In vitro tumor blood vessel model and methods for compound testing thereof

Abstract

The application provides an in-vitro tumor blood vessel model and a method for detecting compounds, and belongs to the technical field of biological tissue engineering. The in-vitro tumor blood vessel model comprises an artificial blood vessel group and a tumor microsphere group. The artificial blood vessel group comprises an artificial blood vessel, an artificial blood vessel inner flow channel for independently culturing and / or perfusing the inside of the artificial blood vessel, and an artificial blood vessel outer flow channel for culturing and / or perfusing the outside of the artificial blood vessel. The tumor microsphere group comprises single or multiple tumor microspheres and a tumor microsphere flow channel for culturing and / or perfusing the single or multiple tumor microspheres, wherein the tumor microsphere flow channel is connected with the artificial blood vessel outer flow channel. The in-vitro tumor blood vessel model can realize the absorption of drugs and nutrients through blood vessels, the construction of a microenvironment for tumor metastasis and invasion, and has the functions of blood circulation and blood vessel filtration.

Description

Technical Field

[0001] This invention belongs to the field of biological tissue engineering technology, specifically relating to an in vitro tumor vascular model and a method for detecting compounds using the in vitro tumor vascular model. Background Technology

[0002] In the study of pharmacokinetics, pharmacodynamics and drug toxicity of anti-tumor drugs, traditional methods mainly involve animal experiments and two-dimensional cell culture. Although these methods have achieved many successes, they are limited by factors such as cycle time, cost, precision, and ethics, making it difficult to accurately and effectively assess the actual effects of drugs.

[0003] Therefore, there is an urgent need to establish effective models for cancer drug screening and evaluation.

[0004] Organ-on-a-chip technology combines methods from multiple disciplines, including cell biology, engineering, and biomaterials, to construct three-dimensional tumor microenvironments in vitro. This serves as a screening model for tumor drugs, and compared to traditional toxicological animal experiments and in vitro 2D cell models, it more accurately reflects the effects and toxicity of drugs on corresponding cells, tissues, and organs. However, constructing single-organ-on-a-chip models for drug screening limits their functionality and application because they cannot reflect the complexity, functional changes, and integrity of organ functions. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art, and to provide an in vitro tumor vascular model and a method for detecting compounds using the in vitro tumor vascular model.

[0006] In one aspect, the present invention provides an in vitro tumor vascular model, comprising:

[0007] Artificial blood vessel group, and

[0008] Tumor microspheres

[0009] The artificial blood vessel group includes an artificial blood vessel, an intravascular flow channel for independently culturing and / or perfusing the interior of the artificial blood vessel, and an extravascular flow channel for culturing and / or perfusing the exterior of the artificial blood vessel.

[0010] The tumor microsphere assembly includes one or more tumor microspheres and tumor microsphere channels for culturing and / or perfusing the one or more tumor microspheres, the tumor microsphere channels being connected to the external channels of the artificial blood vessel.

[0011] Preferably, the artificial blood vessel comprises an endothelial layer and a smooth muscle layer; and / or,

[0012] The width of the artificial blood vessel ranges from 2mm to 40mm.

[0013] Preferably, the length of the in vitro tumor vascular model ranges from 10 mm to 50 mm, and the width ranges from 5 mm to 40 mm.

[0014] Preferably, the diameter of the tumor microspheres in the single or multiple tumor microspheres ranges from 900 μm to 1300 μm.

[0015] Preferably, the in vitro tumor vascular model further includes an independent tumor microsphere assembly.

[0016] The independent tumor microsphere group includes one or more independent tumor microspheres and independent tumor microsphere channels for independent culture and / or perfusion of the one or more independent tumor microspheres.

[0017] Preferably, the independent tumor microspheres are arranged symmetrically with the tumor microspheres.

[0018] Preferably, the artificial blood vessel assembly further includes an artificial blood vessel inlet communicating with the internal flow channel of the artificial blood vessel;

[0019] The tumor microsphere assembly also includes a tumor microsphere inlet that communicates with the tumor microsphere flow channel;

[0020] The independent tumor microsphere assembly also includes an independent tumor microsphere inlet that is connected to the independent tumor microsphere flow channel.

