WO2022183370A1 - 一种基于细胞阻抗传感的抗肿瘤药物筛选的方法 - Google Patents

一种基于细胞阻抗传感的抗肿瘤药物筛选的方法 Download PDF

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WO2022183370A1
WO2022183370A1 PCT/CN2021/078672 CN2021078672W WO2022183370A1 WO 2022183370 A1 WO2022183370 A1 WO 2022183370A1 CN 2021078672 W CN2021078672 W CN 2021078672W WO 2022183370 A1 WO2022183370 A1 WO 2022183370A1
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cell
cells
impedance
conductive
tumor
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French (fr)
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黄建永
姜楠
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance

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  • the present disclosure relates to the technical field of cell impedance sensing detection, in particular to a method for antitumor drug screening based on cell impedance sensing.
  • Drug screening refers to the process of selecting compounds with higher activity on a specific target from a large number of compounds or new compounds through standardized experimental methods.
  • the main technologies include genomics, proteomics, metabolomics, computational biology. Science, biochip technology, microfluidic chip technology and other methods.
  • the experimental process of drug screening takes the form of microplates as the carrier of experimental tools, performs the experimental process with an automated operating system, collects experimental data with sensitive and fast detection instruments, and analyzes and processes the data obtained from the experiments with a computer.
  • Drug screening is a screening at the biochemical level (molecular level) and at the cellular level. Screening at the cellular level includes: ion channel monitoring (including ion concentration, membrane potential, pH value, etc.), reporter gene detection and yeast two-hybrid technology.
  • Cell-level drug screening models have the advantages of less material consumption, relatively clear drug action mechanisms, and large-scale screening. Rapid and high-sensitivity detection technology is one of the key technologies for high-throughput drug screening.
  • the high-throughput screening of antitumor drugs at the cellular level mainly uses the selection of tumor cell lines, cultured cells as experimental models, and detection techniques such as liquid scintillation counting, colorimetry, chemiluminescence, and fluorescence detection. , cytotoxicity, cell cycle regulation and signal transduction are the detection objects for drug screening.
  • the scope and accuracy of drug screening are greatly limited due to the limitations of markers and the use of end-point detection methods.
  • Successful screening can shorten the research and development cycle of innovative drugs, reduce costs, reduce risks, and improve efficiency, so there is a need to accelerate the innovation of in vitro cellular drug screening methods.
  • Biochip technology is a miniature biochemical analysis system that integrates discontinuous analysis processes in the field of life sciences on the surface of silicon chips or glass chips through microscopic technology and based on the principle of specific interaction between molecules, so as to realize the analysis of cells and proteins. , Accurate, rapid and informative detection of genes and other biological components.
  • Biochip-based cell impedance sensing (Electric Cell Impedance Sensing, ECIS for short) is a real-time cell detection system based on electronic impedance, which can perform high-throughput drug screening through phenotypic characteristics such as cell adhesion, morphology and growth. .
  • This method is a label-free, cell-based assay that enables real-time, label-free, non-invasive and dynamic recording of cell proliferation, cytotoxicity, and cytotoxicity in a near-physiological environment from days to weeks through the interaction of cells with electrodes at the bottom of the assay plate. It provides a more accurate and sensitive detection platform for comprehensively understanding the activity and invasiveness of cells, and can synthesize a small amount of lead compounds for cytotoxicity evaluation in the early stage of new drug development, which has good application prospects. .
  • ECIS-based drug screening at the cellular level in in vitro three-dimensional (3D) matrices can integrate a high-throughput, miniaturized screening platform for label-free whole-process dynamic quantitative monitoring, which is easy to operate and will provide new insights into cellular drug interactions. perspective.
  • the present disclosure provides a method for anti-tumor drug screening based on cell impedance sensing, which is used to at least partially solve the technical problems of traditional methods that damage living cells, thereby greatly limiting the dynamic collection of biological information throughout the entire process.
  • the present disclosure provides a method for anti-tumor drug screening based on cell impedance sensing, comprising: S1, performing surface treatment on a conductive chip provided with a microelectrode array; S2, covering the surface of the conductive chip with tumor cells; S3, applying Starvation of tumor cells and subsequent drug addition; S4, forming polymerized hydrogel on tumor cells and adding chemokines; S5, real-time detection of cell impedance, acquisition of cell impedance information, and drug screening based on impedance information .
  • S1 also includes: forming a photoresist layer on the conductive film layer of the conductive substrate; forming a photoresist pattern on the conductive substrate with the photoresist layer; and forming a photoresist pattern on the conductive substrate based on the photoresist pattern A conductive chip with a conductive film pattern is formed thereon, wherein the conductive film pattern is a micro-electrode array.
  • S1 also includes: forming a ring-shaped structure surrounding the micro-electrode array on the conductive chip, wherein the inner surface of the ring-shaped structure and the surface of the conductive chip where the micro-electrode array is located form a cell culture cavity.
  • S1 specifically includes: cleaning the conductive chip with methanol; using 3-aminopropyltriethoxysilane as a silane coupling agent to modify amino groups on the surface of the pre-cleaned conductive chip; using poly(styrene-co-maleic anhydride) ) solution modifies acid anhydride groups on the surface of conductive chips.
  • S2 specifically includes: the selected tumor cell line is human breast cancer cells; the tumor cell suspension is inoculated on the conductive chip after surface pretreatment, and cultured in complete medium.
  • S3 specifically includes: starvation treatment of cells with a medium containing embryonic bovine serum, washing of cells with Dulbecco's phosphate buffered saline; and drug addition to the starved cells with paclitaxel solutions of different concentrations.
  • S4 specifically includes: according to the preset ratio, the collagen type I solution, the modified Eagle's medium, the phosphate buffered saline solution, and the sodium hydroxide solution are mixed to form a polymerized hydrogel, wherein all reagents are Keep on ice and mix; chemokine is embryonic bovine serum in complete medium.
  • the preset concentration of the diluted collagen type I is 3-5.6 mg/mL; the final concentration of collagen type I in the solution mixed according to the preset ratio is 2-4.5 mg/mL; according to the preset The pH of the mixed solution is neutralized to 7.3-7.5.
  • S5 specifically includes: frequency sweep measurement with an output frequency of 10 kHz to 100 kHz and an output sinusoidal voltage of 10 mV to 30 mV for the cell impedance sensing chip that is undergoing colony cell invasion in the three-dimensional matrix after the anti-tumor drug acts;
  • the impedance information is based on the impedance sweep measurement at multiple time points when the chemokine is added to the hydrogel to zero, so as to obtain the cell impedance values at different frequencies and different time points.
  • the drug screening according to the impedance information in S5 specifically includes: obtaining cell impedance values at different frequencies, characterizing the invasion distance of the population cells in the three-dimensional matrix over time with the change of the relative impedance value, and then obtaining the information of the cell invasion process. Drug Screening.
  • An embodiment of the present disclosure provides a method for screening anti-tumor drugs based on cell impedance sensing. By adding drugs to tumor cells, it induces colony cell invasion in collagen type I gel, and effectively realizes the process of cell invasion in a three-dimensional matrix.
  • the high-efficiency and stable cell impedance sensing detection enables the quantitative screening of anti-tumor drugs to achieve the technical effect of real-time, label-free, and continuous dynamic detection.
  • FIG. 1 schematically shows a flow chart of a method for anti-tumor drug screening based on cellular impedance sensing according to an embodiment of the present disclosure
  • FIG. 2 schematically shows a schematic flowchart of preparing a conductive chip with a microelectrode array and a cell culture chamber according to an embodiment of the present disclosure
  • FIG. 3 schematically shows a schematic diagram of the composition of a cell impedance sensing system according to an anti-tumor drug quantitative screening method according to an embodiment of the present disclosure
  • FIG. 4 schematically shows a schematic diagram of performing cell impedance detection on the surface of a conductive chip according to an embodiment of the present disclosure
  • FIG. 5 schematically shows a physical diagram of a cell impedance sensing chip according to an embodiment of the present disclosure
  • FIG. 6 schematically shows a fluorescent labeling diagram of a single cell after the cell is acted on by an anti-tumor drug according to an embodiment of the present disclosure
  • FIG. 7-a schematically shows a physical view of the lower side of the small chamber according to an embodiment of the present disclosure
  • FIG. 7-b schematically shows a cell staining image through an 8 ⁇ m pore size polycarbonate membrane under an inverted microscope IX41 according to an embodiment of the present disclosure
  • Fig. 7-c schematically shows the statistical result diagram of the number of invasive cells after the action of different concentrations of paclitaxel according to the embodiment of the present disclosure
  • Fig. 7-d schematically shows the statistical result graph of absorbance value (cell activity) after the action of different concentrations of paclitaxel according to the embodiment of the present disclosure
  • Figures 8a-d schematically show the morphological features of collagen type I gels according to embodiments of the present disclosure
  • FIGS 8e-f schematically show results of quantitative topology analysis according to an embodiment of the present disclosure
  • FIG. 9 schematically shows the corresponding relationship diagram of the invasion distance and speed of cells in a three-dimensional matrix after the action of an anti-tumor drug according to an embodiment of the present disclosure
  • Fig. 10-a schematically shows the corresponding relationship between invasion distance and time obtained by impedance sensing detection of cells after the action of antitumor drugs in an embodiment of the present disclosure
  • Fig. 10-b schematically shows the corresponding relationship between the relative impedance value and the time obtained by the impedance sensing detection of the cells after the action of the anti-tumor drug according to the embodiment of the present disclosure.
  • Cytotoxicity is the key to the effect of anticancer drugs, and cell death caused by cytotoxicity can be divided into programmed and non-programmed cells, the latter being necrosis. Drugs can regulate the corresponding cell death signaling pathways, thereby inhibiting or inducing cell death. In-depth research on cell death signaling pathways can provide new targets for drug development. On the contrary, in the process of researching the mechanism of antitumor drugs In this study, paying close attention to its effects on cell death pathways can further understand cell death.
  • cytotoxicity assay methods include trypan blue staining, clone (colony) formation, 3H radioisotope incorporation, MTT, and ATP detection.
  • end-point detection methods which only provide a final result for the experiment, and often require labeling, thereby causing damage to cells. Since cells are living bodies and biological cell processes are dynamic rather than static, end-point detection methods greatly limit the dynamic collection of biological information throughout the entire process.
  • Biochip-based cell impedance sensing technology can realize real-time, label-free, continuous and dynamic detection of cell phenotype changes in the form of electrical impedance.
