CN113713866A - Human bone microfluidic chip embedded in cancer cell membrane, preparation method thereof and application of chip in separation of circulating tumor cells - Google Patents
Human bone microfluidic chip embedded in cancer cell membrane, preparation method thereof and application of chip in separation of circulating tumor cells Download PDFInfo
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Abstract
The invention discloses a human bone microfluidic chip embedded with cancer cell membranes, a preparation method thereof and application thereof in separating circulating tumor cells, wherein the method comprises the following steps: culturing cell suspension of the cancer cell strain, and washing and fixing the cancer cell after the cancer cell adheres to the wall to obtain a cancer cell membrane substrate; adding BSA solution and NHS-S-S-Biotin solution for incubation, soaking the cancer cell membrane substrate in streptavidin solution, washing with PBS, adding biotinylated anti-EpCAM antibody solution for incubation, and obtaining the cancer cell membrane substrate modified by the EpCAM antibody; and integrating the cancer cell membrane substrate modified by the EpCAM antibody with human bone PDMS to obtain a human bone microfluidic chip embedded in the cancer cell membrane, which is called MHB-chip for short. The chip can be used for specifically capturing and releasing trace CTCs in a simulated blood sample and a clinical patient blood sample in vitro, and has high capturing efficiency and high capturing purity.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a human bone microfluidic chip with embedded cancer cell membranes, a preparation method thereof and application thereof in separating circulating tumor cells.
Background
Circulating tumor cells are tumor cells which are shed from solid tumors into peripheral blood, the circulating tumor cells can spread to other positions along with the peripheral blood, and the metastatic proliferation of cancer cells is a main reason for death of tumor patients. The circulating tumor cells carry all gene information of tumor patients and can be used as one of important means of liquid biopsy, which provides important basis for diagnosis, personalized treatment and postoperative evaluation of the patients. However, we know that there may be only a few CTCs per ml of blood, and there is an urgent need to develop techniques to isolate rare circulating tumor cells from blood. Over the past decades, many experts have developed ways to enrich CTCs based on their physicochemical properties. Such as magnetic separation, size separation, microfluidic separation, and the like. Among them, microfluidic devices have important advantages in the research of CTC enrichment because of their advantages of easy manufacturing, low cost, and ability to efficiently process complex cellular fluids.
Microfluidic devices combining antibody-modified substrates with geometric images are considered to be a promising device for efficient separation of CTCs. The human bone chip can enhance the contact between the sample and the substrate modified by the antibody, thereby enhancing the separation efficiency of CTC. Meanwhile, microfluidics of a substrate of a nanostructure modified with an antibody is widely studied in order to more increase the contact chance of both. Substrates such as nanospheres, nano columns and nano spinning not only increase the roughness of the substrate, but also increase the contact area between the substrate and cells, thereby achieving the purpose of improving the cell capture efficiency. However, the nano-substrate improves the capture efficiency of target cells and also causes a large amount of background cells to be non-specifically adhered to the nano-substrate. Traditional methods for releasing cells en bloc often achieve enrichment of CTCs by acting on the entire substrate.
However, the release of target cells along with non-specifically adherent background cells in this manner results in low purity of the enriched CTCs, which is not conducive to downstream analysis of the CTCs. There are also techniques for recovering single cells from a substrate, such as laser cutting, micromanipulation, flow cytometry sorting, etc., however, the existing methods for single cell separation often require the use of external equipment, and are costly and inefficient in cell capture.
Therefore, there is a need for a method or material tool for enriching CTCs by simultaneously ensuring cell capture efficiency and capture purity, and a technical problem to be solved is urgently needed.
Disclosure of Invention
The invention aims to provide a human bone microfluidic chip with embedded cancer cell membranes, a preparation method thereof and application thereof in separating circulating tumor cells.