[0021] In another aspect, the present invention provides a method for detecting compounds using the aforementioned in vitro tumor vascular model, comprising the following specific steps:

[0022] Culture media are introduced into each flow channel to culture or perfuse tumor microspheres and / or artificial blood vessels;

[0023] The test compound was introduced into the artificial blood vessel assembly;

[0024] Obtain the regulatory effects of the test compound on tumor microspheres and / or artificial blood vessels.

[0025] Preferably, obtaining the regulatory results of the test compound on tumor microspheres and / or the artificial blood vessel includes:

[0026] To obtain cell viability and / or cell migration results of the tumor microspheres cultured via artificial blood vessels; and / or,

[0027] Obtain the morphology of the artificial blood vessel;

[0028] Based on the cell activity and / or cell migration results of the tumor microspheres, to screen for test compounds that match the tumor; and / or,

[0029] Based on the morphology of the artificial blood vessel, the regulatory effect of the test compound on the artificial blood vessel is obtained.

[0030] Preferably, obtaining the regulatory results of the test compound on tumor microspheres and / or artificial blood vessels further includes:

[0031] To obtain cell viability and / or cell migration results for independent tumor microspheres;

[0032] Based on the cell activity and / or cell migration results of the independent tumor microspheres and the tumor microspheres, the regulatory effect of the test compound on the tumor microspheres after absorption through artificial blood vessels is obtained.

[0033] This invention establishes an in vitro tumor vascular model, enabling the absorption of drugs and nutrients through blood vessels and the construction of a microenvironment conducive to tumor metastasis and invasion, while also providing functions such as blood flow and vascular filtration. Utilizing this in vitro tumor vascular biological model, on the one hand, drugs and nutrients can be absorbed through blood vessels and exert their effects on the tumor, and / or directly affect the tumor, allowing for more accurate, efficient, and convenient testing of anti-tumor drugs; on the other hand, it enables accurate, efficient, and convenient testing of the effects of anti-tumor drugs on artificial blood vessels (e.g., the potential impact of anti-tumor drugs on blood vessels, leading to vasculitis, hypertension, thrombosis, and other vascular-related diseases). Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the overall structure of an in vitro tumor vascular model according to an embodiment of the present invention;

[0035] Figure 2 This is a schematic diagram of the structure of the culture layer according to an embodiment of the present invention;

[0036] Figure 3 This is a schematic diagram of the structure of a sealing layer according to an embodiment of the present invention;

[0037] Figure 4 This is a schematic diagram of the connection layer according to an embodiment of the present invention;

[0038] Figure 5 This is a schematic diagram of the structure of a cultivation system according to an embodiment of the present invention;

[0039] Figure 6 An image of NCI-H23 tumor microspheres according to an embodiment of the present invention;

[0040] Figure 7 This is a graph showing the change in cell activity of NCI-H23 tumor microspheres with the number of days of drug treatment according to an embodiment of the present invention.

[0041] Figure 8 This is an image showing the change of NCI-H23 tumor microspheres with the number of days of drug treatment according to an embodiment of the present invention. Detailed Implementation

[0042] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0043] like Figures 1 to 5 As shown, one aspect of the present invention provides an in vitro tumor vascular model, comprising: an artificial blood vessel group and a tumor microsphere group, wherein the artificial blood vessel group includes an artificial blood vessel, an artificial blood vessel internal flow channel for independently culturing and / or perfusing the interior of the artificial blood vessel, and an artificial blood vessel external flow channel for culturing and / or perfusing the exterior of the artificial blood vessel; the tumor microsphere group includes one or more tumor microspheres and a tumor microsphere flow channel for culturing and / or perfusing the one or more tumor microspheres, the tumor microsphere flow channel being connected to the artificial blood vessel external flow channel, i.e., tumor microspheres with vascular function can be formed based on this model.

[0044] The tumor vascular model in this embodiment enables the absorption of drugs and nutrients through blood vessels, and has the functions of blood circulation and vascular filtration, so as to construct the microenvironment for tumor metastasis and invasion. It can realistically reflect the complexity, functionality and integrity of the body's organ functions, more realistically simulate the in vivo microenvironment, and improve the accuracy of experimental data in the application of the model.

