cells (MCF-7 cells) and human cervical cancer cells (Hela cells) are cultured in a DMEM cell culture medium, and human lung cancer cells (A549 cells) and human liver cancer cells (HepG2 cells) are cultured in an RPMI-1640 cell culture medium. Taking 100 mu L of each cell suspension respectively, counting by using a cell counter and calculating the concentration of the cell suspension; aspirating a defined amount of the above cell suspension, diluting to 1X 10 with the respective cell culture medium5Individual cells/mL.
4) Capturing circulating tumor cells in a sample to be tested:
placing the cell re-etched PDMS surface modified with the anti-EpCAM specific antibody in the step 2) into a cell culture plate, then dropwise adding 0.5mL of each cell suspension uniformly mixed in the step 2) onto the PDMS surface, and then placing the PDMS surface in CO at 37 DEG C2In the cell incubator, the capture time was 60 minutes.
5) Evaluation of capturing effect:
after 60 minutes, the captured circulating tumor cells were fixed with 4% by mass aqueous paraformaldehyde solution for 20 minutes and washed three times with Phosphate Buffered Saline (PBS); then staining the cells with 10 mu g/mL fluorescent dye DAPI solution for 30 minutes; and finally, respectively photographing under 10 times of an Olympus inverted fluorescence microscope, randomly photographing 20 fluorescence photographs under different visual fields, counting the captured circulating tumor cells by using ImageJ software, and calculating the capturing efficiency.
The experimental result shows that the capture rate of the cell replica PDMS surface to anti-EpCAM positive cancer cells (MCF-7 cells and HepG2 cells) can reach 90%, and the capture rate to anti-EpCAM negative cancer cells (Hela cells and A549 cells) can reach 72% (FIG. 4). The cell replica PDMS surface prepared by the method can realize the high-efficiency capture of the circulating tumor cells and has broad spectrum.
Example 3
In the embodiment, a PDMS surface is re-etched by anti-EpCAM specific antibody cells modified by a chemical coupling method to serve as a capture system, and MCF-7 cells with different quantities are added into whole blood to serve as CTCs artificial blood sample models to be captured; and the captured cancer cells and leucocytes are observed by a scanning electron microscope, and the capturing system of the invention is further elaborated and verified, which comprises the following steps:
1) the preparation method of the cell replica PDMS surface is the same as that of example 1.
2) Chemical coupling to anti-EpCAM specific antibodies:
and (3) carrying out ultraviolet ozone treatment on the surface of PDMS for 5 minutes, putting the PDMS into a cell culture plate, adding a n-octadecyl triethoxysilane ethanol solution with the mass concentration of 4%, soaking the PDMS for 45 minutes at room temperature, washing the PDMS once with absolute ethanol and water once, and drying the PDMS once with nitrogen. Adding 1 mmol/L4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS, dimethyl sulfoxide for dilution), soaking at room temperature for 45 minutes, and washing with dimethyl sulfoxide (DMSO), absolute ethyl alcohol, water and PBS solution respectively. After adding 10. mu.g/mL avidin (PBS dilution) and soaking at room temperature for 30min, PBS was washed 3 times. Finally, biotinylated anti-EpCAM (PBS) containing 10 mu g/mL is added for dilution, the mixture is reacted at room temperature for 30 minutes and then washed for 3 times by PBS, and the cell replica PDMS surface modified by the anti-EpCAM specific antibody is obtained.
3) Preparing a circulating tumor cell sample to be tested:
taking 100 μ L MCF-7 cell suspension, counting by a cell counter, diluting to 1 × 10 by DMEM cell culture medium3Concentration per mL of individual cells for use; 9mL of fresh anticoagulated blood is equally divided into 9 parts, MCF-7 cells with the cancer cell numbers of 10, 20, 50, 100, 200, 400, 600, 800 and 1000 are respectively added into the 9 parts, and the mixture is uniformly mixed. Adding erythrocyte lysate (Beijing Solebao biotechnology) into CTCs artificial blood sample, and centrifuging to separate impurities to obtain peripheral blood mononuclear cell suspension containing MCF-7 cells with leukocyte concentration of about 0.8 × 106Individual cells/mL.