In a first aspect of the present invention, there is provided a method for preparing a cancer cell membrane embedded human bone microfluidic chip, the method comprising:
culturing cell suspension of the cancer cell strain, and washing and fixing the cancer cell after the cancer cell adheres to the wall to obtain a cancer cell membrane substrate;
incubating the cancer cell membrane substrate in BSA solution and NHS-S-S-Biotin solution, soaking in streptavidin solution, washing with PBS, adding biotinylated anti-EpCAM antibody solution, and incubating to obtain a cancer cell membrane substrate modified by an EpCAM antibody;
and integrating the cancer cell membrane substrate modified by the EpCAM antibody with human bone PDMS to obtain a human bone microfluidic chip embedded in the cancer cell membrane, which is called MHB-chip for short.
Further, the fixing method adopts 2-3% of glutaraldehyde solution by mass.
Further, the cancer cell line comprises one of a breast cancer cell line, a prostate cancer cell line, a colon cancer cell line, a liver cancer cell line, a gastric cancer cell line and a lung cancer cell line.
Further, the BSA solution is a bovine serum albumin solution with the concentration of 5-20 mg/mL; the NHS-S-S-Biotin solution is obtained by dissolving activated lipid disulfide bond linked Biotin in DMSO, and the concentration of the NHS-S-S-Biotin solution is 0.05-0.5 mg/mL.
Further, the concentration of the streptavidin solution is 40-60 mug/mL; the concentration of the biotinylated anti-EpCAM antibody solution is 10-30 mu g/mL.
In a second aspect of the invention, a cancer cell membrane embedded human bone microfluidic chip prepared by the method is provided.
In a third aspect of the invention, the application of the human bone microfluidic chip with the embedded cancer cell membrane in the separation of circulating tumor cells is provided.
In a fourth aspect of the present invention, there is provided a method for separating circulating tumor cells, the method using the human bone microfluidic chip with embedded cancer cell membrane, comprising:
extracting peripheral blood mononuclear cell suspension from a blood sample containing circulating tumor cells;
introducing the peripheral blood mononuclear cell suspension into a human bone microfluidic chip embedded in the cancer cell membrane so as to capture a plurality of circulating tumor cells;
and (3) introducing a DTT solution into the chip for capturing the plurality of circulating tumor cells to release the captured cells to obtain the circulating tumor cells.
Further, the flow rate of the peripheral blood mononuclear cell suspension added to the human bone microfluidic chip embedded in the cancer cell membrane is 0.25-0.5 mL/h.
Further, the concentration of the DTT solution is 45-55 uM.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
1. the invention provides a preparation method of a human bone microfluidic chip embedded with a cancer cell membrane, which comprises the following steps that (1) a cancer cell membrane substrate is adopted, NHS-S-S-biotin (activated lipid disulfide bond biotin) can be introduced through BSA, and then an EpCAM antibody is successfully modified on the substrate through a chemical reaction; the PDMS layer is a human bone structure, so that the interaction between cells and a substrate can be increased, and the aim of enhancing capture is fulfilled. (2) The cancer cell membrane substrate can be prepared by simple culture and fixation, and has simple operation and low cost. (3) The surface of the cancer cell membrane is of a nano structure, so that the interaction with a target cell can be enhanced, and circulating tumor cells can be captured more firmly and efficiently; (4) the surface of the cancer cell membrane is electronegative and can be used as a natural antifouling layer to prevent nonspecific adhesion of background cells, so that the aim of improving the enrichment purity is fulfilled;
2. when the human bone microfluidic chip embedded in the cancer cell membrane is used for separating circulating tumor cells, the biocompatible DTT is used for breaking S-S bonds to achieve the purpose of releasing the cells, and the activity of the released cells is ensured.
