Drug screening device and drug screening method for inhibiting vascular permeability of cancer cells

Abstract

The invention relates to a drug screening device and a drug screening method for inhibiting vascular permeability of cancer cells. A drug screening device for inhibiting vascular permeability of cancer cells is a micro-fluidic chip, the micro-fluidic chip is of a two-layer structure and comprises a cancer tissue area and a blood vessel area, the cancer tissue area is provided with a cancer tissue area inlet and a cancer tissue area inlet, and the blood vessel area is provided with a blood vessel area inlet and a blood vessel area inlet; a blood vessel wall model is arranged between the cancer tissue area and the blood vessel area, the blood vessel wall model comprises a row of flatly-laid elliptic cylinders, the elliptic cylinders are fixed to the cancer tissue area and the blood vessel area on the two sides, and the space between every two adjacent elliptic cylinders is a slit. Compared with the prior art, the method has the beneficial effects that cancer cells penetrating through a narrow space can be counted, so that the blood vessel infiltration capacity of the cancer cells is detected.

Description

Technical Field

[0001] The present invention relates to the fields of microfluidics, cancer cell intravasation, extravasation, and the like, and in particular to a drug screening device for inhibiting cancer cell vascular permeability, as well as preparation and application thereof. Background Art

[0002] Cancer cell metastasis is the key to cancer's lethality. If the cancerous tissue of the primary focus is not in a critical part, it can be cured through surgical resection. Once the cancer cells break away from the primary focus and enter the blood through intravasation, they reach the distal end through the external circulation of the blood, and then reach the distal tissue by adhering to the wall and extravasating out of the blood vessels, secondary lesions (metastatic lesions) will be formed. This process is also the process of cancer spread. Therefore, the intravasation and extravasation ability of cancer cells is the key to cancer's lethality.

[0003] Drug screening is screening at the biochemical and cellular levels, which refers to the process of using appropriate methods to evaluate the biological activity, pharmacological effects and medicinal value of substances (samples) that may be used as drugs.

[0004] The detection of cancer cells' vascular permeability is of great significance for both early cancer research and clinical drug screening. Traditional methods use transwell to detect cancer cells' permeability. The device consists of two chambers, separated by a layer of microporous membrane, and counts the number of cells that penetrate from one side of the membrane to the other to detect their permeability. Although this method is effective, due to its structural limitations, it is impossible to directly observe the morphological characteristics of cells, which limits related research.

[0005] Therefore, there is an urgent need to develop a multifunctional cancer cell intravasation and extravasation detection device to provide effective strategies from aspects such as cell counting, morphology, and protein labeling. Summary of the invention

[0006] The technical problem to be solved by the present invention is: how to design a drug screening device for inhibiting the vascular permeability of cancer cells, which can directly observe the morphological characteristics of cells and provide convenience for drug screening.

[0007] The specific plan is as follows: A drug screening device for inhibiting the vascular permeability of cancer cells is a microfluidic chip. The microfluidic chip has a two-layer structure, including a cancer tissue area and a blood vessel area. The cancer tissue area is provided with a cancer tissue area entrance and a cancer tissue area inlet, and the blood vessel area is provided with a blood vessel area entrance and a blood vessel area inlet; a blood vessel wall model is provided between the cancer tissue area and the blood vessel area, and the blood vessel wall model includes a row of flat elliptical cylinders, the elliptical cylinders are fixed to the cancer tissue areas and the blood vessel areas on both sides, and the space between two adjacent elliptical cylinders is a slit.

[0008] The microfluidic chip is made of a PDMS chip layer with a slit and elliptical column structure and a glass substrate layer bonded together using plasma cleaning technology. The light transmittance of the material meets the requirements of optical microscopy.

[0009] The microfluidic chip uses polydimethylsiloxane and a glass slide as substrates, which have high light transmittance, good biocompatibility and low cost. The channel pattern is cast on the PDMS side by soft etching, and the PDMS module is permanently bonded to the glass slide by plasma bonding technology, so that the slit becomes a channel with both ends transparent.

