Analysis device and method for evaluating invasion of tumor cells to matrix
By using fluorescent probes to label matrix gel in an analysis device for evaluating tumor cell invasion and combining laser filters, the problem of numerous steps and difficulty in real-time observation in the prior art is solved, and a rapid and economical assessment of tumor cell invasion capability is achieved.
Patent Information
- Application Number
- CN202510448595.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-08-01
AI Technical Summary
The prior art has the limitations of many steps and is difficult to observe and simulate complex 3D tumor microenvironment in real time when evaluating tumor cell invasion capabilities in vitro. In particular, Transwell experiments require microscopy observation and are complex in operation.
The cell loading pool and chemokine loading pool are connected through vertical invasion channels, and the matrix gel is labeled with fluorescent probes. The tumor cell invasion is observed through fluorescent signals. This is simplified into three steps: equipment placement, gelling and cell inoculation, and direct observation is achieved with laser filters.
A rapid, economical and accurate assessment of tumor cell invasion ability is achieved, the operation steps are simplified, the microscopic observation needs are reduced, and a clear invasion path analysis is provided.
Smart Images
Figure CN120404680A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to an analysis device and method for evaluating the invasion of tumor cells into the stroma. Background Art
[0002] Tumor cells destroy the surrounding stroma by generating and secreting MMP (matrix metalloproteinase, an enzyme that degrades ECM), migrate through the vasculature, and then invade the surrounding tissues. The invasion potential of tumor cells is significantly affected by the surrounding stromal factors.
[0003] Currently, cell scratch and Transwell assays are commonly used to evaluate tumor cell invasion in vitro. These methods have significant limitations in simulating the complex 3D tumor microenvironment and real-time observation of the dynamic interaction between cancer cells and the extracellular matrix (ECM). For example, the cell scratch assay may interfere with the results due to scratch differences and ECM damage. The Transwell assay not only has numerous steps, making it difficult to observe cells in real time, but also requires microscopic observation. The steps of the Transwell assay include: coating with matrix gel, diluting Matrigel, solidifying Matrigel gel, hydrating the chamber, and adding serum-free medium; preparing cell suspension, resuspending and adjusting cell density. Inoculating cells, upper chamber: adding 200 μL of cell suspension to the upper chamber of the Transwell. Lower chamber: adding 500 - 600 μL of medium containing 10% FBS to the lower 24-well plate. Control group: setting a control group containing only serum-free medium. Incubating the two groups of cells for 12 - 48 hours; gently wiping off the non-invasive cells in the upper chamber with a cotton swab or cell scraper, and then fixing with methanol; staining and microscopic observation: counting one by one field under the microscope.
[0004] Therefore, we need a more simple, accurate and real-time in vitro experimental model to evaluate the analysis of tumor cell invasion into the stroma. Summary of the Invention
[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide an analysis method for evaluating the invasion of tumor cells into the stroma, so as to accurately and real-time evaluate the invasion ability of tumor cells into the stroma.
[0006] To achieve the above object, the present invention provides an analysis method for evaluating the invasion of tumor cells into the stroma, which uses a cell loading pool and a chemokine loading pool arranged below the cell loading pool, and the two are connected by a vertically arranged invasion channel, including the following steps: S1: Prepare a matrix gel simulating human tissue, and add a fluorescent probe to the matrix gel. The fluorescent probe can react with the enzyme secreted after the degradation of the matrix gel by invasion and emit fluorescence; S2: Add the matrix gel to the invasion channel and incubate at 36-37°C for 30-40 minutes; S3: Take a cell suspension and inoculate it in the cell loading pool, and statically incubate at 36-37°C for 2-3 hours; S4: Add a chemokine to the chemokine loading pool; S5: Set a laser source on one side of the invasion channel, an excitation filter is arranged between the laser source and the invasion channel, and an emission filter is arranged on the side of the invasion channel away from the excitation filter; S6: Collect the image of the matrix gel in the invasion channel, and judge the invasion potential of tumor cells according to the fluorescence emitted by the degradation channel left in the matrix gel by invasion.