  • a microelectrode array is integrated at the bottom of the conductive glass chip, and the obtained cell impedance value can be output in the form of cell index (CI), which can quantitatively evaluate the physiological state of cells, including cell number, survival rate, and cell morphological changes.
  • Impedance-based kinetic profiles can provide information on the transient effects of compound-induced cytotoxicity.
  • ECIS detection can accurately determine the time point at which the cytotoxicity mediated by the compound exerts the maximum effect, which is helpful for the research and disclosure of the mechanism of drug action.
  • An embodiment of the present disclosure provides a method for anti-tumor drug screening based on cell impedance sensing, please refer to FIG. 1 , including: S1, performing surface treatment on a conductive chip provided with a microelectrode array; S2, laying on the surface of the conductive chip Tumor cells; S3, starvation of tumor cells and subsequent drug addition; S4, formation of polymerized hydrogel on tumor cells and addition of chemokines; S5, real-time cell impedance detection to obtain cell impedance information, Drug screening based on impedance information.
  • the conductive chip of the type I collagen (type I collagen, abbreviated as Coll I) gel with three-dimensional culturable cells disclosed above is used as a detection electrode to be electrically connected to an impedance spectrometer to construct a cell impedance
  • tumor cells were inoculated on the microelectrode array of the conductive chip, and after the cells were overgrown and different concentrations of drugs were added, a Coll I gel suitable for the tumor invasion model was prepared on the cell layer through a self-assembly fibrosis process.
  • the impedance spectrometer is connected to the detection electrode (ie, the cell impedance sensing chip) where the cell to be detected is located through the wire through the metal clip, so as to measure the impedance value of the cell to be detected.
  • FIG. 2 is a flow chart of preparing a conductive chip with a microelectrode array and a cell culture chamber, which specifically includes: forming a photoresist layer on the conductive film layer of the conductive substrate; forming a photoresist layer on the conductive substrate with the photoresist layer A photoresist pattern; and forming a conductive chip with a conductive film pattern on a conductive substrate based on the photoresist pattern, wherein the conductive film pattern is a microelectrode array.
  • FIG. 3 is a schematic diagram of the composition of the cell impedance sensing system for the quantitative screening method of antitumor drugs.
  • FIG. 4 is a schematic diagram of the cell impedance detection on the surface of the conductive chip.
  • it further includes: forming a photoresist layer on the conductive film layer of the conductive substrate; forming a photoresist pattern on the conductive substrate with the photoresist layer; and based on the photoresist The pattern forms a conductive chip with a conductive film pattern on the conductive substrate, wherein the conductive film pattern is a micro-electrode array.
  • ITO conductive glass can be used as the substrate of the conductive chip for preparation.
  • the conductive chip can also select other substrates with a conductive thin film structure.
  • it can be formed according to the following preparation process:
  • ITO conductive glass with a size of 40mm ⁇ 40mm ⁇ 0.4mm and a conductive film thickness of about 185nm was selected as the substrate of the conductive chip, and the UV negative photoresist was spin-coated on the substrate at a low speed of 300rpm for 20s and a high speed of 1000rpm for 30s.
  • a photoresist layer is formed on the conductive film layer of the ITO conductive glass, that is, on the conductive film layer of the conductive substrate (ITO conductive glass), in preparation for forming a photoresist pattern in the next step.
  • Pre-baking The above-mentioned ITO conductive glass with a photoresist layer is placed on a heating plate, and it is baked at a temperature of 110° C. for 60s to preliminarily cure the photoresist layer.
  • the ITO conductive glass with the cured photoresist layer was exposed to ultraviolet light (UV) for 20 s through the printed mask using a mask aligner.
  • UV ultraviolet light
  • the electrodes used in the mask are interdigitated electrodes, and the size can be 1 cm long, 30-100 ⁇ m wide, and the distance between adjacent electrodes can be 30-100 ⁇ m;
  • the exposure machine used is: UV depth lithography machine, and the exposure UV light intensity can be : 18 mW/cm 2 , that is, a photoresist pattern is formed on a conductive substrate with a photoresist layer.
  • the ITO conductive glass with the photoresist layer that has been exposed above is placed on a heating plate, and the exposed ITO conductive glass is baked at a temperature of 145° C. for 60s to further cure the photoresist layer.
  • a conductive chip with a conductive film pattern is formed on the substrate, wherein the conductive film pattern is a micro-electrode array.
  • Degumming Use photoresist degumming solution to ultrasonically remove the remaining photoresist for 8 minutes, rinse with deionized water, and dry with nitrogen for the next step of surface pretreatment of the conductive chip.
  • S1 further includes: forming a ring structure surrounding the microelectrode array on the conductive chip, wherein the inner surface of the ring structure and the surface of the conductive chip where the microelectrode array is located form a cell culture cavity.
  • a cell culture chamber is formed around the microelectrode array. Specifically, after preparing the microelectrode array ITO conductive glass chip, a quartz glass ring is selected as the annular structure of the present disclosure, and the microelectrode array surrounding the conductive chip is bonded to the conductive chip to form a preliminary cell culture chamber.
  • the bonded cell culture cavity structure was placed in a 70° C. oven to dry to form the final cell culture cavity.
  • Figure 5-A is a physical photo of a cell impedance sensing chip used to monitor the invasion of colony cells in a three-dimensional matrix
  • Figure 5-B is the overall electrode after ITO conductive glass etching.
  • Fig. 5-C is the partial magnified view of the ITO conductive glass interdigital electrode array under the fluorescence inverted microscope IX71
  • Fig. 5-D is the part of the ITO conductive glass interdigital electrode array under the stereo microscope Enlarge the image.
  • the upper and lower layers of the printed circuit board (PCB) with conductive lines are used as fixtures to fix the ITO conductive glass chip with the cell culture chamber, and the diameter of the ITO glass chip is fixed by soldering.
  • the 0.2mm copper wire electrically connects the conductive surface of the ITO conductive glass with the conductive lines of the PCB board.
  • the PCB board printed with the conductive circuit is electrically connected with the external impedance spectrometer by means of soldering, which is used for the subsequent cell impedance sensing detection for monitoring the invasion of the colony cells in the three-dimensional matrix.
  • the orange strip is the conductive film layer of the ITO glass
  • the white strip is the lower glass after the conductive film layer of the ITO glass is wet-etched.
  • the width of the interdigital electrodes is 100 ⁇ m, adjacent to The electrode spacing was 100 ⁇ m.
  • the microelectrode array is 47 pairs of long strip-shaped interdigital electrode arrays with a length of 10 mm.
  • S1 specifically includes: cleaning the conductive chip with methanol; using 3-aminopropyltriethoxysilane as a silane coupling agent to modify amino groups on the surface of the pre-cleaned conductive chip; using poly(styrene- Co-maleic anhydride) solution modifies acid anhydride groups on the surface of conductive chips.
  • the conductive chip photoetched with the microelectrode array needs to be surface-treated for the subsequent formation of the polymerized hydrogel in the glass dish.
  • the presence enables lysine side chains to covalently bond to the gel surface, thereby immobilizing type I collagen gels suitable for tumor invasion models on the surface of conductive chip dishes with reactive copolymer coatings.
  • PSMA poly(styrene-co-maleic anhydride)
  • PSMA Poly(styrene-alt-maleic anhydride)
  • S2 specifically includes: the selected tumor cell line is human breast cancer cells; the tumor cell suspension is inoculated on the conductive chip after surface pretreatment, and cultured in complete medium.
  • the surface of the pretreated conductive chip is covered with selected tumor cell lines, and the selected tumor cell lines can be triple-negative human breast cancer cells MDA-MB-231 and MDA-MB-436.
  • MDA-MB-231 and MDA-MB-436 cells were digested with 0.25% Trypsin-EDTA solution for 1 min.
  • 40 ⁇ 10 4 number of MDA-MB-231 and the cell suspension of MDA-MB-436 cells were seeded into conductive chip dishes with surface pretreatment, and placed in a 37°C 5% CO 2 cell incubator for 24 h in modified Eagle (DMEM) high-glucose complete medium. For subsequent starvation of tumor cells and subsequent drug addition.
  • DMEM modified Eagle
  • S3 specifically includes: starvation treatment of cells with medium containing embryonic bovine serum, washing of cells with Dulbecco's phosphate buffered saline; and drug addition to the starved cells with paclitaxel solutions of different concentrations.
  • the tumor cells in the conductive chip dish were starved and drug-added afterward.
  • the selected tumor cells were cultured in complete medium for 24 hours, the cells were washed twice with 1 ⁇ DPBS, and then the cells were starved for 24 hours with medium containing 0.5% embryonic bovine serum (FBS). 90% to 100% saturation should be achieved.
  • FBS embryonic bovine serum
  • the cells were washed twice with 1 ⁇ DPBS to remove cell impurities, and then the starved cells were added with different concentrations of paclitaxel (PTX) solution for 2 h.
  • PTX paclitaxel
  • the culture medium containing embryonic bovine serum is the modified Eagle's high glucose medium supplemented with penicillin-streptomycin mixed double antibodies; the paclitaxel solutions of different concentrations are in the concentration range of 10 nmol/L to 30 nmol/L. L.
  • the above-mentioned medium containing 0.5% FBS is DMEM high glucose medium supplemented with 1% double antibody (mixed double antibody to penicillin-streptomycin), and the paclitaxel solution of different concentrations is a concentration gradient of 10nmol/L, 20nmol/L, 30nmol/L of paclitaxel solution.
  • paclitaxel solution first dissolve 10 mg PTX powder recovered to room temperature with 1.17 mL of DMSO and vortex to obtain 10 mmol/L stock solution, and then dilute the stock solution with 0.5% FBS-containing medium to the working concentration.
  • Dosing for breast cancer cells are shown in Figure 6.
  • a, b, c, and d are the control group and the monotherapy after adding the paclitaxel solution with concentrations of 10 nmol/L, 20 nmol/L and 30 nmol/L for 2 hours. Fluorescent labeling of cells. Therefore, it can be seen that with the increase of the concentration of paclitaxel, the spreading area of the cytoskeleton gradually decreases, and the invasiveness and activity of the cells decrease accordingly.
  • Paclitaxel can debalance tubulin and tubulin dimers that make up microtubules, induce and promote tubulin polymerization, microtubule assembly, and prevent depolymerization, thereby stabilizing microtubules and inhibiting cancer cell mitosis and triggering Cell apoptosis, and then effectively prevent the proliferation of cancer cells, play an anti-cancer effect.
  • the short-term disintegration of spindle microtubules can preferentially kill abnormally dividing cells.