3. The MHB-chip for efficiently and specifically recognizing and capturing the circulating tumor cells can be used for specifically capturing and releasing trace CTCs in a simulated blood sample and a clinical patient blood sample in vitro so as to enrich the circulating tumor cells with high efficiency and high purity; thereby being expected to play an important role in the fields of early warning and prevention of cancer metastasis.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic diagram of a cancer cell membrane embedded human bone microfluidic chip of the present invention in separating circulating tumor cells;
FIG. 2 is a photograph of cell membranes of MHB-chip of example 1; wherein, fig. 2(a) is a bright field image of MCF-7 cell membrane substrate wherein the scale bar is 1 cm; fig. 2(b) is an SEM image of MCF-7 cell matrix, wherein the scale bar is 100 μm; fig. 2(c) is an SEM image of an enlarged structure, wherein the scale bar is 5 μm;
FIG. 3 shows the anti-leukocyte adhesion results; wherein, fig. 3(a) is a fluorescent microscope image of leukocytes adhering to a plate glass with a scale bar of 100 μm; fig. 3(b) is a fluorescent microscope image of MCF-7 cell-substrate-adhered leukocytes at a scale of 100 μm; FIG. 3(c) is a graph showing the adhesion rate of leukocytes to plate glass and MCF-7 cell substrate;
FIG. 4 is an SEM image of MCF-7 cells captured on an MHB-chip and an HB-chip; wherein, FIG. 4(a) is an SEM image of MCF-7 cells on an MHB-chip with a scale bar of 10 μm; FIG. 4(b) is an SEM image of MCF-7 cells on HB-chip with a scale bar of 10 μm;
FIG. 5 is a graph of MHB-chip capture efficiency at different flow rates;
FIG. 6 is a study of the capture efficiency of MHB-chip and HB-chip;
FIG. 7 is a study of the cell release properties of MHB-chip; fig. 7(a) -fig. 7(b) fluorescence images before and after release of MCF-7 cells captured on an MHB-chip, scale bar 100 μm; fig. 7(c) is a graph of fluorescence of MCF-7 cells released live-dead cells at a scale of 100 μm;
FIG. 8 is a flow chart of a method for preparing a cancer cell membrane embedded human bone microfluidic chip according to the present invention;
FIG. 9 is a flow chart of a method for isolating circulating tumor cells according to the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
The embodiment of the invention provides a human bone microfluidic chip with embedded cancer cell membranes, which has the following general idea:
according to an exemplary embodiment of the present invention, there is provided a method for preparing a cancer cell membrane embedded human bone microfluidic chip, as shown in fig. 8, the method including:
step S101, culturing cell suspension of a cancer cell strain, and washing and fixing the cancer cell after the cancer cell adheres to the wall to obtain a cancer cell membrane substrate;
in this embodiment, the cancer cell lines should be selected from those with high research concentration, high postoperative metastasis and high recurrence degree, and the following cancer cell lines can be used but are not limited to: breast cancer cell line, prostate cancer cell line, colon cancer cell line, liver cancer cell line, gastric cancer cell line, lung cancer cell line. The breast cancer cell line (i.e., MCF-7 cells) is preferably selected as the in vitro circulating tumor cell model. The breast cancer cell line (namely MCF-7 cells) has moderate metastasis and recurrence degree and low risk; and the biomarker EpCAM is highly expressed on the cell surface stably, so that the breast cancer cell line (namely MCF-7 cell) is selected as an in vitro CTCs model, which is favorable for the smooth development of the research.
As an optional embodiment, a glutaraldehyde solution with the mass fraction of 2-3% is used for fixing.
In an optional embodiment, the BSA solution is a bovine serum albumin solution with a concentration of 5-20 mg/mL; the BSA solution is obtained by dissolving bovine serum albumin in PBS (pH value of 7.2-7.4).
Step S102, incubating the cancer cell membrane substrate in BSA solution and NHS-S-S-Biotin solution, soaking in streptavidin solution, washing with PBS, adding biotinylated anti-EpCAM antibody solution, and incubating to obtain a cancer cell membrane substrate modified by an EpCAM antibody;
in an optional embodiment, the BSA solution is a bovine serum albumin solution with a concentration of 5-20 mg/mL; the BSA solution is obtained by dissolving bovine serum albumin in PBS (pH value of 7.2-7.4). Preferably, the concentration of the BSA solution is 10 mg/mL.
The NHS-S-S-Biotin solution is obtained by dissolving activated lipid disulfide bond linked Biotin in DMSO, and the concentration of the NHS-S-S-Biotin solution is 0.05-0.5 mg/mL. Preferably, the concentration of the NHS-S-S-Biotin solution is 0.1 mg/mL.
The concentration in the range is favorable for successfully obtaining the EpCAM antibody modified cancer cell membrane substrate.