[0007] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. With this method, the invasion ability of tumor cells can be directly observed and recorded visually by the naked eye or directly by a CCD camera (a digital camera with a charge-coupled device), so as to realize a rapid and economical preliminary analysis of the invasion ability of tumor cells; the principle is as follows: in the prior art, the nucleus is stained by Transwell and then the invasion ability is observed under a microscope, which belongs to the microscopic level; in this method, a fluorescent probe is added to the matrix gel, and the fluorescent probe can react with the enzyme secreted after the invasion of the matrix gel to emit fluorescence, that is, the channel after the invasion of tumor cells emits fluorescence, and the length of this channel is relatively long, which can be directly observed without using a microscope; it belongs to the macroscopic level.
[0008] 2. The invasion channel of the present invention is vertically arranged, so that the tumor cells in the upper cell suspension invade downward at the same height, providing a clear starting point for observing the invasion path of tumor cells and helping to accurately analyze the subsequent invasion process.
[0009] 3. Compared with the prior art Transwell experiment, the steps of this method are simpler: the experiment observation is integrated, without additional fluorescence staining and a large amount of image analysis, and there is no need to count one by one field of view under a microscope like the Transwell experiment; the experimental operation is simplified. The Transwell experiment requires 7 steps such as gel preparation and cell inoculation, while this method only requires 3 steps of installing the equipment, gel preparation, and cell inoculation.
[0010] As a preferred embodiment of the present invention, in the step S1, the fluorescent probe includes a fluorescent group and a quencher.
[0011] Principle of the technical solution: During the invasion of tumor cells, under the hydrolysis of MMP-2 or MMP-9, the fluorescent group of the fluorescent probe is separated from the quencher, resulting in enhanced fluorescence signal. After the excitation filter allows light of a specific wavelength to pass through, it excites the fluorescent probe; the emission filter only allows the light emitted by the fluorescent probe to pass through, and the observer can directly observe the fluorescence emitted by the fluorescent probe under the excitation of the excitation filter. The specific results are directly observed by the human eye or photographed by a CCD camera.
[0012] As a preferred embodiment of the present invention, in step S1, the Matrigel can be one of simulated liver invasion Matrigel, simulated bone invasion Matrigel, and simulated lung invasion Matrigel.
[0013] As a preferred embodiment of the present invention, in step S3, the cell suspension is prepared as follows: Tumor cells are cultured in a culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 36 - 37 °C and 5% CO2. When the cells grow to the logarithmic growth phase, they are digested with trypsin and then centrifuged. The cells are resuspended in the culture medium, and the concentration is adjusted to 0.8 - 1.2×10 5 cells / mL.
[0014] As a preferred embodiment of the present invention, in step S4, the chemokine is one of 10% fetal bovine serum, C, CC, CXC, and CX3C.
[0015] On the other hand, the present invention also provides an analysis device for evaluating the invasion of tumor cells into the matrix. Using the above method, it includes a bottom box, and a chemokine loading pool is provided in the bottom box; a first side plate is fixedly connected above the bottom box, and a top box is fixed above the first side plate. A cell loading pool is provided in the top box; a second side plate is bonded to the side of the first side plate, and an invasion channel is formed between the first side plate and the second side plate.
[0016] Principle and beneficial effects of the technical solution: The chemokine loading pool is used to add chemokines, and the cell loading pool is used to add cell suspension, that is, the cultured tumor cell solution; the invasion channel is connected to both of them. When in use, first open the second side plate, add Matrigel and let it solidify, then add the cell suspension into the top box above. After culturing for 2 - 3 h, add the chemokine into the bottom box below to allow the tumor cells to invade the Matrigel.
[0017] As a preferred embodiment of the present invention, openings are provided at the upper ends of the bottom box and the top box.
[0018] Principle and beneficial effects of the technical solution: The openings are used to facilitate the addition of cell suspension and chemokines.
[0019] As a preferred embodiment of the present invention, the invasion channel has a width of 5 μm, a length of 1 cm, and a height of 1 cm.
[0020] Principle and beneficial effects of the technical solution: The overall shape of the invasion channel is a cuboid, and its length and height are relatively long, which is convenient for displaying the invasion channel.
[0021] As a preferred embodiment of the present invention, a plurality of limiting pieces are provided at the upper end of the bottom box and the lower end of the top box, and the plurality of limiting pieces are arranged in pairs for installing an excitation filter and an emission filter.