[0045] In some preferred embodiments, the artificial blood vessel includes an endothelial layer and a smooth muscle layer, that is, the inner wall of the artificial blood vessel is an endothelial layer composed of a single layer of endothelial cells, and the outer side of the inner wall is a smooth muscle layer composed of at least one layer of smooth muscle cells.

[0046] In this embodiment, the endothelial cells of the artificial blood vessel are arranged closely (without gaps between them), and the arrangement is directional and has a complete cell morphology.

[0047] In this embodiment, the endothelial layer of the blood vessel wall has the ability to sense the flow rate and direction of fluid within the blood vessel; the smooth muscle cells on the outer side of the blood vessel wall can sense stimuli (such as adrenaline, which causes contraction), thus realizing the function of vasoconstriction and vasodilation. This allows for the assessment of the toxic effects of drugs on blood vessels. For example, when a drug causes excessive vasoconstriction, it can lead to hypertension; when a drug causes excessive vasodilation, it can lead to decreased blood pressure; and when a drug causes excessive proliferation of smooth muscle cells, it can lead to vasculitis, plaque formation, etc.

[0048] In some other preferred embodiments, the width of the artificial blood vessel ranges from 2 mm to 40 mm.

[0049] As a further preferred option, the width of the artificial blood vessel is preferably in the range of 2mm to 7mm to simulate human veins.

[0050] This embodiment achieves a high degree of simulation of human blood vessels by adjusting the width of the artificial blood vessels to simulate the thickness of real human blood vessels.

[0051] Furthermore, in some preferred embodiments, the length of the in vitro tumor vascular model ranges from 10 mm to 50 mm, and the width ranges from 5 mm to 40 mm.

[0052] The model in this embodiment is relatively small, which improves the convenience of experimental operation.

[0053] Furthermore, in some preferred embodiments, the single or multiple tumor microspheres can be spherical structures, with the diameter of the tumor microspheres ranging from 900 μm to 1300 μm.

[0054] The tumor microspheres in this embodiment have functional characteristics, specifically, a darker color in the center and a lighter, more clearly defined edge.

[0055] It should be noted that this embodiment does not specifically limit the type of single or multiple tumor microspheres. For example, one or more of the following can be selected: lung cancer tumor microspheres, liver cancer tumor microspheres, colorectal cancer tumor microspheres, skin cancer tumor microspheres, and pharyngeal cancer tumor microspheres.

[0056] Specifically, such as Figures 1 to 3 As shown, the tumor microsphere flow channel includes a tumor microsphere culture inlet B2, a tumor microsphere inlet flow channel B2-1, a vascular culture chamber B13, a vascular culture chamber outlet flow channel B2-2, a micro-mixed flow channel B10, a first group of culture chambers B12, a tumor microsphere outlet flow channel B1-1, and a tumor microsphere culture outlet B1, arranged sequentially on the culture layer B in the direction of fluid flow.

[0057] Furthermore, such as Figures 1 to 3 As shown, the internal flow channel of the artificial blood vessel includes, in the direction of fluid flow, a blood vessel inlet C3, a blood vessel inlet channel C3-1, a blood vessel inlet connection hole C3-2, a blood vessel outlet connection hole C4-2, a blood vessel outlet channel C4-1, and a blood vessel outlet C4, arranged sequentially on the sealing layer C. The sealing layer C is stacked below the culture layer B. The artificial blood vessel E is disposed in the blood vessel culture chamber B13 on the culture layer B, and both ends of the artificial blood vessel E are connected to the blood vessel outlet connection hole C4-2 through the blood vessel inlet connection hole C3-2, forming an internal flow channel for culturing and / or perfusing the interior of the artificial blood vessel. The remaining space in the blood vessel culture chamber, excluding the artificial blood vessel, forms an external flow channel for the artificial blood vessel.

[0058] It should be noted that this embodiment does not specify how the artificial blood vessel is set in the blood vessel culture room, as long as fluid flow can be achieved in the internal and external channels of the artificial blood vessel.