  • paclitaxel works by stabilizing tubulin, and paclitaxel has been found to show good effects on a variety of solid tumors. Thus, uncharted territory of cellular activity and new methods of discovering new anticancer drugs can be explored by paclitaxel.
  • S4 specifically includes: according to the preset ratio, the collagen type I solution, the modified Eagle's medium, the phosphate buffered saline solution, and the sodium hydroxide solution are mixed to form a polymerized hydrogel, Wherein, all reagents were kept and mixed on ice; chemokine was embryonic calf serum in complete medium.
  • forming a polymeric hydrogel on a microelectrode array covered with a cell layer includes: diluting a high-concentration type I collagen (Coll I) with an acetic acid solution to a preset concentration; according to a preset ratio, The diluted type I collagen (Coll I) solution, modified Eagle's medium, phosphate buffered saline (PBS), and sodium hydroxide solution (NaOH) were mixed to form a polymeric hydrogel, wherein all reagents were in Keep and mix on ice (4°C) to prevent self-polymerization of type I collagen monomers.
  • PBS phosphate buffered saline
  • NaOH sodium hydroxide solution
  • the so-called polymeric hydrogel may be a type I collagen gel with many nano- and micro-structural features of the extracellular matrix (ECM) in vivo formed through a self-assembled fibrosis process, specifically by in situ Self-assembled fibrosis is formed.
  • ECM extracellular matrix
  • the type I collagen with a concentration of 8.9 to 10.9 mg/mL, an acetic acid solution with a molar concentration of 0.02 mol/L, a DMEM medium with a concentration of 1 ⁇ , and a A phosphate buffered saline solution with a concentration of 10 ⁇ and a sodium hydroxide solution with a molar concentration of 0.5 mol/L are mixed evenly by vortexing to obtain a mixed hydrogel.
  • the molar concentration of the weighed or prepared acetic acid solution may be 0.02 mol/L; the concentration of high-concentration Coll I may be 8.9-10.9 mg/L mL; the concentration of DMEM medium may be 1 ⁇ ; the concentration of phosphate buffered saline may be 10 ⁇ ; and the molarity of the solution of sodium hydroxide may be 0.5 mol/L.
  • the preset concentration of collagen type I after dilution is 3-5.6 mg/mL; the final concentration of collagen type I in the solution mixed according to the preset ratio is 2-4.5 mg/mL mL; the pH of the mixed solution is neutralized to 7.3-7.5 according to the preset ratio.
  • the acetic acid solution can dilute the high-concentration type I collagen (Coll I) to a preset concentration; the sodium hydroxide solution can neutralize the pH of the solution mixed according to the preset ratio to 7.3-7.5.
  • type I collagen gel through the in-situ self-assembly fibrosis process can be formed according to the following preparation process, including: after mixing the above-weighed constituent materials or solutions, rapidly Sonicate for 30 s, pipette 1 mL of solution into the chip dish, replace the medium containing 0.5% FBS, and cover the MDA-MB-231 or MDA-MB-436 cells attached to the microelectrode array at the same time.
  • NHS-Rhodamine fluorescent labeling was performed on the gelated type I collagen gel fibers to characterize the topological features of the three-dimensional Coll I matrix.
  • the fluorescently labeled protein reagent used was NHS-Rhodamine (5-(and-6)-carboxytetramethylrhodamine succinimidyl ester, 5(6)-TAMRA-SE) solution with a molar concentration of 50 ⁇ mol/L.
  • NHS-Rhodamine (5-(and-6)-carboxytetramethylrhodamine succinimidyl ester, 5(6)-TAMRA-SE) solution with a molar concentration of 50 ⁇ mol/L.
  • the three-dimensional Coll I matrix was stained with 50 ⁇ mol/L NHS-Rhodamine at room temperature for 1 h and rinsed 3 times with 1 ⁇ PBS solution for 3 min each.
  • Three-dimensional Coll I substrates were imaged by confocal laser scanning microscopy with a 20 ⁇ objective at 4 ⁇ zoom.
  • the acquired images were 12-bit deep with a resolution of 1024 ⁇ 1024 pixels and a vertical stack size of 41 images (equivalent to 20 ⁇ m).
  • the voxel size of the acquired image was 0.15 ⁇ 0.15 ⁇ 0.5 ⁇ m (x ⁇ y ⁇ z).
  • the stacking was obtained at the approximately vertical center of the Coll I gel layer of thickness about 90 ⁇ m.
  • the fluorescent labeling results of Coll I fibers are shown in Figure 8-a, b, c, and d. It can be seen that with the increase of Coll I monomer concentration, the fibers obtained by self-assembly increase.
  • the topological parameters of different concentrations of Coll I gels were quantitatively analyzed by a MATLAB script custom-written in the laboratory to calculate the average pore size and pore size distribution of the matrix.
  • the above MATLAB script consists of 3 main parts, namely the erosion algorithm, the number of pixels and the autocorrelation algorithm with rotated Gaussian fitting. Through binary conversion and image segmentation, the average pore size and pore size distribution can be obtained by using the erosion algorithm.
  • Three independent experiments were performed, each time topological analysis was performed on at least 4 positions of each Coll I gel. The results of quantitative topology analysis are shown in Fig.
  • Embodiments of the present disclosure provide an easily accessible biomimetic in vitro platform for topologically defined Coll I proteins to dissect cellular behavior under various conditions in vitro.
  • cells were induced to invade in a three-dimensional gel matrix with 10% FBS in complete medium as a chemokine.
  • Cell migration through 3D ECM is an essential feature of physiological and pathological processes such as embryogenesis, immune monitoring, and wound healing. Efficient motility depends on the precise coordination of cell protrusion, adhesion and retraction mechanisms.
  • 3D migration is also sensitive to local extracellular signals that can integrate with established intracellular signals to influence migration patterns and efficiency and confer migration directionality by inducing cell polarity.
  • Directed cell migration is primarily caused by asymmetry in extracellular cues, including soluble factors (chemotaxis), fluid flow (taxis), electric fields (electrotaxis), stiffness (rigidaxis), and adhesion Ligand (Haptotaxis).
  • chemotaxis soluble factors
  • taxis fluid flow
  • electric fields electro fields
  • stiffness rigidaxis
  • adhesion Ligand Adesion Ligand
  • S5 specifically includes: sweep frequency measurement with the cell impedance sensing chip in the three-dimensional matrix undergoing colony cell invasion after the output frequency is 10 kHz-100 kHz and the output sinusoidal voltage is 10 mV-30 mV after the anti-tumor drug is acted on ;
  • the impedance sweep measurement is performed at multiple time points when the chemokine is added to the hydrogel to zero, so as to obtain the cell impedance values at different frequencies and different time points.
  • Cell impedance detection including: the effect of two-terminal impedance measurement mode (2-Terminal Impedance Measurement, referred to as 2-Term Z) with an output sinusoidal voltage of 10mV to 30mV and an output frequency of 10kHz to 100kHz on different concentrations of PTX in the three-dimensional matrix.
  • the swept frequency measurement was performed during the invasion process of the population of cells to obtain the cell impedance values at different frequencies; the cell impedance information was obtained by adding the complete medium to the Coll I gel at the time of zero, and performing the impedance sweep frequency measurement of multiple time periods.
  • the time interval of impedance sweep frequency measurement is 10min, 20min, 30min, 60min, 90min, 120min, 150min, 180min respectively.
  • the impedance information collected at the corresponding time point and detection frequency value was further analyzed and processed with the MATLAB script written by the laboratory, and the relative impedance value change, that is, the cell index (CI), was used to characterize the population cells in the three-dimensional matrix. Invasion distance, the larger the cell index, the larger the invasion distance of cells in the three-dimensional matrix.
  • the cell index (CI) was calculated according to the following formula:
  • N is the number of frequency points set for impedance measurement
  • R t ( fi ) and R 0 ( fi ) are the frequency-dependent impedance values of the conductive chip at time t and time zero.
  • Impedance sweep frequency measurement was performed on the conductive chips inoculated with 40 ⁇ 10 4 MDA-MB-231 cells treated with different concentrations of PTX to induce colony cell invasion. The frequency range was 10 kHz to 100 kHz, and the step size was 1 kHz. The applied sine The voltage is 30mV. Among them, the results are shown in Figure 9. At the same time point, with the increase of PTX concentration, the cell index (CI) gradually decreased; when the PTX concentration was the same, with the increase of invasion time, the CI tended to plateau stable phase.
  • the drug screening according to the impedance information in S5 specifically includes: obtaining cell impedance values at different frequencies, characterizing the invasion distance of the population cells in the three-dimensional matrix over time by relative impedance value changes, and then obtaining the cell invasion process. information for drug screening.
  • Quantitative cell impedance information is obtained by electrical connection with an impedance spectrometer for cell impedance detection.
  • ECIS technology to detect different cell lines will obtain different cell proliferation profiles, which can be used for high-throughput drug screening based on phenotypic characteristics such as cell adhesion, morphology and growth.
  • ECIS technology to detect cytostatics with different mechanisms of action can obtain characteristic cellular effect profiles, which can predict the mechanism of action of drugs in large-scale screening.
  • the pharmacodynamic characteristics of compounds detected by ECIS technology had a good correlation with traditional endpoint methods.
  • ECIS could prompt the optimal detection time for end-point detection.
  • ECIS technology provides a high-throughput, long-term method for compound screening and mechanism-based cluster analysis for compounds with different biological functions.
  • the following is an example of further characterizing the invasion distance of cells in a three-dimensional gel matrix with a live cell tracking probe after fluorescent labeling to verify the effectiveness of the method disclosed.
  • the breast cancer cells treated with drug for 2 h were digested with 0.25% Trypsin-EDTA solution, resuspended in serum-free medium, and the cell density was adjusted to 5 ⁇ 10 4 /mL.
  • the chamber was carefully taken out with tweezers, the liquid in the upper chamber was sucked dry, and transferred to a well pre-added with 800 ⁇ L of paraformaldehyde (PFA), and fixed at room temperature for 30 min.
  • PFA paraformaldehyde
  • the chamber was taken out, the fixative in the upper chamber was sucked dry, and transferred to a well pre-added with about 800 ⁇ L of 0.1% crystal violet solution, and stained at room temperature for 30 min. Gently rinse and soak 3 times with DPBS, remove the chamber, aspirate the upper chamber liquid, and carefully wipe off the cells on the membrane surface of the upper chamber bottom with a damp cotton swab. Randomly select 9 fields of view under the microscope to calculate and count the results.