[0022] Principle and beneficial effects of the technical solution: The limiting pieces are used for installing the excitation filter and the emission filter. During use, the excitation filter and the emission filter are temporarily installed, so that they can be conveniently removed, realizing the effects of repeated use, improving the utilization rate, and reducing the cost. Description of the Drawings
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.
[0024] Figure 1 It is a schematic flow chart of the first embodiment of the analysis method for evaluating the invasion of tumor cells into the matrix of the present invention.
[0025] Figure 2 It is a schematic structural diagram of the second embodiment of the analysis device for evaluating the invasion of tumor cells into the matrix of the present invention.
[0026] Figure 3 It is a cross-sectional view of the second embodiment of the analysis device for evaluating the invasion of tumor cells into the matrix of the present invention.
[0027] The reference numerals in the drawings of the specification include: 1 bottom box, 2 chemokine loading pool, 3 first side piece, 4 top box, 5 cell loading pool, 6 second side piece, 7 invasion channel, 8 opening, 9 limiting piece, 10 excitation filter, 11 emission filter, 12 limiting groove. Detailed Embodiments
[0028] The following details the embodiments of the present invention. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below with reference to the drawings are exemplary and are intended to explain the embodiments of the present invention, and should not be construed as a limitation of the present invention.
[0029] In the description of the embodiments of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.
[0030] In addition, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of the described features. In the description of the embodiments of the present invention, the meaning of "a plurality" is two or more unless otherwise specifically defined.
[0031] The present invention will be described in detail below with reference to the drawings and in conjunction with the embodiments.
[0032] Embodiment 1 As shown in the attached Figure 1 drawings, the present invention provides an analysis method for evaluating the invasion of tumor cells into the stroma: a cell loading pool 5 and a chemokine loading pool 2 provided below the cell loading pool 5 are used, and the two are connected by an invasion channel 7 arranged vertically, including the following steps: S1: Prepare a matrix gel simulating human tissue: The matrix gel can be one of a simulated liver invasion matrix gel, a simulated bone invasion matrix gel, and a simulated lung invasion matrix gel. Among them: The simulated liver invasion matrix gel is composed of 0.5 - 5% gelatin, 10 - 20% type I collagen, and 1 - 2% silicate; the simulated bone invasion matrix gel is composed of 5 - 10% nano-hydroxyapatite, 2 - 5% type I collagen, 2 - 5% hyaluronic acid, and 5 - 10% alginate; the simulated lung invasion matrix gel is composed of 10 - 20% elastin fiber protein, 1 - 5% type I collagen, and 1 - 2% alginate.
[0033] While preparing the matrix gel, a fluorescent probe is added thereto. The fluorescent probe can react with the enzyme secreted by the degradation of the matrix gel after invasion and emit fluorescence; the fluorescent probe includes a fluorescent group and a quencher, and the concentration of the fluorescent probe is 0.2 μmol / L; the principle of the fluorescent probe: When MMP-2 or MMP-9 recognizes and cleaves the peptide segment (Gln-Ile site in GPQG↓IWGQ) of the probe (NIRF), the Cy5.5 molecule is released from the carrier; the cleaved Cy5.5 dye molecule can freely generate a fluorescent signal, and the Cy5.5 molecule escapes from the quenching environment, and the fluorescent signal is significantly enhanced.
[0034] S2: Add Matrigel to the invasion channel 7 and incubate at 36 - 37 °C for 30 - 40 min to solidify the Matrigel into a three-dimensional collagen gel within the invasion channel 7.
[0035] S3: Take the cell suspension and inoculate it into the cell loading chamber 5, and statically incubate at 36 - 37 °C for 2 - 3 h. Preparation method of the cell suspension: Tumor cells are cultured in a culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 36 - 37 °C under 5% CO2 conditions. When the cells grow to the logarithmic growth phase, they are digested with trypsin and then centrifuged. The cells are resuspended with the culture medium, and the concentration is adjusted to 0.8 - 1.2×10 5 cells / mL.
[0036] S4: Add a chemokine to the chemokine loading chamber 2. The chemokine is one of 10% fetal bovine serum, C, CC, CXC, and CX3C.