In an alternative embodiment, the concentration of the streptavidin solution is 40-60 μ g/mL (preferably 50 μ g/mL); the soaking conditions are as follows: soaking for 6-15 h at 2-6 ℃; the concentration of the biotinylated anti-EpCAM antibody solution is 10-30 [ mu ] g/mL (preferably 20 [ mu ] g/mL); this range facilitates subsequent capture of circulating tumor cells;
and S103, integrating the cancer cell membrane substrate modified by the EpCAM antibody with human bone PDMS to obtain a human bone microfluidic chip embedded in the cancer cell membrane, which is called MHB-chip for short.
In step S103, the EpCAM antibody-modified cancer cell membrane substrate may be specifically integrated with human bone PDMS by using a jig.
The human bone PDMS can be obtained by adopting a conventional method in the prior art, and specifically comprises the following steps:
firstly, a first layer of negative photoresist is dripped on a silicon wafer, then the silicon wafer is uniformly spin-coated by a spin coater at the speed of 2000rpm/min, then the silicon wafer is sequentially placed on a baking table at 65 ℃ and 95 ℃ for baking for 5min, after the temperature of the silicon wafer is reduced, the silicon wafer is placed on a photoetching machine for ultraviolet exposure, and then the silicon wafer is placed on the baking table for baking for 2h to cure the surface photoresist. At this point, the first layer of photoresist has been completed. After the photoresist is recovered to the room temperature, a second layer of photoresist is dripped on a silicon wafer, the silicon wafer is uniformly spun by a spin coater at the speed of 2000rpm/min, then the silicon wafer is sequentially placed on baking tables at 65 ℃ and 95 ℃ for baking for 5min, after the silicon wafer is cooled to the room temperature, the silicon wafer is aligned with a grinding plate of another pattern for ultraviolet exposure, and the silicon wafer is placed on the baking tables for heating and curing. Then the silicon chip is placed in a developing solution for treatment for 5min, and then is washed by acetone and the developing solution, and finally is dried by nitrogen. And (3) baking the blow-dried silicon wafer on a baking table at 165 ℃ for 30min to finally obtain the template of the human bone snake-shaped chip.
Glue a and glue B from laboratory were mixed at 10: 1, pouring the mixture on a silicon wafer with a pattern, standing overnight, placing a baking table after bubbles disappear, and baking for 2 hours at 80 ℃. And slightly uncovering the cured PDMS layer, then cutting and punching to obtain the PDMS layer with the pattern for subsequent use.
In summary, the preparation method of the human bone microfluidic chip embedded in the cancer cell membrane provided by the invention comprises the following steps of (1) adopting a cancer cell membrane substrate, introducing NHS-S-S-Biotin (activated lipid disulfide bond connected Biotin) through BSA, and then successfully modifying an EpCAM antibody on the substrate through a chemical reaction; the PDMS layer is a human bone structure, so that the interaction between cells and a substrate can be increased, and the aim of enhancing capture is fulfilled. (2) The cancer cell membrane substrate can be prepared by simple culture and fixation, and has simple operation and low cost. (3) The surface of the cancer cell membrane is of a nano structure, so that the interaction with a target cell can be enhanced, and circulating tumor cells can be captured more firmly and efficiently; (4) the surface of the cancer cell membrane is electronegative and can be used as a natural antifouling layer to prevent nonspecific adhesion of background cells, thereby achieving the purpose of improving the enrichment purity.
According to another exemplary embodiment of the present invention, a cancer cell membrane embedded human bone microfluidic chip prepared by the method is provided.
The human bone microfluidic chip MHB-chip embedded in the cancer cell membrane consists of two functional parts, including a human bone Polydimethylsiloxane (PDMS) layer and a cancer cell membrane substrate modified by an EpCAM antibody.
The MHB-chip for efficiently and specifically recognizing and capturing the circulating tumor cells can be used for specifically capturing and releasing trace CTCs in a simulated blood sample and a clinical patient blood sample in vitro so as to enrich the circulating tumor cells with high efficiency and high purity; thereby being expected to play an important role in the fields of early warning and prevention of cancer metastasis.