  • Figure 7-a is the physical image of the lower side of the chamber
  • Figure 7-b is the stained image of cells passing through the 8 ⁇ m pore size polycarbonate membrane under the inverted microscope IX41
  • Figure 7-c is Statistical results of the number of invasive cells after different concentrations of paclitaxel. Therefore, it can be seen that as the concentration of paclitaxel increases, the number of cells that pass through the 8 ⁇ m pore polycarbonate membrane after digestion of Matrigel is also less, that is, the invasiveness of cells gradually decreases.
  • the results of this traditional endpoint detection method need to be further compared with the pharmacodynamic characteristics of the compounds detected by ECIS technology.
  • MTT cytotoxicity experiments were also performed on tumor cells after drug addition to evaluate the effect of paclitaxel on the viability of breast cancer cells.
  • 5000 cells were added to each well of a 96-well plate. According to the experimental requirements, the cells were first cultured in complete medium for 24 hours and then starved for 24 hours in medium containing 0.5% FBS.
  • Paclitaxel solution with concentration gradient of 10nmol/L, 20nmol/L, 30nmol/L and the control group were used for 2h, then 10 ⁇ L of MTT solution was added to each well, and the cells were incubated in a cell incubator for 4h.
  • fluorescently labeling the tumor cells after drug addition with a live cell tracking probe includes: removing the medium containing different concentrations of paclitaxel; washing the cells twice with DPBS; Hot probe working solution; incubate for 45 min at 37°C 5% CO 2 incubator; change to fresh medium containing 0.5% FBS and continue to incubate for 30 min, for the formation of type I suitable for tumor invasion model on subsequent cell layers collagen gel.
  • the probe working solution is a green live cell tracking probe (Cell-Tracker Green, CMFDA for short) solution with a concentration of 5-25 ⁇ mol/L.
  • CMFDA powder was taken out and returned to room temperature, centrifuged briefly to ensure that the powder fell to the bottom of the tube, and 50 ⁇ g of CMFDA was dissolved with 10.8 ⁇ L DMSO, and mixed thoroughly to obtain a 10 mmol/L storage solution.
  • CMFDA reagents are fluorescent chloromethyl derivatives that diffuse freely through living cell membranes. Once in cells, these mild thiol-reactive probes react with intracellular components to form fluorescent cells that survive at least 24 h after loading for subsequent tracking of colony cell invasion in three-dimensional matrices.
  • the invasion distance of the measured cells in the three-dimensional gel matrix is the invasion distance in the Z direction.
  • the Coll I gel was gelatinized for 3 hours by adding complete medium to immerse the Coll I gel, and immediately transferred to the stage incubator of the laser confocal microscope, and the multi-slice scanning mode of the imaging software was set.
  • the surface of the conductive chip of the microelectrode array is set to 0 ⁇ m, 4 different (x, y) sites are selected each time, the scanning direction is set to upward, and pictures are captured at a Z interval of 10 ⁇ m, and the total length of the scanning interval is 300 ⁇ m, among which,
  • the laser confocal microscope uses a 10 ⁇ objective lens and 488 channels to perform multi-point slice scanning; and at this time zero time, the time interval of time-lapse confocal imaging is set to 30min, and the total time is 3h, a total of 7 time points, at least 3 independent sample experiments.
  • the stage incubator provides an environmental control with a temperature of 37°C and a CO concentration of 5% for the culture of MDA-MB-231 or MDA-MB-436 cells.
  • the above-mentioned two-dimensional image stack obtained by time-lapse confocal imaging in the multi-slice scanning mode can quantitatively calculate the invasion distance in the Z direction of the cell layer through a MATLAB script customized by the laboratory. and rate, including: using MATLAB script to calculate the gray value of each picture in the picture stack, and performing curve fitting through interpolation method to obtain the gray value curve corresponding to different Z-axis positions, and the position of the gray peak value is the current cell layer
  • the invasion distance in the Z direction in the three-dimensional Coll I gel; by dividing the invasion distance of the cell layer by its corresponding time, the average invasion rate of the cell layer in this time period was obtained. The results are shown in Figure 10.
  • Figure 10-a shows the invasion distance of MDA-MB-231 cells in the Z direction corresponding to different concentrations of PTX
  • Figure 10-b shows the MDA with different concentrations of PTX.
  • Figure 10-a at the same time point, with the increase of the applied PTX concentration, the invasive distance of the cells in the Z direction decreased, that is, the invasiveness of the cells gradually decreased. This result is consistent with the endpoint count of the Boyden chamber experiment.
