CN102539485B - Cell migration high-resolution impedance real-time tracking measurement and control device and its manufacturing method and application method - Google Patents

Abstract

The invention relates to a cell migration high-resolution impedance real-time tracking, measurement and control device, and a manufacturing method and an application method thereof, and is particularly suitable for cell impedance measurement, cell migration, cell detection, medicine screening, microfluidics and a nano technology. The tracking, measurement and control device comprise a cavity; a chamber in which liquid is accommodated is formed in the middle of the cavity; the middle of the chamber is divided into an upper chamber and a lower chamber by a microporous membrane; an upper chamber electrode is inserted into the electrode hole of an upper cavity; a lower chamber electrode is inserted into the electrode hole of a lower cavity; the two electrodes are connected with an analysis device respectively; and the lower part of the lower cavity is provided with a liquid inlet and outlet channel which is communicated with the external. The invention has the advantages that: 1, cells are detected in real time, and a transmembrane migration process of cells cultured in vitro is reliably tracked, measured, controlled and analyzed in real time at high resolution in a non-destructive way; 2, detection sensitivity is improved and the transmembrane migration of a single cell is tracked; 3, the effect of a medicine on the cell can be precisely determined through a microflow pipeline; 4, manual measurement and control errors are reduced; 5, the tracking, measurement and control device is easy and convenient to operate and high in accuracy; and 6, the tracking, measurement and control device is easy to manufacture and low in cost.

Description

Cell migration high-resolution impedance real-time tracking measurement and control device and its manufacturing method and application method

Technical field:

The invention relates to a cell migration high-resolution impedance real-time tracking measurement and control device and its manufacturing method and application method, which is especially suitable for cell electrical impedance measurement, cell migration, cancer detection, drug screening, microfluidic control and nanotechnology applications, and belongs to biological detection technology field.

Background technique:

Cell electrical impedance sensing technology is a technology that measures changes in the electrical impedance of a detection system due to changes in cell morphology, cell movement, or mutual contact between cells. This technology has the advantages of long-term, real-time, fast, non-destructive, and simple analysis of in vitro cultured cells, and has been widely used in the fields of cell morphology change tracking, drug screening, biochemical detection, and disease diagnosis, and has become a promising tool for biosensor research. an important content. the

The process of cell growth, proliferation and migration plays a key role in many biological processes such as embryonic development, wound healing, immune response and so on. In particular, the process of cell migration, which has been widely concerned, plays an important role in various diseases such as tumor metastasis and inflammatory response. For tumor cells, their migration characteristics have become an important indicator for evaluating antitumor drugs, and drugs that can slow down tumor cell migration may play an important role in curbing tumor metastasis. Rapid, high-throughput drug screening can be achieved by detecting changes in cell activity states. The drug efficacy test based on the cell impedance sensor can not only determine the effect of the drug on the cell through the response of the cell, but also determine the cell specificity of the drug response, which can play a huge role in drug screening and disease diagnosis.

Currently, electrical impedance sensing techniques for cell migration analysis can be divided into two categories: surface and transmembrane migration. The electrical impedance measurement used for the analysis of cell transmembrane migration is the Boyden chamber method. Cells move across the membrane under the action of drugs, which causes changes in the number of basal cells and changes the electrical impedance of the system. Although this method can also monitor cell transmembrane migration in real time, its resolution is very limited, and the drug delivery and drug-induced time response are not optimized. the

Invention content:

The purpose of the cell migration high-resolution impedance real-time tracking measurement and control device and its manufacturing method and application method provided by the present invention is: 1. Real-time detection of cells: by integrating electrical impedance sensing technology and microfluidic chip technology to realize in vitro culture High-resolution, real-time, non-destructive, reliable tracking, measurement, control, and analysis of cell transmembrane migration; 2. Improve detection sensitivity to track the transmembrane migration of single cells; 3. Accurately determine the effect of drugs on cells through microfluidic channels; 4. Real-time, quantitative and reduce human error; 5. Easy operation and high accuracy; 6. The device is simple to manufacture and low in cost; 7. It is especially used for rapid and high-throughput drug screening and disease diagnosis.