According to another exemplary embodiment of the present invention, there is provided an application of the human bone microfluidic chip with embedded cancer cell membrane in separating circulating tumor cells.
When the human bone microfluidic chip embedded in the cancer cell membrane is used for separating circulating tumor cells, the human bone microfluidic chip embedded in the cancer cell membrane is firstly used for capturing, and then the biocompatible DTT is used for breaking S-S bonds so as to achieve the purpose of releasing the cells, and meanwhile, the activity of the released cells is ensured. In conclusion, the microfluidic device can enrich the circulating tumor cells with high efficiency and high purity, and shows that the safe and efficient mode has certain clinical application potential.
The research means for capturing CTCs in blood samples of clinical patients by the microfluidic chip can adopt but is not limited to the method: blood samples of tumor patients are obtained, and after a lymphocyte layer is obtained through percoll cell separating medium, the lymphocyte layer is introduced into an MHB-chip for a capture experiment. It is preferred to use the present invention by obtaining blood samples of cancer patients in oncology hospitals, performing triple fluorescent staining of the captured cells, and then analyzing the captured cells.
According to another exemplary embodiment of the present invention, there is provided a method for separating circulating tumor cells, the method using the human bone microfluidic chip with embedded cancer cell membrane, as shown in fig. 9, comprising:
step S201, extracting and obtaining peripheral blood mononuclear cell suspension from a blood sample containing circulating tumor cells;
the blood sample containing circulating tumor cells of the present invention is taken from a patient with advanced cancer who has high postoperative metastasis and recurrence, and is not limited to the following cancer patients: colon cancer patients, breast cancer patients, liver cancer patients, prostate cancer patients, stomach cancer patients, and lung cancer patients.
The extraction method adopts the conventional method in the prior art, and particularly adopts lymphocyte separation fluid to extract and wash from a blood sample.
Step S202, introducing the peripheral blood mononuclear cell suspension into a human bone microfluidic chip embedded in the cancer cell membrane to obtain a chip for capturing a plurality of circulating tumor cells;
preferably, the flow rate of the peripheral blood mononuclear cell suspension added to the human bone microfluidic chip embedded in the cancer cell membrane is 0.25-0.5 mL/h. When the flow rate is 0.25-0.5 mL/h, the trapping efficiency can reach more than 80%.
Step S203, introducing a DTT solution into the chip for capturing the plurality of circulating tumor cells to release the captured cells, and obtaining the circulating tumor cells.
In this embodiment, biocompatible DTT is used to break S-S bonds for the purpose of releasing cells while ensuring the activity of the released cells.
In a preferred embodiment, the concentration of the DTT solution is 45-55 uM. Too high a concentration of the DTT solution results in decreased cell activity, while too low a concentration results in low cell release efficiency.
The following will explain in detail a cancer cell membrane embedded human bone microfluidic chip, a preparation method thereof and an application thereof in separating circulating tumor cells according to the present application with reference to examples and experimental data.
Example 1 human bone microfluidic chip with embedded cancer cell membrane and method for preparing the same
1. The cancer cell membrane embedded human bone microfluidic chip MHB-chip is composed of two functional components, including a Polydimethylsiloxane (PDMS) layer with human bone patterns on the surface and a transparent MCF-7 cell Membrane Substrate (MS).
2. Preparation method of human bone microfluidic chip embedded in cancer cell membrane
(1) To prepare MS, we will mix 1X 106MCF-7 cell suspension was added to a petri dish (clean glass was placed in the petri dish) for culture. When the cells were fully grown on glass, the medium was removed and washed with PBS. Next, cells on MS were fixed with 2.5% glutaraldehyde, thereby maintaining the nanostructure of MCF-7 cell membrane. (ii) a
(2) A10 mg/mL solution of bovine serum albumin was introduced into the MCF-7 cell membrane. MCF-7 cell membranes were treated with a solution of NHS-S-S-Biotin (0.1mg/mL in DMSO) for 2h at room temperature, followed by incubation of the cancer cell membrane substrate with MS 4 ℃ overnight using a 50. mu.g/mL solution of streptavidin. Before cell capture experiments are carried out, a 20 mu g/mL biotinylated anti-EpCAM antibody solution in PBS is added into MS, the MS is washed for 3 times by PBS at room temperature for 2h to obtain a cancer cell membrane substrate modified by the EpCAM antibody;
(3) the PDMS layer was prepared according to the prior art method with a humanoid pattern on top of the PDMS layer to obtain Herringbone Bone (HB) PDMS. Integrating the Herringbone Bone (HB) PDMS and the EpCAM antibody modified cancer cell membrane substrate MS through a clamp to form the MHB-chip.