[0059] For example, to facilitate the installation of artificial blood vessels, such as Figure 1 and Figure 3 As shown, both ends of the artificial blood vessel E are fitted with connecting tubes D2, and the other ends of the connecting tubes D2 are fitted onto fixing blocks D1. The two fixing blocks D1 are fixedly mounted on the culture layer B, and are located at opposite ends of the blood vessel culture chamber B13. Furthermore, each fixing block D1 contains a flow channel communicating with the connecting tubes D2, the blood vessel inlet connection hole C3-2, or the blood vessel outlet connection hole C4-2, to form an internal flow channel within the artificial blood vessel.

[0060] Based on the specific structures of the tumor microsphere assembly and the artificial blood vessel assembly, the principles of culture or perfusion of the artificial blood vessels and tumor microspheres are as follows: (Combined) Figure 5 As shown, a first culture medium and a second culture medium are introduced into the tumor microsphere channel and the artificial blood vessel channel, respectively, for culturing or perfusing the tumor microspheres and artificial blood vessels. Since the second culture medium in the artificial blood vessel channel contains the test compound, the culture media in the two channels exchange substances in the blood vessel culture chamber due to concentration and pressure differences. Specifically, the test compound in the artificial blood vessel channel is absorbed and filtered by the endothelial and smooth muscle layers of the artificial blood vessel and then seeps into the blood vessel culture chamber of the artificial blood vessel external channel. The test compound is then fully mixed with the first culture medium in the micro-mixed channel and flows into the first group of culture chambers to act on the tumor microspheres in the first group of culture chambers, thus obtaining tumor microspheres with vascular function. These microspheres can be used to screen tumor drugs and also to obtain results on the effects of drugs on blood vessels.

[0061] In some other preferred embodiments, in order to provide a control for tumor microspheres with vascular function, the in vitro tumor vascular model further includes an independent tumor microsphere group, which includes one or more independent tumor microspheres and independent tumor microsphere channels for independent culture and / or perfusion of the one or more independent tumor microspheres.

[0062] Specifically, such as Figure 2 As shown, the independent tumor microsphere flow channel includes an independent culture inlet B5, an independent inlet channel B5-1, a second group of culture chambers B7, an independent outlet channel B6-1, and an independent culture outlet B6, which are arranged sequentially on the culture layer B in the direction of fluid flow.

[0063] Furthermore, the second group of culture chambers is set up in correspondence with the first group of culture chambers, including corresponding positions and numbers. The two groups of culture chambers are symmetrically arranged on both sides of the culture layer along its width direction, and each group of culture chambers includes five sub-culture chambers to achieve co-culture of multiple tumor microspheres.

[0064] For example, such as Figure 2 As shown, the first group of culture chambers B12 includes five equally spaced sub-culture chambers, each sub-culture chamber being connected to sub-flow channel B12-2 at intervals. The second group of culture chambers B7 also includes five equally spaced sub-culture chambers, each sub-culture chamber being connected to sub-flow channel B7-2 at intervals.

[0065] It should be noted that when multiple tumor microspheres are tumor microspheres of one organ, the principle of parallel replication of the experiment can be guaranteed. Of course, multiple tumor microspheres can also be tumor microspheres of multiple organs to achieve co-culture of multiple organs.

[0066] Furthermore, when the tumor microsphere channels and the channels in the independent tumor microsphere channels are set on different planes of the culture layer, fluid conversion holes are also formed on the culture layer to change the direction of the fluid and connect the channels located on different surfaces.

[0067] For example, please refer to Figure 2 When the outlet flow channel B2-2 of the vascular culture chamber is located on the upper surface of the culture layer B, and the micro-mixing flow channel B10 is located on the lower surface of the culture layer B, a first fluid conversion hole B9 is formed between the two flow channels. When the inlet flow channel B12-2 of the first group of culture chambers B12 is located on the upper surface of the culture layer B, a second fluid conversion hole B11 is also formed between this flow channel and the micro-mixing flow channel B10. When the outlet flow channel B7-2 of the second group of culture chambers B7 is located on the upper surface of the culture layer B, and the independent outlet flow channel B6-1 is located on the lower surface of the culture layer, a third fluid conversion hole B8 is formed between the two flow channels.

[0068] In other preferred embodiments, to improve the convenience of adding the test compound, the artificial blood vessel group further includes an artificial blood vessel inlet communicating with the flow channel of the artificial blood vessel; the tumor microsphere group further includes a tumor microsphere inlet communicating with the flow channel of the tumor microsphere; the independent tumor microsphere group further includes an independent tumor microsphere inlet communicating with the flow channel of the independent tumor microsphere, so as to introduce the corresponding culture medium through each inlet. Of course, an outlet should also be provided to draw out the culture medium from each flow channel.