  • the detection results of CI are consistent with the results of quantitatively characterizing the invasion distance of the cell layer in Figure 10, and the dynamic detection of cell invasiveness based on impedance is consistent with the corresponding results obtained by the Boyden chamber end-point cell count and MTT method. Therefore, the cell impedance sensing system of the present disclosure can complete the application of monitoring the migration and invasion of population cells in a three-dimensional matrix after the action of anti-tumor drugs, so as to be used for quantitative screening of anti-tumor drugs.
  • the present disclosure discloses a quantitative screening method for antitumor drugs based on cell impedance sensing. Specifically, the present disclosure acts on tumor cells with different concentrations of PTX to prepare Coll I gels suitable for tumor invasion models, and further adds chemokines to induce the invasion of population cells in a three-dimensional matrix.
  • the conductive chip of the glue is combined with an impedance spectrometer to construct a cell impedance sensing detection system and an online analysis system, which realizes real-time and label-free monitoring of cell impedance information during the invasion of population cells under the action of anti-tumor drugs in a three-dimensional matrix.
  • a new method for quantitative screening of antitumor drugs is provided.
  • the real-time monitoring method of cell invasion after drug addition of the present disclosure provides important information on the mechanism of drug action, off-target effects of drug cytotoxicity, and the like.
  • the label-free assay used in the present disclosure can directly measure cellular function without the use of labeled molecules, and its advantages include a simple homogeneous assay format, non-invasive measurement, Less interference with normal cell function, kinetic assays and reduced assay development time.
  • the development of a more informative 3D in vitro assay of tumor cell invasion after drug addition, enabling quantitative and label-free drug screening, may advance the current development of cell-level drug screening methods.
  • the quantitative antitumor drug screening method based on cellular impedance sensing disclosed in the present disclosure is not only of great significance for drug screening at the cellular level and clinical drug toxicity detection, but also has important commercial promotion value, and is expected to be used in new drug screening and drug safety. It plays a characteristic role in evaluation and other aspects, and produces good social and economic value.

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Abstract

提供一种基于细胞阻抗传感的抗肿瘤药物筛选的方法，包括：S1，对设有微电极阵列的导电芯片进行表面处理；S2，在导电芯片表面铺设肿瘤细胞；S3，对肿瘤细胞进行饥饿处理及其后的加药作用；S4，在肿瘤细胞上形成聚合水凝胶并加入趋化因子；S5，实时进行细胞阻抗检测，获取细胞的阻抗信息，根据阻抗信息进行药物筛选。通过将不同浓度梯度的紫杉醇作用于人乳腺癌细胞，诱导胶原蛋白I型凝胶中群体细胞侵袭，有效实现了三维基质中细胞侵袭过程中高效稳定的细胞阻抗传感检测，使得抗肿瘤药物定量筛选达到了实时、无标记、持续动态检测的技术效果。

Description

一种基于细胞阻抗传感的抗肿瘤药物筛选的方法 技术领域
本公开涉及细胞阻抗传感检测技术领域，具体涉及一种基于细胞阻抗传感的抗肿瘤药物筛选的方法。
背景技术
药物筛选系指通过规范化的实验手段从大量化合物或者新化合物中选择对某一特定作用靶点具有较高活性的化合物的过程，主要的技术有基因组学、蛋白质组学、代谢组学、计算生物学、生物芯片技术、微流控芯片技术等方法。药物筛选的实验流程是以微板形式作为实验工具载体，以自动化操作系统执行实验过程，以灵敏快速的检测仪器采集实验数据，以计算机对实验获得的数据进行分析处理。药物筛选是生化水平(分子水平)和细胞水平的筛选。细胞水平的筛选则包括：离子通道监测(包括离子浓度、膜电位、pH值等)、报告基因检测以及酵母双杂交技术。细胞水平的药物筛选模型具有材料用量少、药物作用机制比较明确和大规模筛选等优点。快速、高灵敏度的检测技术是高通量药物筛选的关键技术之一。目前，在细胞水平上对抗肿瘤药物的高通量筛选主要是采用选取肿瘤细胞系，以培养细胞为实验模型，采用液闪计数、比色法、化学发光及荧光检测等检测技术，以细胞增殖、细胞毒作用、细胞周期调控及信号转导等为检测对象进行药物筛选。然而，由于标记物的限制以及采用终点检测的方法，大大限制了药物筛选的范围和准确性。成功的筛选能够缩短创新药物的研究与开发的周期、降低成本、减少风险和提高效率，因而需要加速体外细胞药物筛选方法的创新。
生物芯片技术是通过缩微技术，根据分子间特异性地相互作用的原理，将生命科学领域中不连续的分析过程集成于硅芯片或玻璃芯片表面的微型生物化学分析系统，以实现对细胞、蛋白质、基因及其它生物组分的准确、快速、大信息量的检测。基于生物芯片的细胞阻抗传感技术(Electric Cell Impedance Sensing，简称ECIS)是一种基于电子阻抗的 实时细胞检测系统，可通过细胞粘附、形态及生长等表型特点进行高通量的药物筛选。该方法是基于细胞的无标记分析方法，可通过细胞与检测板底部电极的相互作用，在近似生理环境下连续几天至几周实时、无标记、非侵入性动态地记录细胞增殖、细胞毒性和细胞形态的变化，为全面了解细胞的活性和侵袭性提供了一个更加准确、敏感地检测平台，可在新药研发的早期，合成出少量先导化合物进行细胞毒性的评价，具有很好的应用前景。除了无标记应用的技术优势外，ECIS的另一个特点是实时动态检测，相比于传统的终点法优势明显，能够以定量数据分析细胞增殖、形态变化、凋亡和坏死等生物学特点。因此，基于ECIS的用于体外三维(3D)基质中细胞水平的药物筛选可整合高通量、小型化筛选平台进行无标记全程动态定量监测，该方法易于操作且将为细胞药物相互作用提供新的视角。
发明内容
(一)要解决的技术问题
针对上述问题，本公开提供了一种基于细胞阻抗传感的抗肿瘤药物筛选的方法，用于至少部分解决传统方法对细胞活体产生破坏，进而大大局限了生物信息的全程动态收集等技术问题。
(二)技术方案
本公开提供了一种基于细胞阻抗传感的抗肿瘤药物筛选的方法，包括：S1，对设有微电极阵列的导电芯片进行表面处理；S2，在导电芯片表面铺满肿瘤细胞；S3，对肿瘤细胞进行饥饿处理及其后的加药作用；S4，在肿瘤细胞上形成聚合水凝胶并加入趋化因子；S5，实时进行细胞阻抗检测，获取细胞的阻抗信息，根据阻抗信息进行药物筛选。
进一步地，S1之前还包括：在导电衬底的导电膜层上形成光刻胶层；在具有光刻胶层的导电衬底上形成光刻胶图案；以及基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
进一步地，S1之前还包括：在导电芯片上形成围设微电极阵列的环形结构，其中，环形结构的内表面和微电极阵列所在的导电芯片表面形成细胞培养腔。
进一步地，S1具体包括：以甲醇清洗导电芯片；以3-氨基丙基三乙氧基硅烷为硅烷偶联剂对预清洗的导电芯片表面修饰氨基；以聚(苯乙烯-co-马来酸酐)溶液对导电芯片表面修饰酸酐基团。
进一步地，S2具体包括：所选肿瘤细胞系为人乳腺癌细胞；将肿瘤细胞悬液接种到表面预处理后的导电芯片上，以完全培养基培养。
进一步地，S3具体包括：用含胚牛血清的培养基饥饿处理细胞，用杜氏磷酸盐缓冲液清洗细胞；以不同浓度的紫杉醇溶液对饥饿处理后的细胞进行加药作用。
进一步地，S4具体包括：依据预设配比，将胶原蛋白I型溶液、改良伊格尔培养基、磷酸盐缓冲盐溶液、氢氧化钠溶液进行混合形成聚合水凝胶，其中，所有试剂均在冰上保持并混合；趋化因子为完全培养基中的胚牛血清。
进一步地，稀释后的胶原蛋白I型的预设浓度为3～5.6mg/mL；依据预设配比混合后的溶液中胶原蛋白I型的终浓度为2～4.5mg/mL；依据预设配比混合后的溶液pH中和至7.3～7.5。