A photograph of a cell membrane of an MHB-chip according to an embodiment of the present invention is shown in FIG. 2, and it can be seen from FIG. 2 that FIG. 2(a) shows that the substrate surface is overgrown with MCF-7 cells, and FIGS. 2(b) and 2(c) show that the prepared substrate surface is a nanostructure. Example 2 human bone microfluidic chip with embedded cancer cell membrane and method for preparing the same
1. The cancer cell membrane embedded human bone microfluidic chip MHB-chip is composed of two functional components, including a Polydimethylsiloxane (PDMS) layer with human bone patterns on the surface and a transparent MCF-7 cell Membrane Substrate (MS).
2. Preparation method of human bone microfluidic chip embedded in cancer cell membrane
(1) To prepare MS, we will mix 1X 106MCF-7 cell suspension was added to a petri dish (clean glass was placed in the petri dish) for culture. When the cells were fully grown on glass, the medium was removed and washed with PBS. Next, fixing the cells on MS with 2.5% glutaraldehyde, thereby maintaining the nanostructure of the MCF-7 cell membrane, obtaining a cancer cell membrane substrate;
(2) MCF-7 cell membranes were soaked in a 5mg/mL bovine serum albumin solution, then MCF-7 cell membranes were treated with a solution of NHS-S-S-Biotin (0.05mg/mL in DMSO) at room temperature for 2h, and then the cancer cell membrane substrates were incubated with MS 4 ℃ overnight using a 40. mu.g/mL solution of streptavidin. Before cell capture experiments are carried out, a biotinylated anti-EpCAM antibody solution of 10 mu g/mL in PBS is added into MS, the MS is washed for 3 times by PBS at room temperature for 2h to obtain a cancer cell membrane substrate modified by the EpCAM antibody;
(3) the PDMS layer was prepared according to the prior art method with a humanoid pattern on top of the PDMS layer to obtain Herringbone Bone (HB) PDMS. Integrating the Herringbone Bone (HB) PDMS and the EpCAM antibody modified cancer cell membrane substrate MS through a clamp to form the MHB-chip. The capture efficiency of the MHB-chip of the present invention was about 80%, and the capture purity was substantially the same as that of example 1.
Example 3 human bone microfluidic chip with embedded cancer cell membrane and preparation method thereof
1. The cancer cell membrane embedded human bone microfluidic chip MHB-chip is composed of two functional components, including a Polydimethylsiloxane (PDMS) layer with human bone patterns on the surface and a transparent MCF-7 cell Membrane Substrate (MS).
2. Preparation method of human bone microfluidic chip embedded in cancer cell membrane
(1) To prepare MS, we will mix 1X 106MCF-7 cell suspension was added to a petri dish (clean glass was placed in the petri dish) for culture. When the cells were fully grown on glass, the medium was removed and washed with PBS to obtain a cancer cell membrane substrate;
(2) MCF-7 cell membranes were soaked in 20mg/mL bovine serum albumin solution, then MCF-7 cell membranes were treated with NHS-S-S-Biotin (0.05mg/mL in DMSO) solution for 2h at room temperature, and then the cancer cell membrane substrates were incubated with MS 4 ℃ overnight using 60. mu.g/mL streptavidin solution. Before cell capture experiments are carried out, a biotinylated anti-EpCAM antibody solution with the concentration of 30 mu g/mL in PBS is added into MS, the solution is washed for 3 times by PBS at room temperature for 2h, and a cancer cell membrane substrate modified by the EpCAM antibody is obtained;
(3) the PDMS layer was prepared according to the prior art method with a humanoid pattern on top of the PDMS layer to obtain Herringbone Bone (HB) PDMS. Integrating the Herringbone Bone (HB) PDMS and the EpCAM antibody modified cancer cell membrane substrate MS through a clamp to form the MHB-chip. The capture efficiency of the MHB-chip of the present invention was about 80%, and the capture purity was substantially the same as that of example 1.