The technical solution of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention is: it includes a cavity, a cavity capable of containing liquid is arranged in the middle of the cavity, and a microporous membrane is used to separate the cavity into upper and lower parts of the cavity. The chamber and the lower chamber, the upper part of the microporous film is the upper chamber, and the lower part is the lower chamber; the lower part of the upper chamber and the upper part of the lower chamber are close to the microporous film respectively, and there are electrode holes communicating from the outside to the inner cavity The electrode hole of the upper chamber is inserted into the electrode of the upper chamber, and the electrode hole of the lower chamber is inserted into the electrode of the lower chamber. .

Further technical solutions are:

The cell migration high-resolution impedance real-time tracking measurement and control device, the chamber is a combined structure, that is, the upper chamber, the lower chamber and the base that closes the bottom of the lower chamber are three independent components; the upper chamber and the lower chamber Processed at one time or separately; the microporous film is sandwiched between the lower end surface of the upper cavity and the upper end surface of the lower cavity; the joint between the upper cavity and the lower cavity is sealed; the lower end surface of the lower cavity is connected to an independent base Sealed connection.

The high-resolution impedance real-time tracking measurement and control device for cell migration, the upper and lower chamber materials are polymer materials used in the field of microfluidics; the base material is selected from glass slides, or used in microfluidics. The polymer material in the field of fluid control; the sealing connection between the upper cavity and the lower cavity, and the sealing connection between the lower end surface of the lower cavity and the independent base are all bonded by high vacuum oxygen plasma method.

The described cell migration high-resolution impedance real-time tracking measurement and control device, the cavity is an integral structure, that is, the upper cavity, the lower cavity and the base that closes the lower end of the lower cavity are an integral component; the aperture of the lower cavity is smaller than that of the upper cavity. Chamber aperture, the microporous film set is embedded in the upper chamber on the microporous film ring.

The high-resolution impedance real-time tracking measurement and control device for cell migration, the upper cavity, the lower cavity and the base of the cavity of the overall structure are processed from an integral material, and the material is a polymer material used in the field of microfluidics.

In the high-resolution impedance real-time tracking measurement and control device for cell migration, the diameters of the upper chamber and the lower chamber are less than or equal to 8 mm.

The high-resolution impedance real-time tracking measurement and control device for cell migration, the upper chamber is a cell culture medium chamber, the electrode in the upper chamber is a working electrode; the lower chamber is an inducer chamber, and the electrode in the lower chamber is a reference electrode; The analysis device mentioned above is an impedance analyzer, or a multipurpose electric meter, or an electrochemical workstation.

The high-resolution impedance real-time tracking measurement and control device for cell migration, the thickness of the microporous film is less than or equal to 200 microns, the area is less than or equal to 5 square centimeters, and the diameter of the micropores on the microporous film is less than or equal to 8 microns; the material of the microporous film It is a polymer material or photoresist SU8 used in the field of microfluidics.

The technical scheme of the manufacturing method of the cell migration high-resolution impedance real-time tracking measurement and control device with a combined cavity in the present invention is: the cell migration high-resolution impedance real-time tracking measurement and control device includes a combined structure cavity, that is, the upper cavity and the lower cavity. The cavity and the base that closes the bottom of the lower cavity are three independent components; the manufacturing method includes the following steps:

The first step is to prepare the cavity raw materials: prepare the cavity raw materials into a liquid state;

The second step, raw material solidification: pour the liquid cavity raw material on the silicon wafer or glass slide, and bake it in the oven for a certain time, so that the cavity raw material is solidified into a cavity blank;

The third step is to make the cavity: cut the cavity blank into the shape of the upper cavity and the lower cavity; then process the upper cavity and the lower cavity, the electrode hole and the liquid inlet and outlet channel; the electrode hole is close to the lower cavity of the upper cavity. end face, and another upper end face close to the lower cavity;

The fourth step, assembly: spread the microporous film on the upper end surface of the lower cavity, then place the upper cavity on the microporous film on the upper end surface of the lower cavity, and then place the lower end surface of the lower cavity on the base , and finally use the high vacuum oxygen plasma method to bond the upper end surface of the lower cavity, the lower end surface of the upper cavity and the microporous film together; then bond the lower end surface of the lower cavity to the base in the same way;

The fifth step is to insert the upper chamber electrode and the lower chamber electrode into the upper chamber electrode hole and the lower chamber electrode hole respectively.