[0037] S5: Set a laser source on one side of the invasion channel 7. An excitation filter 10 is arranged between the laser source and the invasion channel 7, and an emission filter 11 is arranged on the side of the invasion channel 7 away from the excitation filter 10. Principle of the laser filter: When a fluorescent probe is excited by light of a specific wavelength, the electrons in its molecule will transition from the ground state to the excited state. The electrons in the excited state are unstable and will return to the lowest vibrational energy level of the first electronic excited state through vibrational relaxation and internal energy conversion, and then transition to the ground state, releasing energy in the form of photons to generate fluorescence. The wavelength of this fluorescence is usually longer than that of the excitation light, and this phenomenon is called the Stokes shift.
[0038] The excitation filter 10 allows light within a specific wavelength range to pass through to excite the fluorescent probe. The emission filter 11 blocks unwanted spectral components, such as the autofluorescence of biological tissues, and only allows light within a specific wavelength range emitted by the fluorescent probe to pass through, thereby improving the signal-to-noise ratio and sensitivity of imaging. The fluorescent signal filtered by the excitation filter 10 and the emission filter 11 can clearly show the shape, length, etc. of the channel left after invasion. The maximum excitation wavelength of Cy5.5 is 675 nm, and the maximum emission wavelength is 694 nm.
[0039] S6: Collect the image of the Matrigel in the invasion channel 7, and judge the invasion potential of tumor cells based on the fluorescence emitted by the degradation channel left by invasion in the Matrigel. The image can be directly taken using a CCD camera.
[0040] Example 2 As shown in the appendix Figure 2 and the appendix Figure 3As shown: Based on the first embodiment, the present invention further provides an analysis device for evaluating the invasion of tumor cells into the stroma. Using the method for evaluating the invasion of tumor cells into the stroma described above, it includes a bottom box 1 and a first side piece 3 fixed on the bottom box 1. A chemokine loading pool 2 is provided in the bottom box 1, and the chemokine loading pool 2 is used to add chemokines; a top box 4 is fixed above the first side piece 3. A cell loading pool 5 is provided in the top box 4, and the cell loading pool 5 is used to add a cell suspension, that is, a cultured tumor cell solution; a second side piece 6 is bonded to the side of the first side piece 3, and bonding means fitting; an invasion channel 7 is formed between the first side piece 3 and the second side piece 6. The invasion channel 7 has a width of 5 μm, a length of 1 cm, and a height of 1 cm. The larger width of the drawing is for easy understanding; the invasion channel 7 communicates with the chemokine loading pool 2 and the cell loading pool 5 and is used to add matrix gel; in this embodiment, as Figure 1 shown, a limiting groove 12 is provided above the bottom box 1 for placing the second side piece 6. The second side piece 6 can be slid into the limiting groove 12. One side of it is bonded to the first side piece 3, and the other side is supported by the limiting groove 12, and thus is relatively fixed to the first side piece 3.
[0041] As shown in the attached Figure 3 figure, in this embodiment, openings 8 are provided at the upper ends of both the bottom box 1 and the top box 4, and the openings 8 are used to facilitate the addition of the cell suspension and chemokines.
[0042] As shown in the attached Figure 3 figure, in this embodiment, a plurality of limiting pieces 9 are provided at the upper end of the bottom box 1 and the lower end of the top box 4. The plurality of limiting pieces 9 are arranged in pairs and are used to install an excitation filter 10 and an emission filter 11. During use, the excitation filter 10 and the emission filter 11 are temporarily slid into the limiting pieces 9 from the side, so that they can be conveniently removed, achieving the effects of repeated use, improving the utilization rate, and reducing the cost.
[0043] When using this device for tumor cell invasion analysis, first open the second side piece 6, add matrix gel into the invasion channel 7 of the first side piece 3 and let it solidify; after solidification, bond the second side piece 6, then add a cell suspension into the upper top box 4. After culturing for 2 - 3 h, add chemokines into the lower bottom box 1 to allow tumor cells to invade the matrix gel. Then slide the excitation filter 10 and the emission filter 11 into the limiting pieces 9 from the side, align one side of the excitation filter 10 with the laser source, and then observe from one side of the emission filter 11 to observe the fluorescence generated by the degraded channel after invasion, and analyze the invasion ability of tumor cells.