Comparative example 1
This comparative example is an HB-chip, a Herringbone Bone (HB) PDMS of the prior art.
Application example 1 method for separating circulating tumor cells Using MHB-chip of example
1. MHB-chip capture of MCF-7 cells
Extracting peripheral blood mononuclear cell suspension from a blood sample containing circulating tumor cells; introducing the cell suspension into the human bone microfluidic chip embedded in the cancer cell membrane in the embodiment 1 to obtain a chip for capturing a plurality of circulating tumor cells;
to optimize the capture performance of the MHB-chip, we first selected EpCAM-positive MCF-7 cells as samples and investigated the capture efficiency at different flow rates (0.25mL/h, 0.5mL/h, 1mL/h, 2mL/h, 4 mL/h). 1mL MCF-7 cell samples were introduced into the chip at different flow rates (0.25mL/h, 0.5mL/h, 1mL/h, 2mL/h, 4 mL/h). Then the MHB-chip was gently rinsed with PBS. The captured cells were observed and counted under a fluorescent microscope.
As shown in FIG. 5, it was found that the collection efficiency gradually decreased with the increase of the flow rate, and that the collection efficiency reached 80% or more at a flow rate of 0.25 to 0.5 mL/h. Therefore, we selected 0.25mL/h as the optimal condition for the subsequent experiment.
2. Release of circulating tumor cells
To release the captured CTCs, a release experiment (50uM, 30min) was performed on the captured cells using DTT, and after PBS washing, the chip cells were collected from the outlet of the device. Residual cells on the microfluidic device were imaged and counted using a fluorescence microscope. FDA/PI staining was used to identify cell activity after the release process.
As shown in FIG. 7, it can be seen from FIG. 7 that FIG. 7(a) is a picture of the captured cells and FIG. 7(b) is a picture of the cells subjected to DTT treatment, and that the number of the cells in FIG. 7(b) is significantly reduced. Next, we performed mirror image mortalities on the released cells, using FDA/PI solutions for staining. The results show that cells can be successfully released, and the activity of the released cells is ensured.
Anti-adhesion capability of MS of Experimental example 1, comparative example and GS of comparative example 1
Preparing the substrate of MHB-chip in example 1 (i.e., the EpCAM antibody-modified cancer cell membrane substrate, MS for short, in example 1); and Substrate (GS) of HB-chip in comparative example 1.
PBMCs were extracted from blood by lymphocyte separation and washed three times with PBS. Then, we stained the collected cells with FDA (10. mu.g/mL) for 10 minutes at room temperature to obtain a stained cell suspension;
will be 1 × 105FDA-stained leukocyte suspensions were added to MS in example 1 and GS in comparative example 1, respectively, and incubated at room temperature for 1h, and gently rinsed with PBS. Leukocyte adhesion on MS and GS was observed under a fluorescent microscope (IX-81, Olympus, Japan).
As a result, as shown in FIGS. 3(a) and 3(b), the number of adhered GS leukocytes was significantly greater than that of adhered MS leukocytes, with the number of adhered GS leukocytes being about 7% and the number of adhered MS leukocytes being about 0.5% (FIG. 3 (c)). Thus, the MS of the present invention has good effect of resisting the adhesion of leucocytes.
Capture efficiency and capture purity of chips of Experimental example 2, comparative example 1 and comparative example 1
1. To a concentration of 105one/mL of FDA-stained MCF-7 cells were passed through the MHB-chip of example 1 and the HB-chip of comparative example 1 at a rate of 0.25 mL/h. Then, the MHB-chip and the HB-chip were gently washed with PBS. And (4) observing and counting under a fluorescence microscope.