[0069] For example, such as Figures 1 to 4As shown, the connecting layer A, stacked above the culture layer B, is provided with an artificial blood vessel inlet A3, an artificial blood vessel outlet A4, a tumor microsphere inlet A2, a tumor microsphere outlet A1, and independent tumor microsphere inlets A5 and A6. Specifically, the artificial blood vessel inlet A3 is connected to the blood vessel flow inlet C3 via the artificial blood vessel culture inlet B3 on the culture layer B, and the artificial blood vessel outlet A4 is connected to the blood vessel flow outlet C4 via the artificial blood vessel culture inlet B4 on the culture layer B. Next, the tumor microsphere inlet A2 is connected to the tumor microsphere culture inlet B2, and the tumor microsphere outlet A1 is connected to the tumor microsphere culture outlet B1. Furthermore, the independent tumor microsphere inlet A5 is connected to the independent culture inlet B5, and the independent tumor microsphere outlet A6 is connected to the independent culture outlet B6.

[0070] This embodiment provides separate inlets and outlets for each flow channel, facilitating the introduction of culture medium and test compounds into the corresponding flow channels, thereby improving the convenience of obtaining results on the effects of different drugs on tumor microspheres and artificial blood vessels.

[0071] In another aspect, the present invention provides a method for detecting compounds using the in vitro tumor vascular model described above, comprising the following specific steps:

[0072] First, introduce culture media into each flow channel to culture or perfuse tumor microspheres and / or artificial blood vessels;

[0073] Second, introduce the test compound into the artificial blood vessel group;

[0074] Third, obtain the regulatory results of the test compound on tumor microspheres and / or artificial blood vessels.

[0075] It should be understood that when using the model described above to culture or perfuse tumor microspheres, it is also necessary to connect the model to the culture tubing, such as... Figure 5 As shown, the culture tubing includes a first culture flask 1, a second culture flask 2, a third culture flask 3, and a first culture medium, a second culture medium, and a third culture medium corresponding to the contents of each culture flask. Each culture flask is equipped with a pump M1, a pump M2, and a pump M3. The first culture flask 1 is connected to the tumor microsphere channel, the second culture flask 2 is connected to the artificial blood vessel channel, and the third culture flask 3 is connected to the independent tumor microsphere channel. The pumps on each culture tubing introduce the culture medium from each culture flask into each channel.

[0076] In some preferred embodiments, the detection method specifically includes:

[0077] First, introduce a first culture medium into the tumor microsphere group to culture or perfuse the tumor microspheres;

[0078] Second, a second culture medium is introduced into the artificial blood vessel group to culture or perfuse the artificial blood vessel, while the test compound is introduced into the artificial blood vessel group at the same time.

[0079] Third, the tumor microspheres in the tumor microsphere group were analyzed to obtain the cell activity and cell migration results of the tumor microspheres cultured in artificial blood vessels; and / or, the morphology of the artificial blood vessels was analyzed to obtain the morphological changes of the artificial blood vessels.

[0080] Based on the cell activity and / or cell migration results of tumor microspheres, to screen for test compounds that match the tumor, and / or, based on the morphological changes of artificial blood vessels, to obtain the regulatory effect of test compounds on artificial blood vessels.

[0081] It should be noted that, to ensure the tumor microspheres possess the pre-defined functional characteristics, a first activity analysis can be performed on the artificial blood vessels and tumor microspheres cultured in the second culture medium without the test compound. This ensures that the vascular endothelial cells are tightly packed and directional, and that the tumor microspheres exhibit good functional characteristics. Subsequently, a second activity analysis is performed on the tumor microspheres and artificial blood vessels cultured in the second culture medium with the test compound added, to obtain the regulatory effects of the test compound on tumors and blood vessels.

[0082] It should be understood that, since the test compound is added to the second culture medium in this embodiment, drug exchange occurs in the vascular culture chamber, and the drug acts on the tumor microspheres along with the first culture medium. The activity of tumor cells decreases after drug administration, and the tumor cells solidify or scatter after drug administration. The morphology of blood vessels also changes after drug administration.