The application of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention is in the fields of cell biology research, drug screening and disease diagnosis.

The high-resolution impedance real-time tracking measurement and control device for cell migration of the present invention is applied to the real-time measurement and control method for cell transmembrane migration. A chamber, the middle of the chamber is divided into an upper chamber and a lower chamber by a microporous membrane; the method of real-time measurement and control comprises the following steps:

A. Disinfect and sterilize the cavity;

B. Fill the upper chamber with a sufficient amount of culture solution containing cells from the inlet of the upper chamber. The cell content is: 10 ⁵ -10 ⁷ cells per milliliter of the culture solution containing cells.

C. Sufficient perfusion of cell-free culture solution and/or drug solution for experiments from the liquid inlet and outlet channels of the lower chamber;

D. After the cells have been cultured for a certain period of time, inject a culture solution containing an appropriate concentration of platelet-derived growth factor to replace the original cell-free culture solution in the lower chamber as an inducer for cell migration; under the action of the inducer, the cells pass through the micropore The film migrates from the upper chamber to the lower chamber successively;

E. The electrode in the upper chamber is the working electrode, and the lower chamber is the reference electrode, which are closely distributed on the upper and lower sides of the microporous film. The two electrodes are connected to the analysis device for real-time measurement and control of the cell migration process, including Detection of impedance changes caused by migration of single cells.

The high-resolution impedance real-time tracking measurement and control device for cell migration of the present invention is applied to the real-time measurement and control method of cell transmembrane migration. In step B of the above method, the culture solution containing cells injected into the upper chamber in sufficient amount contains 10 ⁵ ~ Cell-containing culture solution of 10 ⁷ cancer cells U-87.

The high-resolution impedance real- ^time tracking measurement and control device for cell migration of the present invention is applied to the real-time measurement and control method of cell transmembrane migration ^. Cell-containing culture medium of a cancer cell HCT-116; and:

F. At the selected time, the cells that migrated to the lower chamber were incubated with the calcein-AM stock solution and then used for fluorescence imaging with an inverted optical microscope;

G. For comparison, at different times, the cells migrating to the lower chamber were observed and counted with an optical microscope, and the data were collected under the action of different concentrations of chemical inducers, and compared with the results obtained by impedance measurement.

The technical effect of the present invention is remarkable: 1. The change of the system impedance caused by the process of cell attachment, proliferation and migration can be detected in real time through the electrodes distributed on both sides of the film; Membrane migration tracking; 3. Accurately timing and quantitatively introducing drugs through microfluidic channels and determining the effect of drugs on cells according to the response of cells; 4. Real-time, quantitative and reducing human error; 5. Easy operation and high accuracy , and the device is simple to manufacture and low in cost; 6. It is used for rapid and high-throughput drug screening and disease diagnosis. This method realizes high-resolution, real-time, non-damaging, reliable tracking, measurement, control, and analysis of the transmembrane migration process of cultured cells in vitro by integrating electrical impedance sensing technology and microfluidic chip technology.

Description of drawings

Fig. 1 is an A-A cross-sectional view of an embodiment of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention; the cavity of this embodiment is a combined structure;

Fig. 2 is the top view of Fig. 1;

Fig. 3 is a B-B cross-sectional view of another embodiment of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention; the cavity of this embodiment is an integral structure;

Fig. 4 is the top view of Fig. 3;

Fig. 5 is microporous film scanning electron microscope picture;

Figure 6 is a scanning electron micrograph of U-87 cells attached to the microporous film;

Fig. 7 is a graph of the change in electrical impedance corresponding to the migration of U-87 cells through an 8 micron diameter hole; the graph was measured after injection of a chemical inducer at a frequency of 80 kHz. The big picture is the record within 60 minutes, and the illustration is the change of electrical impedance in the next 120 minutes;

Fig. 8 is a spectrum diagram of electrical impedance changes corresponding to the transmembrane migration of HCT-116 cells through a pore with a diameter of 8 microns. The graph was measured after the injection of the chemical inducer at a frequency of 80 kHz; the large graph is the recording within 60 minutes, and the inset is the electrical impedance change graph for the next 120 minutes;

Figure 9 is a fluorescent image of HCT-116 cells on the base of the lower chamber after transmembrane migration for 3 hours;

Fig. 10 is a diagram showing the change of HCT-116 cell density on the base of the lower chamber over time under the action of different concentrations of chemical inducers.