4) Capturing circulating tumor cells in a sample to be tested:
placing the cell re-etched PDMS surface modified by the anti-EpCAM specific antibody in the step 2) into a cell culture plate, then dropwise adding 0.5mL of the uniformly mixed cell suspension in the step 2) onto the PDMS surface, and then placing the PDMS surface in CO at 37 DEG C2In the cell incubator, the capture time was 60 minutes.
5) Evaluation of capturing effect:
after the capturing time is over, fixing the captured cells for 20 minutes by using a paraformaldehyde aqueous solution with the mass concentration of 4%, and washing the cells for three times by using Phosphate Buffer Solution (PBS); staining with 10. mu.g/mL fluorescent dye DAPI solution for 30min (DAPI dye can make captured cancer cells and leukocytes fluorescent blue), and then staining with 10. mu.g/mL Cytokeratin-FITC phosphate buffer for 60 min (Cytokeratin-FITC dye can only make captured cancer cells fluorescent green); and finally, respectively photographing under 10 times of an Olympus inverted fluorescence microscope, randomly photographing 20 fluorescence photographs under different visual fields, counting the captured circulating tumor cells by using ImageJ software, and calculating the capturing efficiency and the capturing purity.
6) And (3) morphological analysis:
the captured cells were fixed with a paraformaldehyde aqueous solution having a mass concentration of 4% for 20 minutes, and then dehydrated with a gradient of ethanol solutions having volume concentrations of 10, 30, 50, 70, 90, and 100%, respectively. The morphology of the captured cancer cells and the morphology of the captured leukocytes are respectively observed under a scanning electron microscope.
The experimental result shows that the capture efficiency of the cell replica PDMS surface modified by the anti-EpCAM specific antibody to cancer cells in CTCs artificial blood samples is high and can reach 85% on average (FIG. 5A); background cell adhesion was low and cancer cell capture purity was up to 92% (fig. 5B). The scanning electron microscope picture shows that the spread area of the cancer cells on the surface of the material is larger, the pseudopodia is longer in extension, and the adhesion effect of the cancer cells on the surface of the material is increased (fig. 6A); while the white blood cells were relatively round, the pseudopodia was hardly protruded, the spread area was small, and therefore, there were few white blood cells adhered to the surface of the material (fig. 6B). The method is shown to realize the efficient and specific capture of the circulating tumor cells from the CTCs artificial blood samples.
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (5)
1. The cell re-etching surface is characterized by having a micro-nano topological structure surface which is complementary with a template cell structure, wherein the template cell is a tissue cell with a pseudopodous structure;
the surface of the micro-nano topological structure is also modified with a rare cell specific recognition antibody to be captured, the rare cell specific recognition antibody is connected and modified on the surface of the micro-nano topological structure by a chemical coupling method or a biological affinity method, and the rare cell to be captured is selected from stem cells, circulating endothelial cells, residual diseased cells and circulating tumor cells;
the preparation method of the micro-nano topological structure surface comprises the following steps: fixing template cells on a cell culture surface through paraformaldehyde, acetone, methanol or glutaraldehyde, dehydrating and shrinking the template cells, pouring a high-molecular prepolymer on the surfaces of the template cells, and stripping the high-molecular prepolymer after thermosetting, crosslinking and curing to obtain a micro-nano topological structure surface;
the material of the surface of the micro-nano topological structure is selected from polydimethylsilane, polypyrole peroxide and polyurethane;
the template cell is selected from cancer cells, leukocytes, and phagocytes.
2. The cell replication surface of claim 1, wherein the template cells are dehydrated and shrunk by dehydration of anhydrous ethanol, anhydrous crystalline salts, or polyhydroxy compounds.
3. The cell replication surface according to claim 1, wherein the micro-nano topological structure surface is obtained by a soft lithography method or a cell imprinting method.
4. The cell replication surface of claim 1, wherein the micro-nano topological structure complementary to the template cell occupies an area of not less than 65% of the total area.
5. Use of a cell replica surface according to any one of claims 1-4 for efficient capture of rare cells in blood for non-disease diagnosis and treatment.
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