进一步地，S5具体包括：以输出频率为10kHz～100kHz、输出正弦电压为10mV～30mV对抗肿瘤药物作用后三维基质中正在进行群体细胞侵袭的细胞阻抗传感芯片进行扫频测量；获取定量的细胞阻抗信息是以在水凝胶加入趋化因子为零时刻，进行多时间点的阻抗扫频测量，以获得不同频率不同时间点的细胞阻抗值。
进一步地，S5中根据阻抗信息进行药物筛选具体包括：获取不同频率下细胞阻抗值，以相对阻抗值变化表征三维基质中群体细胞随时间的侵袭距离，进而得到细胞侵袭过程的信息，由此进行药物筛选。
(三)有益效果
本公开实施例提供的一种基于细胞阻抗传感的抗肿瘤药物筛选的方法，通过加药作用于肿瘤细胞，诱导胶原蛋白I型凝胶中群体细胞侵 袭，有效实现了三维基质中细胞侵袭过程中高效稳定的细胞阻抗传感检测，使得抗肿瘤药物定量筛选达到了实时、无标记、持续动态检测的技术效果。
附图说明
图1示意性示出了根据本公开实施例基于细胞阻抗传感的抗肿瘤药物筛选的方法的流程图；
图2示意性示出了根据本公开实施例制备具有微电极阵列的导电芯片及细胞培养腔的流程示意图；
图3示意性示出了根据本公开实施例抗肿瘤药物定量筛选方法的细胞阻抗传感系统的组成示意图；
图4示意性示出了根据本公开实施例导电芯片表面进行细胞阻抗检测的示意图；
图5示意性示出了根据本公开实施例细胞阻抗传感芯片的实物图；
图6示意性示出了根据本公开实施例中细胞在抗肿瘤药物作用后单细胞的荧光标记图；
图7-a示意性示出了根据本公开实施例中小室下侧面的实物图；
图7-b示意性示出了根据本公开实施例中倒置显微镜IX41下穿过8μm孔径聚碳酸酯膜的细胞染色图；
图7-c示意性示出了根据本公开实施例中不同浓度紫杉醇作用后侵袭细胞数目的统计结果图；
图7-d示意性示出了根据本公开实施例中不同浓度紫杉醇作用后吸光度值(细胞活性)统计结果图；
图8a～d示意性示出了根据本公开实施例中胶原蛋白I型凝胶的形貌特征图；
图8e～f示意性示出了根据本公开实施例中定量拓扑分析的结果图；
图9示意性示出了根据本公开实施例中细胞在抗肿瘤药物作用后在三维基质中侵袭距离和速度与时间的对应关系图；
图10-a示意性示出了根据本公开实施例中细胞在抗肿瘤药物作用后阻抗传感检测获得的侵袭距离与时间的对应关系图；
图10-b示意性示出了根据本公开实施例中细胞在抗肿瘤药物作用后阻抗传感检测获得的相对阻抗值与时间的对应关系图。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开进一步详细说明。
细胞毒性是抗癌药物作用的关键所在，细胞毒性引起的细胞死亡有程序性和非程序性之分，后者即坏死。药物可以针对相应的细胞死亡信号通路进行调控，从而抑制或诱导细胞的死亡，对细胞死亡信号通路的深入研究，可为药物研发提供新的靶点，反之，在抗肿瘤药物作用机制的研究过程中，密切关注其对细胞死亡通路影响，则可进一步认识细胞的死亡。
传统细胞毒性测定方法有台盼蓝染色法、克隆(集落)形成法、3H放射性同位素掺入法、MTT法以及ATP检测法等。但这些细胞学研究检测形式多是终点检测法，仅给实验提供一个最终结果，且经常需要标记，从而对细胞产生破坏。由于细胞是活体，生物细胞进程是动态地而非静态的，终点检测法大大局限了生物信息的全程动态收集。
基于生物芯片的细胞阻抗传感技术(ECIS)通过电阻抗的形式，可以实现实时、无标记、持续动态检测细胞表型变化。在导电玻璃芯片的底部整合有微电极阵列，获得的细胞阻抗值可以细胞指数(Cell index，简称CI)的形式输出，可定量评估细胞生理状态包括细胞数量、存活率及细胞形态变化等。基于阻抗检测得到的动力学谱图能够提供化合物引起的细胞毒性作用的瞬时效应信息。此外，在细胞药物毒性试验中，ECIS检测能够精确确定化合物介导的细胞毒性作用发挥最大效应的时间点，有助于药物作用机理的研究及揭示。
本公开的实施例提供一种基于细胞阻抗传感的抗肿瘤药物筛选的 方法，请参见图1，包括：S1，对设有微电极阵列的导电芯片进行表面处理；S2，在导电芯片表面铺设肿瘤细胞；S3，对肿瘤细胞进行饥饿处理及其后的加药作用；S4，在肿瘤细胞上形成聚合水凝胶并加入趋化因子；S5，实时进行细胞阻抗检测，获取细胞的阻抗信息，根据阻抗信息进行药物筛选。
根据本公开的实施例，将上述公开的具有可三维培养细胞的胶原蛋白I型(type I collagen，缩写为Coll I)凝胶的导电芯片作为检测电极与阻抗谱仪电连接，构建一细胞阻抗传感系统，对不同浓度抗肿瘤药物作用的群体细胞侵袭进行实时定量的细胞阻抗检测。具体地，在导电芯片的微电极阵列上接种肿瘤细胞，待细胞长满加不同浓度加药作用后在细胞层上经自组装纤维化过程制备了适于肿瘤侵袭模型的Coll I凝胶。然后将阻抗谱仪通过导线经金属夹片与待检测细胞所在的检测电极(即细胞阻抗传感芯片)相连，以测量待检测细胞的阻抗值。
图2为制备具有微电极阵列的导电芯片及细胞培养腔的流程图，具体包括：在导电衬底的导电膜层上形成光刻胶层；在具有光刻胶层的导电衬底上形成光刻胶图案；以及基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。图3为抗肿瘤药物定量筛选方法的细胞阻抗传感系统的组成示意图。图4为导电芯片表面进行细胞阻抗检测的示意图。
在上述实施例的基础上，S1之前还包括：在导电衬底的导电膜层上形成光刻胶层；在具有光刻胶层的导电衬底上形成光刻胶图案；以及基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
关于上述具有微电极阵列的导电芯片可以采用ITO导电玻璃作为该导电芯片的衬底进行制备，具体地，该导电芯片还选择其他具有导电薄膜结构的衬底。另外，关于形成具有微电极阵列的导电芯片，具体地，如图2所示，可以依据如下制备过程形成：
旋涂：选取尺寸为40mm×40mm×0.4mm、导电膜层厚度约为185nm的ITO导电玻璃作为导电芯片的衬底，以低速300rpm 20s、高速1000 rpm 30s将紫外负性光刻胶旋涂在ITO导电玻璃的导电膜层上，即在导电衬底(ITO导电玻璃)的导电膜层上形成光刻胶层，以为下一步形成光刻胶图案做准备。
前烘：将上述具有光刻胶层的ITO导电玻璃设置在加热板上，以温度110℃对其烘烤60s，以将光刻胶层进行初步固化。
曝光：使用掩膜对准器通过打印的掩膜将进行固化了光刻胶层的ITO导电玻璃暴露于紫外光(UV)下20s。其中，掩膜的使用电极为叉指电极，尺寸可以为长1cm，宽30～100μm，相邻电极间距可以为30～100μm；所用曝光机为：紫外深度光刻机，曝光紫外光强可以为：18mW/cm 2，即在具有光刻胶层的导电衬底上形成光刻胶图案。
中烘：将上述进行了曝光操作的具有光刻胶层的ITO导电玻璃设置在加热板上，以温度145℃将曝光后的ITO导电玻璃烘烤60s，以将光刻胶层作进一步固化。
显影：将上述中烘后的ITO导电玻璃浸入光刻胶显影液中显影75s，用去离子水清洗后氮气吹干，再次浸入光刻胶显影液中显影20s，再次水洗吹干以除去上述未被曝光的光刻胶层的部分，以露出未被光刻胶层图案保护的ITO导电膜层。
刻蚀：以比例HCl∶DDI Water∶FeCl3·6H2O＝4L∶1L∶50g配制刻蚀液，其中FeCl 3·6H 2O可以为分析纯，含量≥99.0％。使用上述刻蚀液摇晃刻蚀上述显影后的ITO导电玻璃8min，以除去未被光刻胶层保护的ITO导电膜层，用去离子水清洗后氮气吹干，即基于光刻胶图案在导电衬底上形成具有导电膜图案的导电芯片，其中，导电膜图案为微电极阵列。
去胶：使用光刻胶去胶液超声去除残留的光刻胶8min，用去离子水清洗后氮气吹干以备下一步导电芯片表面预处理。
在上述实施例的基础上，S1之前还包括：在导电芯片上形成围设微电极阵列的环形结构，其中，环形结构的内表面和微电极阵列所在的导电芯片表面形成细胞培养腔。
为更好的在具有微电极阵列的导电芯片表面制备适于肿瘤侵袭模 型的type I collagen凝胶，同时为形成细胞阻抗传感检测过程中细胞的接种和培养条件，需要在导电芯片的导电面上围绕微电极阵列形成一细胞培养腔。具体地，在制得微电极阵列ITO导电玻璃芯片之后，选择石英玻璃圆环作为本公开的环形结构，围设导电芯片上的微电极阵列粘结在导电芯片上，形成初步的细胞培养腔。其中，粘结剂选择主剂∶固化剂＝20∶1的PDMS粘接剂，石英玻璃圆环的尺寸为内径27mm、外径30mm、壁厚1.5mm、深10mm。将粘结完毕的细胞培养腔结构放入70℃烘箱烘干，形成最终的细胞培养腔。
按照光刻和湿法刻蚀工艺制备上述具有微电极阵列的导电芯片及其对应的细胞培养腔。其中，结果如图5所示，其中图5-A为用于监测三维基质中群体细胞侵袭的细胞阻抗传感芯片的实物照片，图5-B为ITO导电玻璃刻蚀后的整体电极在体式显微镜下的局部放大图，图5-C为ITO导电玻璃的叉指电极阵列在荧光倒置显微镜IX71下的局部放大图，图5-D为ITO导电玻璃的叉指电极阵列在体式显微镜下的局部放大图。在实物照片5-A中，用上下两层具有导电线路的印刷电路板(Printed circuit board，简称PCB)作为夹具以固定粘有细胞培养腔的ITO导电玻璃芯片，并通过锡焊的方式用直径0.2mm铜丝将ITO导电玻璃的导电面与PCB板的导电线路电连接。以及，通过锡焊的方式用电线将印有导电线路的PCB板与外部的阻抗谱仪电连接，用于后续的监测三维基质中群体细胞侵袭的细胞阻抗传感检测。在局部放大图5-C中，橙色条带是ITO玻璃的导电膜层，白色条带是ITO玻璃导电膜层被湿法刻蚀后下层的玻璃，其中叉指电极的宽度为100μm，相邻电极间距为100μm。在局部放大图5-D中，微电极阵列为47对长度为10mm的长条形叉指电极阵列。
在上述实施例的基础上，S1具体包括：以甲醇清洗导电芯片；以3-氨基丙基三乙氧基硅烷为硅烷偶联剂对预清洗的导电芯片表面修饰氨基；以聚(苯乙烯-co-马来酸酐)溶液对导电芯片表面修饰酸酐基团。
根据本公开的实施例，需要对光刻有微电极阵列的导电芯片进行表面处理，以用于后续在玻璃皿中形成聚合水凝胶，在type I collagen 凝胶纤维化过程中酸酐基团的存在使得赖氨酸侧链能够共价键合凝胶表面，从而将适于肿瘤侵袭模型的type I collagen凝胶固定在具有反应性共聚物涂层的导电芯片皿表面。
在上述实施例的基础上，甲醇为无水甲醇，清洗次数至少为2次；3-氨基丙基三乙氧基硅烷溶液的配比为3-氨基丙基三乙氧基硅烷∶丙酮＝3∶25～4∶25；聚(苯乙烯-co-马来酸酐)为重均分子量2000～3000，质量百分比0.14～0.15％的酸酐共聚物，其酸酐溶液配比为聚(苯乙烯-co-马来酸酐)∶丙酮＝0.9∶2～1.1∶2。
采用无水甲醇清洗导电芯片2次，每次以摇床100rpm转速清洗3min，氮气吹干。以3-氨基丙基三乙氧基硅烷(3-AminopropylTriethoxySilane，简称APTES)为硅烷偶联剂，加入丙酮配制APTES∶丙酮＝3∶25溶液对预清洗的导电芯片皿表面修饰氨基。具体地，可以在27mm内径的玻璃皿中加入405μL上述溶液，不加盖置于通风橱中等待1h彻底自然晾干，此时，玻璃皿底部呈现白色。然后，用去离子水清洗培养腔2次，每次3min，氮气吹干。再将聚(苯乙烯-co-马来酸酐)(Poly(styrene-alt-maleic anhydride)，简称PSMA)溶液以低速300rpm 20s旋涂在修饰了氨基的导电芯片皿表面以进一步修饰酸酐基团，配比为PSMA-copolymer∶丙酮＝1∶2，其中，PSMA为质量百分比0.14％，重均分子量2000～3000的酸酐共聚物。不加盖置于通风橱中等待30min彻底自然晾干，最后用去离子水清洗培养腔2次，每次3min，氮气吹干。
在上述实施例的基础上，S2具体包括：所选肿瘤细胞系为人乳腺癌细胞；将肿瘤细胞悬液接种到表面预处理后的导电芯片上，以完全培养基培养。