[0010] The outer contour of the microfluidic chip is a rectangular parallelepiped, and it can be placed with the front side facing up or with the side facing up.

[0011] A drug screening method is an intravasation screening method or an extravasation screening method, using the above-mentioned drug screening device for inhibiting vascular permeability of cancer cells, wherein: The intravasation screening method includes the following steps: S11, implanting cancer cells into the cancer tissue area and introducing culture medium, and introducing high-glucose culture medium into the vascular area, with the two areas separated by a vascular wall model; S12. Due to diffusion, a nutrient gradient is formed between the cancer tissue area and the vascular area. The nutrient gradient induces cancer cells in the cancer tissue area to penetrate the vascular wall model along the slit and enter the vascular area, simulating the process of cancer cell intravasation. S13, adding cancer treatment drugs to the cancer cell implantation area, and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S14, then, gradually adjusting the drug concentration to detect the minimum inhibitory concentration of the drug; The extravasation screening method includes the following steps: S21, implanting cancer cells into the vascular area and introducing high-glucose culture medium, introducing culture medium into the cancer tissue area and adding extravasation inducing factors, and separating the two areas by a vascular wall model; S22. Under the action of extravasation inducing factors, cancer cells in the vascular area penetrate the vascular wall model along the slit and enter the cancer tissue area, simulating the extravasation process of cancer cells.

[0012] S23, adding cancer treatment drugs to the cancer cell implantation area, and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S24. Then, the drug concentration is adjusted step by step to detect the minimum inhibitory concentration of the drug.

[0013] Beneficial effects: Compared with the prior art, the present invention can detect the vascular permeability by counting the cancer cells that penetrate a narrow space. It can also be combined with a microscope to observe the cells in situ and conduct in-depth research on the morphology and protein expression of cancer cells. This is of great significance for the functional research of nodes in signal pathways and the efficacy analysis of drugs in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the present invention.

[0015] Figure 2 yes Figure 1 A partial enlarged schematic diagram of .

[0016] Figure 3 This is a partially enlarged schematic diagram of the vascular wall model.

[0017] Figure 4 A schematic cross-sectional view of a capillary.

[0018] in: 10 is the cancer tissue area; 11 is the entrance to the cancer tissue area; 12 is the exit from the cancer tissue area; 20 is the blood vessel area; 21 is the entrance of the tube area; 22 is the inlet of the tube area; 30 is a blood vessel wall model; 31 is an elliptical cylinder; 32 is a slit; 91 is plasma; 92 is tissue fluid; 93 is tissue cells; 94 is blood cells; 95 is capillary wall cells. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention.

[0020] like Figure 1-3 A drug screening device for inhibiting vascular permeability of cancer cells is provided, which is a microfluidic chip. The microfluidic chip has a two-layer structure, including a cancer tissue area 10 and a blood vessel area 20. The cancer tissue area 10 is provided with a cancer tissue area entrance 11 and a cancer tissue area inlet 12, and the blood vessel area 20 is provided with a blood vessel area entrance 21 and a blood vessel area inlet 22.

[0021] A vascular wall model 30 is provided between the cancer tissue area 10 and the vascular area 20 . The vascular wall model 30 includes a row of flat elliptical cylinders 31 . The elliptical cylinders 31 are fixed to the cancer tissue area 10 and the vascular area 20 on both sides, so that the microfluidic chip has a stable structure. The space between two adjacent elliptical cylinders 31 is a slit 32 .

[0022] See also Figure 4The structure of the vascular wall model 30 is similar to that of a real vascular wall, and the situation of "cancer cells infiltrating into the blood through the vascular wall" is also similar to the situation of "cancer cells passing through the slit 32". In this way, the situation of cancer cells passing through the vascular wall can be fed back through the study of "cancer cells passing through the slit 32".