The names of the reference signs in the figure are: the chamber-1; the upper chamber-1.1; the lower chamber-1.2; the base-1.3; the chamber-2; the upper chamber-2.1; Porous membrane-3; micropore-3.1; microporous membrane ring-4; liquid access channel-5; electrode hole-6; upper chamber electrode-6.1; lower chamber electrode-6.2; analysis device-7. the

Detailed ways

The present invention will be further described below in conjunction with drawings and embodiments.

Embodiment 1: It is a basic embodiment of the cell migration high-resolution impedance real-time tracking measurement and control device in which the cavity 1 is a composite structure of the present invention, and accompanying drawing 1 is an example thereof. It includes a chamber 1, a chamber 2 that can hold liquid in the middle of the chamber 1, and a microporous membrane 3 in the middle of the chamber 2 to separate the chamber 2 into an upper chamber 2.1 and a lower chamber 2.2, and the microporous The upper part of the film 3 is the upper cavity 1.1, and the lower part is the lower cavity 1.2; the lower part of the upper cavity 1.1 and the upper part of the lower cavity 1.2 are respectively provided with electrode holes 6 connected from the outside to the inner cavity near the microporous film 3; The electrode hole 6 of the chamber body 1.1 is inserted into the upper chamber electrode 6.1, and the electrode hole 6 of the lower chamber body 1.2 is inserted into the lower chamber electrode 6.2, and the two electrodes are respectively connected with the analysis device 7; The liquid connected to the outside enters and exits the channel 5 .

Embodiment 2: As shown in Figures 1 and 2, it is an embodiment of the high-resolution impedance real-time tracking measurement and control device for cell migration in which the cavity 1 is a composite structure of the present invention. The cavity 1 is a combined structure, that is, the upper cavity 1.1, the lower cavity 1.2 and the base 1.3 closing the bottom of the lower cavity 1.2 are three independent components; the upper cavity 2.1 and the lower cavity 2.2 are processed once or separately The microporous film 3 is sandwiched between the lower end surface of the upper cavity 1.1 and the upper end surface of the lower cavity 1.2; the upper cavity 1.1 and the lower cavity 1.2 are sealed and connected; The base 1.3 is hermetically connected. The material of the upper cavity 1.1 and the lower cavity 1.2 is a polymer material used in the field of microfluidics, which is selected from PDMS, that is, polydimethylsiloxane, or thermoplastic material polymethyl methacrylate PMMA , or polycarbonate PC, or polystyrene PS, PDMS is selected in this embodiment, that is, polydimethylsiloxane; the base 1.3 material is selected from glass slides, or polymer materials used in the field of microfluidics, Or polydimethylsiloxane PDMS, or thermoplastic material polymethyl methacrylate PMMA, or polycarbonate PC, or polystyrene PS, this embodiment is selected as a glass slide; upper chamber 1.1 and lower chamber 1.2 The sealing connection of the joint part, and the sealing connection between the lower end surface of the lower cavity 1.2 and the independent base 1.3 are all bonded by a high vacuum oxygen plasma method. The diameters of the upper chamber 2.1 and the lower chamber 2.2 are less than or equal to 8 mm, and the diameter of this embodiment is selected as 5 mm. The upper chamber 2.1 is a cell culture medium chamber, and the upper chamber electrode 6.1 is a working electrode, and the material is selected from platinum, gold, titanium, copper, iron metal or alloy, or carbon fiber, or graphite-like carbon material, or corresponding The chemically modified electrode is selected as platinum in this embodiment; the lower chamber 2.2 is an inducer chamber, and the lower chamber electrode 6.2 is a reference electrode, and the material is selected from silver/silver chloride, or mercury/mercuric oxide, or mercury/sulfuric acid Mercury, or calomel, or platinum, or gold, or carbon fiber is selected as silver/silver chloride in the present embodiment; the analysis device 7 is an impedance analyzer, or a multipurpose electric meter, or an electrochemical workstation, and the present embodiment is selected as Impedance Analyzer. The thickness of the microporous film 3 is less than or equal to 200 microns, the area is less than or equal to 5 square centimeters, and the diameter of the pores on the microporous film 3 is less than or equal to 8 microns; the material of the microporous film 3 is a polymer used in the field of microfluidics Material, selected from polycarbonate film, or thermoplastic material polymethyl methacrylate PMMA, or polystyrene PS, or polymer polydimethylsiloxane, or photoresist SU8. The present embodiment is chosen as: the thickness of microporous film 3 is equal to 100 microns, and the area is equal to 1 square centimeter, and the microporous aperture on microporous film 3 is equal to or equal to 5 microns; The material of microporous film 3 is selected from polycarbonate film, Or thermoplastic material polymethyl methacrylate PMMA, or polystyrene PS, or polymer polydimethylsiloxane, or optical glue SU8, this embodiment is selected as: thermoplastic material polymethyl methacrylate PMMA.