在预处理的导电芯片表面铺满所选肿瘤细胞系细胞，所选的肿瘤细胞系可以为三阴性人乳腺癌细胞MDA-MB-231和MDA-MB-436。用DPBS清洗细胞2次后，以0.25％的Trypsin-EDTA溶液消化MDA-MB-231和MDA-MB-436细胞1min，用血球计数板计数后，将40×10 4数目的MDA-MB-231和MDA-MB-436细胞的细胞悬液接种到 表面预处理的导电芯片皿中，置于37℃5％CO 2细胞培养箱中以改良伊格尔(DMEM)高糖完全培养基培养24h，以用于后续肿瘤细胞的饥饿处理及其后的加药作用。
在上述实施例的基础上，S3具体包括：用含胚牛血清的培养基饥饿处理细胞，用杜氏磷酸盐缓冲液清洗细胞；以不同浓度的紫杉醇溶液对饥饿处理后的细胞进行加药作用。
进一步地，对导电芯片皿中的肿瘤细胞进行饥饿处理及其后的加药作用。当所选肿瘤细胞用完全培养基培养24h后，用1×DPBS清洗细胞2次后，用含0.5％胚牛血清(FBS)的培养基饥饿处理细胞24h，此时，芯片皿中的细胞量应该达到90％～100％的饱和度。再用1×DPBS清洗细胞2次以除去细胞杂质，接着以不同浓度的紫杉醇(PTX)溶液对饥饿处理后的细胞继续加药作用2h。
在上述实施例的基础上，含胚牛血清的培养基为添加了青霉素链霉素混合双抗的改良伊格尔高糖培养基；不同浓度的紫杉醇溶液为浓度范围为10nmol/L～30nmol/L。
上述含0.5％FBS的培养基为添加了1％双抗(青霉素链霉素混合双抗)的DMEM高糖培养基，不同浓度的紫杉醇溶液为浓度梯度10nmol/L、20nmol/L、30nmol/L的紫杉醇溶液。在紫杉醇溶液的配制过程中，先用1.17mL的DMSO溶解恢复至室温的10mg PTX粉末涡旋振荡得到10mmol/L的储存液，再以含0.5％FBS的培养基稀释储存液至工作浓度，以用于乳腺癌细胞的加药作用。其中，加药作用结果如图6所示，图6中a、b、c、d分别为对照组以及加了浓度为10nmol/L、20nmol/L、30nmol/L的紫杉醇溶液作用2h后的单细胞荧光标记图。因此，可见随着紫杉醇浓度的升高，细胞骨架的铺展面积逐渐减小，细胞的侵袭性和活性也会相应地降低。
在本公开的实施例中，以不同浓度的紫杉醇溶液对饥饿处理后的乳腺癌细胞加药作用。紫杉醇可使微管蛋白和组成微管的微管蛋白二聚体失去动态平衡，诱导与促进微管蛋白聚合、微管装配、防止解聚，从而使微管稳定并抑制癌细胞的有丝分裂和触发细胞凋亡，进而有效阻止癌 细胞的增殖，起到抗癌作用。纺锤体微管短暂的瓦解能优先杀灭异常分裂的细胞，一些重要的抗癌药物如秋水仙碱、长春碱、长春新碱等就是通过阻止微管蛋白重聚合而起到抗肿瘤作用的。与抗有丝分裂的抗肿瘤药物相反，紫杉醇是通过稳定微管蛋白而起作用，同时发现紫杉醇对多种实体瘤显示出良好的作用。因而，可以通过紫杉醇探索细胞活性的未知领域和发现新的抗癌药物的新方法。
在上述实施例的基础上，S4具体包括：依据预设配比，将胶原蛋白I型溶液、改良伊格尔培养基、磷酸盐缓冲盐溶液、氢氧化钠溶液进行混合形成聚合水凝胶，其中，所有试剂均在冰上保持并混合；趋化因子为完全培养基中的胚牛血清。
根据本公开的实施例，在铺满细胞层的微电极阵列上形成聚合水凝胶，包括：以乙酸溶液稀释高浓度的type I collagen(Coll I)到预设浓度；依据预设配比，将稀释后的type I collagen(Coll I)溶液、改良伊格尔培养基、磷酸盐缓冲盐溶液(PBS)、氢氧化钠溶液(NaOH)进行混合形成聚合水凝胶，其中，所有试剂均在冰上(4℃)保持并混合，以防止type I collagen单体自聚。
在本公开的实施例中，所谓聚合水凝胶可以是经自组装纤维化过程形成的具有体内细胞外基质(ECM)的许多纳米和微观结构特征的type I collagen凝胶，具体可以通过原位自组装纤维化过程形成。
表1 type I collagen凝胶的制备配方组成
Figure PCTCN2021078672-appb-000001
依据上述表1，可以分别进行不同配比Coll I水凝胶的制备。具体 地，可以按照上述表1中不同序号对应的配比称取浓度为8.9～10.9mg/mL的type I collagen、摩尔浓度为0.02mol/L的乙酸溶液、浓度为1×的DMEM培养基、浓度为10×的磷酸盐缓冲盐溶液、摩尔浓度为0.5mol/L的氢氧化钠溶液，将其涡旋均匀混合，即可得到混合水凝胶。
根据本公开的实施例，在上述原位自组装纤维化过程前，称取或配制的乙酸的溶液的摩尔浓度可以为0.02mol/L；高浓度的Coll I的浓度可以为8.9～10.9mg/mL；DMEM培养基的浓度可以为1×；磷酸盐缓冲盐溶液的浓度可以为10×；以及氢氧化钠的溶液的摩尔浓度可以为0.5mol/L。
在上述实施例的基础上，稀释后的胶原蛋白I型的预设浓度为3～5.6mg/mL；依据预设配比混合后的溶液中胶原蛋白I型的终浓度为2～4.5mg/mL；依据预设配比混合后的溶液pH中和至7.3～7.5。
其中，乙酸溶液可以稀释高浓度的type I collagen(Coll I)到预设浓度；氢氧化钠溶液可以中和依据预设配比混合后的溶液pH至7.3～7.5。
根据本公开的实施例，关于经原位自组装纤维化过程形成type I collagen凝胶，具体地，可以依据如下制备过程形成，包括：将上述称取的各组成材料或溶液进行混合之后，快速超声振荡30s，用移液枪吸取1mL溶液快速滴于芯片皿中，以替换含0.5％FBS的培养基，同时覆盖附着在微电极阵列上的MDA-MB-231或MDA-MB-436细胞，注意避免在溶液中引入气泡，所有试剂均在冰上(4℃)保持并混合以防止type I collagen单体自聚；以及迅速将导电芯片皿转移至37℃5％CO 295％湿度的细胞培养箱中成胶3h，经自组装纤维化过程制备适于群体细胞侵袭的三维Coll I凝胶。接着用完全培养基浸没Coll I凝胶的顶部，以防止凝胶脱水，并以完全培养基中的10％FBS作为趋化因子，当胚牛血清(FBS)扩散后沿凝胶薄层产生趋化因子梯度，从而诱导三维基质中群体细胞侵袭。
在本公开的实施例中，进一步地，对成胶后的type I collagen凝胶纤维进行NHS-Rhodamine荧光标记，以表征三维Coll I基质的拓扑 形貌特征。所使用的荧光标记蛋白试剂为摩尔浓度50μmol/L的NHS-Rhodamine(5-(and-6)-carboxytetramethylrhodamine succinimidyl ester，简称5(6)-TAMRA-SE)溶液。将NHS-Rhodamine粉末取出回温至室温，用1mL DMSO溶解25mg NHS-Rhodamine，充分超声振荡混匀即得到47.39mmol/L的储存液，使用前用1×PBS溶液稀释上述储存液至工作液浓度(50μmol/L)。为了可视化和分析拓扑参数，将三维Coll I基质在室温下用50μmol/L的NHS-Rhodamine染色1h并用1×PBS溶液漂洗3次，每次3min。通过激光共聚焦扫描显微镜以20×物镜4×zoom对三维Coll I基质成像。所获取的图像为12位深，分辨率为1024×1024像素，垂直堆栈大小为41张图像(相当于20μm)。所获取图像的体素尺寸为0.15×0.15×0.5μm(x×y×z)。在厚度约90μm Coll I凝胶层的近似垂直中心位置处获得堆叠。其中，Coll I纤维荧光标记结果如图8-a、b、c、d所示，可见随着Coll I单体浓度的升高，自组装获得的纤维增多。
在本公开的实施例中，通过实验室自定义编写的MATLAB脚本定量分析了不同浓度Coll I凝胶的拓扑参数，以计算基质的平均孔径尺寸和孔径分布。上述MATLAB脚本包括3个主要部分，即腐蚀算法，像素数和具有旋转高斯拟合的自相关算法。通过二进制转换和图像分割，再用腐蚀算法即可获得平均孔径大小及孔径分布。进行了3次独立的实验，每次至少对每个Coll I凝胶的4个位置做拓扑分析。定量拓扑分析的结果如图8-e、f所示，与纤维荧光染色后共聚焦显微镜扫描的图片结果相一致，即随着Coll I浓度的增加会有更小的孔径分布，平均孔径尺寸也相应降低。已知细胞命运是由来自细胞外基质的线索触发的，包括其化学，生物学和物理特性。特别地，机械和拓扑性质越来越被认为是重要的信号。本公开的实施例为拓扑定义的Coll I蛋白提供了一个易于访问的仿生体外平台，以在体外各种条件下解剖细胞行为。
根据本公开的实施例，以完全培养基中的10％FBS作为趋化因子，诱导细胞在三维凝胶基质中侵袭。通过3D ECM进行细胞迁移是胚胎发生、免疫监测和伤口愈合等生理和病理过程的基本特征。有效的运动性 取决于细胞突起，粘附和收缩机制的精确协调。但是，3D迁移对局部细胞外信号也很敏感，这些信号可以与已建立的细胞内信号整合，从而影响迁移模式和效率，并通过诱导细胞极性来赋予迁移方向性。定向细胞迁移主要是由于细胞胞外线索的不对称性引起的，其中包括可溶性因子(趋化性)，流体流动(趋流性)，电场(趋电性)，刚度(趋硬性)和粘附配体(趋触性)。值得注意的是，肿瘤的进展与ECM的生化，机械和结构变化有关，推测这些变化会影响侵袭性癌细胞的迁移，因此了解这些变化影响肿瘤细胞行为的机制至关重要。
在上述实施例的基础上，S5具体包括：以输出频率为10kHz～100kHz、输出正弦电压为10mV～30mV对抗肿瘤药物作用后三维基质中正在进行群体细胞侵袭的细胞阻抗传感芯片进行扫频测量；获取定量的细胞阻抗信息是以在水凝胶加入趋化因子为零时刻，进行多时间点的阻抗扫频测量，以获得不同频率不同时间点的细胞阻抗值。
进行细胞阻抗检测，包括：以输出正弦电压为10mV～30mV、输出频率为10kHz～100kHz的两端法阻抗测量模式(2-Terminal Impedance Measurement，简称2-Term Z)对三维基质中不同浓度PTX作用的群体细胞侵袭过程进行扫频测量，以获得不同频率下细胞阻抗值；获取细胞阻抗信息是以在Coll I凝胶中加入完全培养基为零时刻，进行多时间段的阻抗扫频测量，其中，阻抗扫频测量的时间间隔分别为10min、20min、30min、60min、90min、120min、150min、180min。对相应时间点和检测频率值采集到的阻抗信息进一步用实验室自定义编写的MATLAB脚本分析处理，以相对阻抗值变化即细胞指数(Cell index，简称CI)表征三维基质中群体细胞随时间的侵袭距离，细胞指数越大表示细胞在三维基质中的侵袭距离越大。细胞指数(CI)根据如下公式计算：
Figure PCTCN2021078672-appb-000002
其中，N是阻抗测量所设频率点的数目，R t(f i)和R 0(f i)是t时刻和零时刻导电芯片取决于频率的阻抗值。对接种了40×10 4MDA-MB-231细胞用不同浓度PTX处理后诱导群体细胞侵袭的导电芯片进行阻抗扫频测量，使用10kHz～100kHz频率范围进行扫频，步长为1kHz，施加的正 弦电压为30mV。其中，结果如图9所示，在相同的时间点，随着PTX浓度的增加，细胞指数(CI)逐渐降低；当PTX浓度相同时，随着侵袭时间的增加，CI趋于平台稳定期。
在上述实施例的基础上，S5中根据阻抗信息进行药物筛选具体包括：获取不同频率下细胞阻抗值，以相对阻抗值变化表征三维基质中群体细胞随时间的侵袭距离，进而得到细胞侵袭过程的信息，由此进行药物筛选。
通过与阻抗谱仪电连接进行细胞阻抗检测，获取定量的细胞阻抗信息。应用ECIS技术检测不同的细胞系会得到不同的细胞增殖图谱，可基于细胞粘附、形态及生长等表型特点进行高通量的药物筛选。应用ECIS技术检测不同作用机制的细胞生长抑制剂可获得特征性细胞效应图谱，从而可在大规模筛选中预测药物的作用机制。ECIS技术检测的化合物药效特征与传统终点法具有良好的相关性。将某一化合物的细胞效应图谱与诱导凋亡的动力学特点相比，两种分析方法的结果具有良好的一致性，即ECIS可为终点法检测提示最优检测时间。ECIS技术为化合物筛选提供高通量、长时效的方法，并为不同生物学功能的化合物进行基于作用机制的聚类分析。