[0023] The microfluidic chip is formed by bonding a PDMS chip layer with a slit 32 and an elliptical column 31 structure and a glass substrate layer through plasma cleaning technology. The light transmittance of the material meets the requirements of an optical microscope.

[0024] The microfluidic chip uses polydimethylsiloxane (PDMS) and a glass slide as substrates, which have high light transmittance, good biocompatibility and low cost. The channel pattern is cast on the PDMS side by soft etching, and the PDMS module is permanently bonded to the glass slide by plasma bonding technology, so that the slit 31 becomes a channel with both ends transparent.

[0025] The major axis of the elliptical column 31 is approximately 10-10 µm, the minor axis thereof is approximately 10-10 µm, the length L thereof is approximately 10-10 µm, and the narrowest part of the slit 32 is approximately 3 µm.

[0026] The outer contour of the microfluidic chip is a rectangular parallelepiped, and it can be placed with the front side facing up or with the side facing up.

[0027] The cancer tissue area 10 is used to culture cancer cells, and a high-sugar culture medium is introduced into the blood vessel area to simulate the nutrient richness of blood vessels.

[0028] The specific working process is: In the present application, the number of cancer cells that penetrate the capillary wall simulation part (vascular wall model 30) from one side to the other side is used as a measure of the intravasation and extravasation ability of cancer cells. The device can be used to detect the intravasation and extravasation ability of different types of cancer cells.

[0029] Drug screening methods include intravasation screening methods and extravasation screening methods, which are described below.

[0030] The intravasation screening method comprises the following steps: S11, implanting cancer cells into the cancer tissue area 10 and introducing culture medium, and introducing high-glucose culture medium into the vascular area 20, which are separated by the vascular wall model 30; S12, due to diffusion, a nutrient gradient is formed between the cancer tissue area 10 and the blood vessel area 20, and the nutrient gradient induces the cancer cells in the cancer tissue area 10 to penetrate the blood vessel wall model 30 along the slit 32 and enter the blood vessel area 20, simulating the process of cancer cell intravasation; S13, adding cancer treatment drugs to the cancer cell implantation area (cancer tissue area 10), and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S14. Then, the drug concentration is adjusted step by step to detect the minimum inhibitory concentration of the drug.

[0031] The extravasation screening method comprises the following steps: S21, implanting cancer cells into the vascular area 20 and introducing a high-glucose culture medium, introducing a culture medium into the cancer tissue area 10 and adding an extravasation inducing factor, and separating the two areas by a vascular wall model 30; S22. Under the action of the extravasation inducing factor, the cancer cells in the blood vessel area 20 penetrate the blood vessel wall model 30 along the slit 32 and enter the cancer tissue area 10, simulating the extravasation process of cancer cells.

[0032] S23, adding cancer treatment drugs to the cancer cell implantation area (cancer tissue area 10), and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S24. Then, the drug concentration is adjusted step by step to detect the minimum inhibitory concentration of the drug.

[0033] In the above step S13 or step S23, a signal pathway intervention factor is added to the "area where cancer cells are implanted", and the functionality of the signal pathway node is verified by detecting the number of cancer cells passing through the blood vessel wall under the action of the intervention factor.

[0034] In the above step S11 or S21, the microfluidic chip can be placed with the front side facing up or with the side facing up: if it is placed with the side facing up, the newly implanted cells can grow against the blood vessel wall, ensuring that the cancer cell group penetrates into the micro-slit at the same initial position; if it is placed with the front side facing up, it can be placed on a microscope stage for cell morphological observation, and further protein labeling can be supported to further study the permeability characteristics of cancer cells.

[0035] Features of this application S1. With respect to the prior art, the present invention can detect the vascular permeability by counting the cancer cells that penetrate a narrow space. It can also be combined with a microscope to observe the cells in situ and conduct in-depth research on the morphology and protein expression of cancer cells. This is of great significance for the functional research of nodes in signal pathways and the efficacy analysis of drugs in clinical practice.