Embodiment 3: Different from the above embodiment 2, as shown in FIGS. 3 and 4 , it is an embodiment of the cell migration high-resolution impedance real-time tracking measurement and control device in which the cavity 1 is an integral structure of the present invention. The cavity 1 is an integral structure, that is, the upper cavity 1.1, the lower cavity 1.2 and the base 1.3 that closes the lower end of the lower cavity 1.2 are an integral component; the aperture of the lower chamber 2.2 is smaller than the aperture of the upper chamber 2.1, and the microporous film 3 is set on the microporous film ring 4 and embedded in the upper chamber 2.1. The upper cavity 1.1, the lower cavity 1.2 and the base 1.3 of the cavity 1 of the overall structure are processed from an integral material. The material is a polymer material used in the field of microfluidics, which is selected from PDMS, that is, polydimethylsiloxane Methyl siloxane, or thermoplastic material polymethyl methacrylate PMMA, or polycarbonate PC, or polystyrene PS, polycarbonate PC is selected in this embodiment.

Example 4: The manufacturing method of the cell migration high-resolution impedance real-time tracking measurement and control device with a combined structure according to the present invention Example: The cell migration high-resolution impedance real-time tracking measurement and control device includes a combined structure cavity 1, namely the upper cavity The body 1.1, the lower cavity 1.2 and the base 1.3 closing the bottom of the lower cavity 1.2 are three independent components; the manufacturing method includes the following steps:

The first step is to prepare cavity raw materials: mix the cavity raw material PDMS monomer/curing agent in a ratio of 10:1 to prepare liquid PDMS;

The second step, raw material curing: pour the liquid cavity raw material PDMS on the silicon wafer or glass slide, and bake it in a 65-90 degree oven for 25-40 minutes to solidify the cavity raw material PDMS into a cavity blank;

The third step is to make the cavity: cut the cavity blank into the shape of the upper cavity 1.1 and the lower cavity 1.2; then process the upper cavity 2.1 and the lower cavity 2.2 and the electrode hole 6 and the liquid inlet and outlet channel 5; the electrode hole 6 One is close to the lower end surface of the upper cavity 1.1, and the other is close to the upper end surface of the lower cavity 1.2;

The fourth step, assembly: spread the microporous film 3 on the upper end surface of the lower cavity 1.2, then place the upper cavity 1.1 on the microporous film 3 on the upper end surface of the lower cavity 1.2, and then place the lower cavity 1.2 The lower end surface is placed on the base 1.3, and finally the upper end surface of the lower cavity 1.2, the lower end surface of the upper cavity 1.1 and the microporous film 3 are bonded together by the high vacuum oxygen plasma method; The lower end surface of 1.2 is bonded to the base 1.3;

The fifth step is to insert the upper chamber electrode 6.1 and the lower chamber electrode 6.2 into the upper chamber electrode hole and the lower chamber electrode hole respectively, and connect the two electrodes with the analysis device 7 when in use.

Embodiment 5: It is the application of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention in the fields of cell biology research, drug screening and disease diagnosis.

Embodiment 6: It is the basic embodiment of the real-time measurement and control device for cell migration high-resolution impedance tracking and control device of the present invention applied to the real-time measurement and control method of cell transmembrane migration: the cell migration high-resolution impedance real-time tracking measurement and control device includes a cavity 1, the cavity The middle part of the body 1 has a chamber 2 capable of holding liquid, and the middle part of the chamber 2 is divided into an upper chamber 2.1 and a lower chamber 2.2 by a microporous film 3 in the middle; the real-time measurement and control method includes the following steps:

A. Disinfect and sterilize the cavity 1;

B. From the inlet of the upper chamber 2.1, the upper chamber 2.1 is perfused with a sufficient amount of cell-containing culture solution, and the cell content is: 10 ⁵ -10 ⁷ cells per milliliter of the cell-containing culture solution.