As can be seen from FIG. 6, the MCF-7 cell capture efficiency of the MHB-chip of the example of the invention was about 80%, and the MCF-7 cell capture efficiency of the HB-chip of comparative example 1 was about 60%. The capture efficiency of the MHB-chip is obviously higher than that of the HB-chip, which probably is because the bottom of the MHB-chip is of a nano structure, and the MHB-chip of the embodiment of the invention enhances the interaction between MS and MCF-7 cells and can realize more firm and more efficient capture of circulating tumor cells.
2. After the counting was complete, it was fixed with 2.5% glutaraldehyde. Samples were dehydrated by 15%, 30%, 50%, 70%, 80%, 90%, 100% alcohol concentration, and dried by supercritical carbon dioxide. The cell morphology captured by MHB-chip and HB-chip was observed by scanning electron microscopy.
As a result, as shown in FIG. 4, it was revealed that MCF-7 cells trapped on the MHB-chip had a smooth surface (FIG. 4(a)), while MCF-7 cells trapped on the HB-chip had many artifacts (FIG. 4(b)), which is probably due to the fact that MS of the MHB-chip was electronegative, indicating that the MHB-chip of the present invention can be used as a natural anti-fouling layer to prevent nonspecific adhesion of background cells for the purpose of increasing the enrichment purity.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A preparation method of a human bone microfluidic chip embedded with cancer cell membranes is characterized by comprising the following steps:
culturing cell suspension of the cancer cell strain, and washing and fixing the cancer cell after the cancer cell adheres to the wall to obtain a cancer cell membrane substrate;
incubating the cancer cell membrane substrate in BSA solution and NHS-S-S-Biotin solution, soaking in streptavidin solution, washing with PBS, adding biotinylated anti-EpCAM antibody solution, and incubating to obtain a cancer cell membrane substrate modified by an EpCAM antibody;
and integrating the cancer cell membrane substrate modified by the EpCAM antibody with human bone PDMS to obtain a human bone microfluidic chip embedded in the cancer cell membrane, which is called MHB-chip for short.
2. The preparation method of the human bone microfluidic chip embedded in the cancer cell membrane according to claim 1, wherein the fixing adopts 2-3% of glutaraldehyde solution by mass.
3. The method for preparing a microfluidic chip of human bone with embedded cancer cell membrane according to claim 1, wherein the cancer cell line comprises one of breast cancer cell line, prostate cancer cell line, colon cancer cell line, liver cancer cell line, gastric cancer cell line and lung cancer cell line.
4. The method for preparing the human bone microfluidic chip embedded in the cancer cell membrane according to claim 1, wherein the BSA solution is a bovine serum albumin solution with a concentration of 5-20 mg/mL; the NHS-S-S-Biotin solution is obtained by dissolving activated lipid disulfide bond linked Biotin in DMSO, and the concentration of the NHS-S-S-Biotin solution is 0.05-0.5 mg/mL.
5. The preparation method of the human bone microfluidic chip with the embedded cancer cell membrane according to claim 1, wherein the concentration of the streptavidin solution is 40-60 μ g/mL; the concentration of the biotinylated anti-EpCAM antibody solution is 10-30 mu g/mL.
6. A cancer cell membrane embedded human bone microfluidic chip obtained by the method of any one of claims 1 to 5.
7. The use of the cancer cell membrane embedded human bone microfluidic chip of claim 6 in separating circulating tumor cells.
8. A method for separating circulating tumor cells, which uses the cancer cell membrane embedded human bone microfluidic chip of claim 6, comprising:
extracting peripheral blood mononuclear cell suspension from a blood sample containing circulating tumor cells;
introducing the peripheral blood mononuclear cell suspension into a human bone microfluidic chip embedded with the cancer cell membrane so as to capture a plurality of circulating tumor cells;
and (3) introducing a DTT solution into the chip for capturing the plurality of circulating tumor cells to release the captured cells to obtain the circulating tumor cells.
9. The method for separating circulating tumor cells according to claim 8, wherein the flow rate of the peripheral blood mononuclear cell suspension added to the human bone microfluidic chip embedded in the cancer cell membrane is 0.25-0.5 mL/h.
10. The method for isolating circulating tumor cells according to claim 8, wherein the concentration of the DTT solution is 45-55 uM.
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