下面以一活细胞示踪探针荧光标记后进一步通过显微镜表征细胞在三维凝胶基质中侵袭距离的实施例以验证本公开方法的有效性。
进一步地，对加药作用后的肿瘤细胞进行了传统的Boyden小室实验，以评价紫杉醇对乳腺癌细胞侵袭性的影响。将标准型Matrigel在4℃过夜融化，在冰上用4℃预冷的无血清培养基按1∶8比例稀释Matrigel，在小室上室底部中央垂直加入100μL稀释后的Matrigel，注意避免产生气泡，37℃的细胞培养箱中温育4h使其干成胶状，使用前进行基底膜水化。以0.25％的Trypsin-EDTA溶液消化加药作用2h的乳腺癌细胞，用无血清培养基重悬，调整细胞密度至5×10 4/mL。取100μL细胞悬液加入上室，24孔板的下室中加入700μL含20％FBS的培养基，特别注意的是，下层培养液与小室间避免有气泡产生，继续在孵箱中培养24h。用镊子小心取出小室，吸干上室液体，转移到预先加入800μL多聚甲醛 (paraformaldehyde，简称PFA)的孔中，室温固定30min。取出小室，吸干上室的固定液，转移到预先加入约800μL 0.1％的结晶紫溶液的孔中，室温染色30min。轻轻用DPBS冲洗浸泡3次，取出小室，吸去上室液体，用湿棉棒小心擦去上室底部膜表面上的细胞。在显微镜下随机取9个视野计算，统计结果。其中，细胞侵袭结果如图7所示，图7-a为小室下侧面的实物图，图7-b为倒置显微镜IX41下穿过8μm孔径聚碳酸酯膜的细胞染色图，图7-c为不同浓度紫杉醇作用后侵袭细胞数目的统计结果图。因此，可见随着紫杉醇浓度的升高，消化Matrigel后穿过8μm孔径聚碳酸酯膜的细胞数量也就越少，也即细胞的侵袭性逐渐降低。此传统终点检测法的结果有待与ECIS技术检测的化合物药效特征做进一步对比。
在本公开的实施例中，也对加药作用后的肿瘤细胞进行了MTT细胞毒性实验，以评价紫杉醇对乳腺癌细胞活性的影响。用5mL MTT溶剂溶解25mg MTT，配制成5mg/mL的MTT溶液。在96孔板中每孔加入5000个细胞，按照实验需求，先用完全培养基培养24h后用含0.5％FBS的培养基继续饥饿处理24h。以浓度梯度为10nmol/L、20nmol/L、30nmol/L的紫杉醇溶液及对照组作用2h后，每孔加入10μL MTT溶液，在细胞培养箱中继续孵育4h。每孔加入100μL Formazan溶解液，适当混匀，在细胞培养箱内继续孵育，直至在普通光学显微镜下观察发现Formazan完全溶解。通常37℃孵育3-4h左右，紫色结晶会全部溶解。如果紫色结晶较小，溶解的时间会短一些。如果紫色结晶较大，溶解的时间会长一些，此时为了加速溶解可以适当摇晃数次。用酶标仪在570nm处测定吸光度，图7-d为不同浓度紫杉醇作用后吸光度值(细胞活性)统计结果图。因此，可见随着紫杉醇浓度的升高，细胞的活性逐渐降低。
根据本公开的实施例，对加药作用后的肿瘤细胞用活细胞示踪探针荧光标记，包括：吸除含不同浓度紫杉醇的培养基；用DPBS清洗细胞2次；轻轻加入37℃预热的探针工作液；于37℃5％CO 2孵箱条件下孵育45min；换用新鲜含0.5％FBS的培养基继续培养30min，用于后续细胞层上形成适于肿瘤侵袭模型的type I collagen凝胶。
在本公开的实施例中，探针工作液为浓度5-25μmol/L的绿色活细胞示踪探针(Cell-Tracker Green，简称CMFDA)溶液。将CMFDA粉末取出回温至室温，短暂离心以保证粉末落入管底，用10.8μL DMSO溶解50μg CMFDA，充分混匀即得到10mmol/L的储存液。使用前用无血清培养基稀释上述储存液至工作液浓度(5～25μmol/L)，并将探针工作液于37℃预热。CMFDA试剂是可透过活细胞膜自由扩散的荧光氯甲基衍生物。进入细胞后，这些温和的硫醇反应性探针就会与细胞内组分反应，形成可在上样后存活至少24h的荧光细胞，以用于后续三维基质中群体细胞侵袭的追踪。
通过激光共聚焦显微镜定量表征细胞在三维凝胶基质中侵袭距离和速度。利用成像软件进行多点层扫模式的延时共聚焦成像和三维重建，所测细胞在三维凝胶基质中的侵袭距离为Z方向上侵袭距离。Coll I凝胶成胶3h后加入完全培养基以浸没Coll I凝胶，并立即转移至激光共聚焦显微镜的载物台培养器中，设置成像软件的多点层扫模式。将微电极阵列的导电芯片表面设为0μm处，每次选取4个不同的(x，y)位点，扫描方向设为向上，以10μm的Z间隔捕获图片，扫描间距总长为300μm，其中，激光共聚焦显微镜采用10×物镜，488通路进行多点层扫；以及以此时刻为零时刻，设置延时共聚焦成像的时间间隔为30min，时间总长为3h，共计7个时间点，至少进行3个独立的样本实验。其中，载物台培养器为MDA-MB-231或MDA-MB-436细胞的培养提供温度为37℃，CO 2浓度为5％的环境控制。
在本公开的实施例中，上述经多点层扫模式的延时共聚焦成像获得的二维的图片栈，可通过实验室自定义编写的MATLAB脚本定量计算细胞层所在Z方向上的侵袭距离和速率，包括：使用MATLAB脚本计算图片栈中每一张图片的灰度值，通过插值法进行曲线拟合从而获得不同Z轴位置对应灰度值曲线，灰度峰值所在位置即为当前细胞层在三维Coll I凝胶中Z方向上侵袭距离；通过将细胞层的侵袭距离除以其对应的时间，即获得细胞层在该时间段内的平均侵袭速率。结果如图10所示，图10-a为不同浓度PTX加药作用的MDA-MB-231细胞在不同时间 所对应的Z方向上侵袭距离，图10-b为不同浓度PTX加药作用的MDA-MB-231细胞在不同时间段内所对应的Z方向上的平均侵袭速率，其中，Coll I凝胶所采用的单体浓度为2.5mg/mL。在图10-a中，在相同的时间点，随着所施加PTX浓度的增加，细胞在Z方向上的侵袭距离递减，也即细胞的侵袭性逐渐降低，此结果与Boyden小室实验的终点计数结果相一致；在图10-b中，在相同的时间段内，PTX浓度越高，平均侵袭速率越低，就单一PTX浓度而言，总的趋势是侵袭过程开始时段速率快，后半部分速率慢。
CI的检测结果与图10定量表征细胞层的侵袭距离结果相吻合，基于阻抗的动态检测细胞侵袭性与相应的Boyden小室实验终点细胞计数及MTT法得到的结果一致。因此，本公开的细胞阻抗传感系统能完成抗肿瘤药物作用后群体细胞在三维基质中迁移和侵袭中的监测应用，从而用于抗肿瘤药物的定量筛选。
本公开公开了一种基于细胞阻抗传感的抗肿瘤药物定量筛选方法。具体而言，本公开以不同浓度PTX作用于肿瘤细胞，制备了适于肿瘤侵袭模型的Coll I凝胶，进一步加入趋化因子以诱导群体细胞在三维基质中的侵袭，将具有上述Coll I凝胶的导电芯片与阻抗谱仪联用构建细胞阻抗传感检测系统和在线分析系统，实现了三维基质中抗肿瘤药物作用的群体细胞侵袭过程中细胞阻抗信息的实时、无标记监测，从而获得了抗肿瘤药物的定量筛选新方法。相比于传统的Boyden小室实验终点计数法，本公开的这种加药作用后细胞侵袭的实时监测方法，提供了药物作用机理、药物细胞毒性作用的脱靶效应等方面的重要信息。与使用荧光、放射性同位素、发光或光吸收的经典检测方法不同，本公开所使用的无标记检测无需使用标记分子即可直接测量细胞功能，其优点包括简单的均相测定形式，无创测量，对正常细胞功能的干扰较小，动力学测定以及缩短测定开发时间。开发了更有信息性的加药作用后肿瘤细胞侵袭的3D体外分析方法，使药物筛选具有定量性和无标记，这可能推动目前细胞水平药物筛选方法的发展。本公开所公开的基于细胞阻抗传感的抗肿瘤药物定量筛选方法，不仅对于细胞水平药物筛选和临床药物毒 性检测具有重要的意义，而且拥有重要的商业推广价值，有望在新药筛选、药物安全性评价等方面发挥特点作用，产生良好的社会和经济价值。
以上所述的具体实施例，对本公开的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

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  1. 一种基于细胞阻抗传感的抗肿瘤药物筛选的方法，包括：
    S1，对设有微电极阵列的导电芯片进行表面处理；
    S2，在所述导电芯片表面铺满肿瘤细胞；
    S3，对所述肿瘤细胞进行饥饿处理及其后的加药作用；
    S4，在所述肿瘤细胞上形成聚合水凝胶并加入趋化因子；
    S5，实时进行细胞阻抗检测，获取所述细胞的阻抗信息，根据所述阻抗信息进行药物筛选。
  2. 根据权利要求1所述的用于监测三维基质中群体细胞侵袭的阻抗传感方法，其特征在于，所述S1之前还包括：
    在所述导电衬底的导电膜层上形成光刻胶层；
    在所述具有光刻胶层的导电衬底上形成光刻胶图案；以及
    基于所述光刻胶图案在所述导电衬底上形成具有导电膜图案的导电芯片，
    其中，所述导电膜图案为所述微电极阵列。
  3. 根据权利要求2所述的用于监测三维基质中群体细胞侵袭的阻抗传感方法，其特征在于，所述S1之前还包括：
    在所述导电芯片上形成围设所述微电极阵列的环形结构，
    其中，所述环形结构的内表面和所述微电极阵列所在的导电芯片表面形成所述细胞培养腔。
  4. 根据权利要求1所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述S1具体包括：
    以甲醇清洗所述导电芯片；
    以3-氨基丙基三乙氧基硅烷为硅烷偶联剂对预清洗的导电芯片表面修饰氨基；
    以聚(苯乙烯-co-马来酸酐)溶液对所述导电芯片表面修饰酸酐基 团。
  5. 根据权利要求1所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述S2具体包括：
    所选肿瘤细胞系为人乳腺癌细胞；
    将所述肿瘤细胞悬液接种到表面预处理后的导电芯片上，以完全培养基培养。
  6. 根据权利要求5所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述S3具体包括：
    用含胚牛血清的培养基饥饿处理细胞，
    用杜氏磷酸盐缓冲液清洗细胞；
    以不同浓度的紫杉醇溶液对饥饿处理后的细胞进行加药作用。
  7. 根据权利要求6所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述S4具体包括：
    依据预设配比，将胶原蛋白I型溶液、改良伊格尔培养基、磷酸盐缓冲盐溶液、氢氧化钠溶液进行混合形成聚合水凝胶，
    其中，所有试剂均在冰上保持并混合；
    所述趋化因子为完全培养基中的胚牛血清。
  8. 根据权利要求7所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述稀释后的胶原蛋白I型的预设浓度为3～5.6mg/mL；
    所述依据预设配比混合后的溶液中胶原蛋白I型的终浓度为2～4.5mg/mL；
    所述依据预设配比混合后的溶液pH中和至7.3～7.5。
  9. 根据权利要求1所述的基于细胞阻抗传感的抗肿瘤药物筛选的 方法，其特征在于，所述S5具体包括：
    以输出频率为10kHz～100kHz、输出正弦电压为10mV～30mV对抗肿瘤药物作用后三维基质中正在进行群体细胞侵袭的细胞阻抗传感芯片进行扫频测量；
    所述获取定量的细胞阻抗信息是以在所述水凝胶加入趋化因子为零时刻，进行多时间点的阻抗扫频测量，以获得不同频率不同时间点的细胞阻抗值。
  10. 根据权利要求9所述的基于细胞阻抗传感的抗肿瘤药物筛选的方法，其特征在于，所述S5中根据所述阻抗信息进行药物筛选具体包括：
    获取不同频率下细胞阻抗值，以相对阻抗值变化表征三维基质中群体细胞随时间的侵袭距离，进而得到所述细胞侵袭过程的信息，由此进行药物筛选。
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