C. The liquid inlet and outlet channel 5 of the lower chamber 2.2 is fully perfused with a cell-free culture solution and/or a medicinal solution for the experiment;

D. After the cells have been cultured for a certain period of time, inject a culture solution containing an appropriate concentration of platelet-derived growth factor to replace the original cell-free culture solution in the lower chamber 2.2 as an inducer for cell migration; under the action of the inducer, the cells pass through The microporous film 3 gradually migrates from the upper chamber 2.1 to the lower chamber 2.2;

E, the upper chamber electrode 6.1 is a working electrode, and the lower chamber 2.2 is a reference electrode, which are closely distributed on the upper and lower sides of the microporous film 3 respectively. These two electrodes are connected with the analysis device 7 for performing the migration process of the cells Real-time measurement and control, including the detection of impedance changes caused by the migration of single cells.

Embodiment 7: The difference from the above-mentioned embodiment 6 is that this embodiment is a better specific embodiment of the real-time measurement and control method of the cell migration high-resolution impedance tracking measurement and control device of the present invention applied to cell transmembrane migration. On the basis of embodiment 6, wherein

C. A sufficient amount of cell-free culture solution is perfused from the liquid inlet and outlet channel 5 of the lower chamber 2.2;

D. After the cells have been cultured for 12 to 18 hours, inject a culture solution containing an appropriate concentration of platelet-derived growth factor to replace the original cell-free culture solution in the lower chamber 2.2 as an inducer for cell migration; the role of cells in the inducer Next, move from the upper chamber 2.1 to the lower chamber 2.2 successively through the microporous membrane 3;

E, the upper chamber electrode 6.1 is a platinum working electrode, and the lower chamber 2.2 is a silver/silver chloride reference electrode, which are closely distributed on the upper and lower sides of the microporous film 3 respectively. These two electrodes are connected with the analysis device 7 for Real-time measurement and control of cell migration process, including detection of impedance changes caused by single cell migration.

Embodiment 8: The difference from the above embodiment 6 is that this embodiment is another embodiment of the application of the cell migration high-resolution impedance real-time tracking measurement and control device of the present invention to the real-time measurement and control method of cell transmembrane migration. In step B of the above-mentioned embodiment 6, the cell-containing culture solution injected into the upper chamber 2.1 in sufficient amount is the cell-containing culture solution containing 10 ⁵ -10 ⁷ cancer cells U-87 per milliliter.

Embodiment 9: The difference from the above embodiment 6 is that this embodiment is another embodiment of the high-resolution impedance real-time tracking measurement and control device for cell migration of the present invention applied to the real-time measurement and control method for cell transmembrane migration. In the above embodiment In step B of 6, the cell-containing culture solution injected into the upper chamber 2.1 in sufficient amount is the cell-containing culture solution containing 10 ⁵ -10 ⁷ cancer cells HCT-116 per milliliter; The cells that migrated to the lower chamber 2.2 were incubated with the calcein-AM stock solution at a given time and then used for fluorescence imaging with an inverted optical microscope; G. For comparison, the cells that migrated to the lower chamber 2.2 were used at different times Observe and count with an optical microscope, and collect data under the action of different concentrations of chemical inducers, and compare with the results obtained by impedance measurement.

In conjunction with accompanying drawing 5-10, technical scheme, principle and remarkable effect of the present invention are further described as follows:

Fig. 5 is a scanning electron microscope image of a microporous film. The surface of the film is smooth, with irregular holes distributed therein, and its diameter is about 8 microns; Fig. 6 is a scanning electron microscope image of U-87 cells attached to the above-mentioned microporous film, It shows that the cells are slightly larger than the pores on the film, and can adhere and spread on the film; Figure 7 is the spectrum of electrical impedance change corresponding to the migration of U-87 cells through the 8-micron diameter hole, indicating that the cell migration can cause the film to double. The change of terminal electrical impedance, the decrease of impedance indicates that cells pass through the micropore of the film, and the increase of impedance indicates that new cells migrate to the micropore, which can be used to detect the migration behavior of cells in real time, and calculate the micropore passing through the film within a specific time. The number of cells in the well. The graph was measured after chemical inducer injection at a frequency of 80 kHz. The large picture is the recording over 60 minutes, the inset shows the change in electrical impedance for the next 120 minutes, where the smallest drop is about 3 ohms and corresponds to a cell passing through the micropore of the membrane, by the change in impedance during this time The integral can be used to calculate the total number of cells passing through the micropores of the film during this time; Figure 8 is the spectrum of electrical impedance changes corresponding to the transmembrane migration of HCT-116 cells through the 8 micron diameter hole, which shows that the cell migration can cause the membrane to double. The change of terminal electrical impedance, the decrease of impedance indicates that cells pass through the micropore of the film, and the increase of impedance indicates that new cells migrate to the micropore, which can be used to detect the migration behavior of cells in real time, and calculate the micropore passing through the film within a specific time. The number of cells in the well. The graph was measured after the injection of the chemical inducer at a frequency of 80 kHz; the large graph is the recording over 60 minutes, and the inset is the graph of the change in electrical impedance for the next 120 minutes, where the smallest drop is about 3 ohms and corresponds to a Cells pass through the micropores of the film, and the total number of cells passing through the micropores of the film can be calculated by integrating the impedance change during this time; Figure 9 shows the transmembrane migration on the substrate of the lower chamber for 3 hours Fluorescence profile of HCT-116 cells, the total number of cells that migrated through the membrane after 3 hours can be determined by counting the number of cells that is consistent with the migration of cells during 3 hours calculated using the assay described in this patent The total number passing through the film is consistent; Figure 10 is a graph showing the change of HCT-116 cell density over time on the substrate of the lower chamber under the action of different concentrations of chemical inducers, indicating that the concentration of chemical inducers is greater than or equal to 10 nanograms In the case of per milliliter, the speed of cell migration through the micropores of the film will not vary depending on its concentration, that is, the present invention can be used to monitor the quantitative research of different chemical inducers on cell migration behavior.

The protection scope of the claims of the present invention is not limited to the above-mentioned embodiments.

Claims (4)

1. a manufacture method for cell migration high-resolution impedance real-time follow-up measure and control device, is characterized in that:

Cell migration high-resolution impedance real-time follow-up measure and control device comprises: draw together cavity (1), there is the chamber (2) of energy receiving fluids at this cavity (1) middle part, its middle part of this chamber (2) is divided into upper chamber (2.1) and lower chambers (2.2) by a microporous membrane (3) by chamber (2), microporous membrane (3) top is upper cavity (1.1), and bottom is lower chamber (1.2); The electrode hole (6) being communicated with to internal cavities from outside is located to have respectively in the top of the bottom of upper cavity (1.1) and lower chamber (1.2) near microporous membrane (3); What the electrode hole (6) of upper cavity (1.1) inserted is upper chamber's electrode (6.1), and what the electrode hole (6) of lower chamber (1.2) inserted is lower chambers electrode (6.2), and two electrodes connect with analytical equipment (7) respectively; Liquid access way (5) with outside UNICOM is arranged at the bottom of lower chamber (1.2); Cavity (1) is unitized construction, and upper cavity (1.1) is three independently members with the base (1.3) of lower chamber (1.2) and sealing lower chamber (1.2) bottom; Upper chamber (2.1) and lower chambers (2.2) once or respectively process; Microporous membrane (3) is clipped between the lower surface of upper cavity (1.1) and the upper surface of lower chamber (1.2); Upper cavity (1.1) is tightly connected with lower chamber (1.2) joint portion; The lower surface of lower chamber (1.2) and independently base (1.3) are tightly connected;

Manufacture method comprises the steps:

The first step, preparation cavity raw material: cavity raw material is mixed with to liquid state;

Second step, raw material solidify: liquid cavity raw material is poured on silicon chip or slide, in baking oven, dries the some time, make cavity raw material be solidified into cavity blank;