[0044] The preferred embodiments of the present application have been described in detail above in conjunction with the accompanying drawings. Typical well-known structures and common general knowledge technologies in the preferred embodiments are not described in detail herein. Those of ordinary skill in the art can, under the inspiration given by this embodiment, complete and implement the technical solution of the present invention in combination with their own capabilities. Some typical well-known structures, well-known methods or common general knowledge technologies should not become an obstacle for those of ordinary skill in the art to implement the present application.
[0045] The scope of protection required by the present application shall be subject to the content of its claims, and the content recorded in the summary of the invention, specific implementation manners and the drawings of the description is used to interpret the claims.
[0046] Within the scope of the technical concept of the present application, several variations can also be made to the specific implementation manners of the present application, and these modified specific implementation manners should also be regarded as within the scope of protection of the present application.
Claims
1. An analytical method for evaluating the invasion of tumor cells into the stroma, characterized in that: A cell loading pool and a chemokine loading pool arranged below the cell loading pool are connected by a vertically arranged invasion channel, and the method includes the following steps: S1: Prepare a matrix gel simulating human tissue, and add a fluorescent probe to the matrix gel. The fluorescent probe can react with the enzyme secreted by the degradation of the matrix gel after invasion to emit fluorescence. S2: Add the matrix gel to the invasion channel and incubate at 36 - 37 °C for 30 - 40 min. S3: Take a cell suspension and inoculate it in the cell loading pool, and statically incubate at 36 - 37 °C for 2 - 3 h. S4: Add a chemokine to the chemokine loading pool. S5: Set a laser source on one side of the invasion channel, an excitation filter is arranged between the laser source and the invasion channel, and an emission filter is arranged on the side of the invasion channel away from the excitation filter. S6: Collect the matrix gel image in the invasion channel, and judge the invasion potential of tumor cells according to the fluorescence emitted by the degradation channel left by the invasion in the matrix gel.
2. The analytical method for evaluating the invasion of tumor cells into the stroma according to claim 1, characterized in that: In the step S1, the fluorescent probe includes a fluorescent group and a quencher.
3. The analytical method for evaluating the invasion of tumor cells into the stroma according to claim 1, characterized in that: In the step S1, the matrix gel can be one of a simulated liver invasion matrix gel, a simulated bone invasion matrix gel, and a simulated lung invasion matrix gel.
4. The analysis method for evaluating the invasion of tumor cells into the stroma according to claim 1, characterized in that: In the step S3, the cell suspension is prepared in the following manner: tumor cells are cultured in a culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 36-37 °C under 5% CO2 conditions until the cells grow to the logarithmic growth phase. After digestion with trypsin and centrifugation, the cells are resuspended in the culture medium, and the concentration is adjusted to 0.8-1.2×10 5 cells / mL.
5. The analysis method for evaluating the invasion of tumor cells into the stroma according to claim 1, characterized in that: In the step S4, the chemokine is one of 10% fetal bovine serum, C, CC, CXC, CX3C.
6. An analysis device for evaluating the invasion of tumor cells into the stroma, using the method according to any one of claims 1 to 5, characterized in that: It includes a bottom box, and a chemokine loading pool is arranged in the bottom box; a first side plate is fixedly connected above the bottom box, a top box is fixed above the first side plate, and a cell loading pool is arranged in the top box; a second side plate is bonded to the side of the first side plate, and an invasion channel is formed between the first side plate and the second side plate.
7. The analytical device for evaluating the invasion of tumor cells into the stroma according to claim 6, characterized in that: Openings are provided at the upper ends of the bottom box and the top box.
8. The analysis device for evaluating the invasion of tumor cells into the stroma according to claim 6, characterized in that: The invasion channel has a width of 5 μm, a length of 1 cm, and a height of 1 cm.
9. The analysis device for evaluating the invasion of tumor cells into the stroma according to claim 8, characterized in that: A plurality of limiting pieces are arranged at the upper end of the bottom box and the lower end of the top box, and the plurality of limiting pieces are arranged in pairs for installing the excitation filter and the emission filter.