[0036] S2. Due to its small size and light transmittance, the present application can be combined with a microscope to perform in situ morphological observation of cancer cells and further protein labeling.

[0037] For other details, please refer to the prior art and will not be described in detail.

[0038] The technical means disclosed in the scheme of the present invention are not limited to the technical means disclosed in the above-mentioned implementation mode, but also include technical schemes composed of any combination of the above-mentioned technical features. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A drug screening device for inhibiting vascular permeability of cancer cells, characterized in that: The microfluidic chip has a two-layer structure, including a cancer tissue area (10) and a blood vessel area (20). The cancer tissue area (10) is provided with a cancer tissue area entrance (11) and a cancer tissue area inlet (12), and the blood vessel area (20) is provided with a blood vessel area entrance (21) and a blood vessel area inlet (22). A blood vessel wall model (30) is provided between the cancer tissue area (10) and the blood vessel area (20). The blood vessel wall model (30) includes a row of flat elliptical columns (31). The elliptical columns (31) are fixed to the cancer tissue areas (10) and the blood vessel areas (20) on both sides, and the space between two adjacent elliptical columns (31) is a slit (32).

2. The drug screening device for inhibiting cancer cell vascular permeability according to claim 1, characterized in that: The microfluidic chip is formed by bonding a PDMS chip layer with a slit (32) and an elliptical column (31) structure and a glass substrate layer through a plasma cleaning technology, and the light transmittance of the material meets the requirements of an optical microscope.

3. The drug screening device for inhibiting cancer cell vascular permeability according to claim 2, characterized in that: The microfluidic chip uses polydimethylsiloxane and a glass slide with high light transmittance, good biocompatibility and low cost as substrates. The channel pattern is cast on the PDMS side by soft etching, and the PDMS module is permanently bonded to the glass slide by plasma bonding technology, so that the slit (31) becomes a channel with both ends transparent.

4. The drug screening device for inhibiting cancer cell vascular permeability according to claim 3, characterized in that: The outer contour of the microfluidic chip is a rectangular parallelepiped, and it can be placed with the front side facing up or with the side facing up.

5. A drug screening method, which is an intravasation screening method or an extravasation screening method, characterized in that: The drug screening device for inhibiting cancer cell vascular permeability as claimed in claim 4, wherein: The intravasation screening method includes the following steps: S11, implanting cancer cells into the cancer tissue area (10) and introducing culture medium, and introducing high-glucose culture medium into the vascular area (20), which are separated by a vascular wall model (30); S12. Due to diffusion, a nutrient gradient is formed between the cancer tissue area (10) and the blood vessel area (20). The nutrient gradient induces cancer cells in the cancer tissue area (10) to penetrate the blood vessel wall model (30) along the slit (32) and enter the blood vessel area (20), simulating the process of cancer cell intravasation; S13, adding cancer treatment drugs to the cancer cell implantation area, and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S14, then, gradually adjusting the drug concentration to detect the minimum inhibitory concentration of the drug; The extravasation screening method includes the following steps: S21, implanting cancer cells into the vascular area (20) and introducing a high-glucose culture medium, introducing a culture medium into the cancer tissue area (10) and adding an extravasation inducing factor, and separating the two areas by a vascular wall model (30); S22. Under the action of the extravasation inducing factor, the cancer cells in the vascular region (20) penetrate the vascular wall model (30) along the slit (32) and enter the cancer tissue region (10), simulating the extravasation process of the cancer cells.

6. S23, adding cancer treatment drugs to the cancer cell implantation area, and screening the effectiveness of the drugs by detecting the number of cancer cells that pass through the blood vessel wall under the action of the drugs; S24. Then, the drug concentration is adjusted step by step to detect the minimum inhibitory concentration of the drug.

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