The 3rd step, making cavity: the shape that cavity blank is cut into upper cavity (1.1) and lower chamber (1.2); Then process upper chamber (2.1) and lower chambers (2.2) and electrode hole (6) and liquid access way (5); (6) lower surfaces near upper cavity (1.1) of electrode hole, another is near the upper surface of lower chamber (1.2);

The 4th step, assembling: microporous membrane (3) is laid on the upper surface of lower chamber (1.2), then upper cavity (1.1) is placed on the microporous membrane (3) on lower chamber (1.2) upper surface, again the lower surface of lower chamber (1.2) is placed in to base (1.3) upper, finally uses high vacuum oxygen plasma method that the lower surface of the upper surface of lower chamber (1.2) and upper cavity (1.1) and microporous membrane (3) are bonded together; In kind the lower surface of lower chamber (1.2) and base (1.3) are bonded together again;

Wu Bu,Jiang upper chamber electrode (6.1) and lower chambers electrode (6.2) insert respectively in epicoele electrode hole and cavity of resorption electrode hole.

2. cell migration high-resolution impedance real-time follow-up measure and control device is applied to a real-time investigating method for cell transmembrane migration, it is characterized in that:

Cell migration high-resolution impedance real-time follow-up measure and control device comprises cavity (1), there is the chamber (2) of energy receiving fluids at this cavity (1) middle part, its middle part of this chamber (2) is divided into upper chamber (2.1) and lower chambers (2.2) by a microporous membrane (3) by chamber (2), microporous membrane (3) top is upper cavity (1.1), and bottom is lower chamber (1.2); The electrode hole (6) being communicated with to internal cavities from outside is located to have respectively in the top of the bottom of upper cavity (1.1) and lower chamber (1.2) near microporous membrane (3); What the electrode hole (6) of upper cavity (1.1) inserted is upper chamber's electrode (6.1), and what the electrode hole (6) of lower chamber (1.2) inserted is lower chambers electrode (6.2), and two electrodes connect with analytical equipment (7) respectively; Liquid access way (5) with outside UNICOM is arranged at the bottom of lower chamber (1.2);

The method of observing and controlling in real time comprises the steps:

A, by cavity (1) sterilizing that carries out disinfection;

The nutrient solution that cell is contained to the enough perfusions of upper chamber (2.1) in B ,Cong upper chamber (2.1) porch, its cell content is: in the nutrient solution that every milliliter contains cell, have 10 ⁵～10 ⁷individual cell;

C, from the not celliferous nutrient solution of the enough perfusions of liquid access way (5) of lower chambers (2.2), and/or the liquid for testing;

D, after cell is cultivated certain hour, inject nutrient solution containing appropriate concentration platelet derived growth factor and replace former not celliferous nutrient solution in lower chambers (2.2) as the derivant of cell migration; Cell, under the effect of derivant, moves to lower chambers (2.2) from upper chamber (2.1) successively by microporous membrane (3);

E, upper chamber's electrode (6.1) are working electrode, lower chambers (2.2) is contrast electrode, tight distribution is in the both sides up and down of microporous membrane (3) respectively, this two electrode is connected with analytical equipment (7), for the transition process of cell is carried out to real-time observing and controlling, comprise that the impedance variation that single celled migration is caused detects.

3. cell migration high-resolution impedance real-time follow-up measure and control device as claimed in claim 2 is applied to the real-time investigating method of cell transmembrane migration, it is characterized in that, the nutrient solution that contains cell of the enough injections of described B step Zhong，Xiang upper chamber (2.1) is every milliliter and contains 10 ⁵～10 ⁷the nutrient solution that contains cell of individual cancer cell U-87.

4. cell migration high-resolution impedance real-time follow-up measure and control device as claimed in claim 2 is applied to the real-time investigating method of cell transmembrane migration, it is characterized in that, the nutrient solution that contains cell of the enough injections of described B step Zhong，Xiang upper chamber (2.1) is every milliliter and contains 10 ⁵～10 ⁷the nutrient solution that contains cell of individual cancer cell HCT-116; Also have:

F, in the selected time, the calcein-AM stoste for cell that moves to lower chambers (2.2) is hatched and with inversion optical microscope, is carried out fluorescence imaging afterwards;

G, be for relatively, at different time, will move to observation by light microscope the counting for cell of lower chambers (2.2), and under the effect of variable concentrations chemical inducer image data, the result obtaining with impedance measurement compares.

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