[0083] This embodiment can screen for suitable drugs targeting tumor cells in different organs based on the cell activity and cell migration results of tumor microsphere groups, and obtain the results of the effects of anti-tumor drugs on artificial blood vessels. For example, the effects of anti-tumor drugs on blood vessels include vasculitis, hypertension, thrombosis, etc.

[0084] In some other preferred embodiments, the detection method further includes:

[0085] First, a third culture medium is introduced into the independent tumor microsphere group to culture or perfuse the independent tumor microspheres;

[0086] Second, the tumor microspheres within the independent tumor microsphere group were analyzed to obtain the cell activity and cell migration results of the independent tumor microspheres;

[0087] Based on the cell activity and / or cell migration results of tumor microspheres and independent tumor microspheres, the effects of the test compound on tumor microspheres after absorption through artificial blood vessels can be obtained.

[0088] It should be understood that, since no drug exchange occurred in this embodiment, no drug acted on the independent tumor microspheres, their cell activity increased, and the independent tumor microspheres proliferated normally, the tumor cell diameter increased or the tumor cells migrated, forming a control group with the tumor microsphere group described above, in order to obtain the effect of the test compound on the tumor.

[0089] This invention establishes a tumor vascular model by simulating the interaction between human blood vessels and single-organ or multi-organ tumor microspheres in vitro. This model can more realistically reflect the effects of the test compounds in the human body, thereby realizing the microenvironment for drug and nutrient absorption through blood vessels and tumor metastasis and invasion. By analyzing the tumor microspheres, it can be used to screen tumor drugs and obtain the results of the effects of tumor drugs on vascular toxicity.

[0090] The in vitro tumor vascular model and its specific applications will be further illustrated below with reference to specific embodiments:

[0091] Example 1

[0092] This example illustrates a method for compound detection using an in vitro tumor vascular model, including the following steps:

[0093] S1. Obtain models with artificial blood vessel groups, tumor microsphere groups, and independent tumor microsphere groups.

[0094] S2. Use Huvec endothelial cells to culture artificial blood vessels until the artificial blood vessels exhibit functional characteristics, such as tightly packed endothelial cells with directionality.

[0095] S3. 3D tumor microspheres were prepared using human lung cancer cells NCI-H23 and cultured until the NCI-H23 tumor microspheres exhibited functional characteristics.

[0096] S4. Under aseptic conditions, take the sterilized model and the cultured artificial blood vessel, and place the cultured artificial blood vessel into the blood vessel culture chamber. Insert the artificial blood vessel into the fixation block and connecting tube to fix the artificial blood vessel.

[0097] S5. Under aseptic conditions, the cultured NCI-H23 tumor microspheres were placed sequentially in the first and second culture chambers.

[0098] It should be noted that this embodiment uses lung cancer cells NCI-H23 as an example. Of course, tumor cells from other organs can also be selected and cultured in different subculture chambers.

[0099] S6. The connecting layer and the culture layer are sealed together using non-biotoxic double-sided adhesive to form closed artificial blood vessel flow channels, closed tumor microsphere flow channels, and closed independent tumor microsphere flow channels.

[0100] In a sterile environment, the assembled model is connected to the culture tubing, and ethylene oxide is used to sterilize the model and the culture tubing to form a culture system.

[0101] like Figure 5 As shown, the first culture medium in the first culture flask 1 is connected to the tumor microsphere channel via pump M1, the second culture medium in the second culture flask 2 is connected to the artificial blood vessel channel via pump M2, and the third culture medium in the third culture flask 3 is connected to the independent tumor microsphere channel via pump M3.

[0102] S7. Place 15 mL of the corresponding culture medium into each culture flask (1 / 2 / 3), start the culture system, and when each culture medium fills the corresponding culture chamber and forms a flow perfusion loop, place the model along with the culture tubing into a 37°C sterile incubator; continue perfusion culture for 24 hours, then remove the model along with the culture tubing and use a high-content image analysis system to perform activity analysis on the artificial blood vessels and tumor microspheres within the model, ensuring that the artificial blood vessels and tumor microspheres have good functional characteristics, with tightly packed vascular endothelium and a certain directionality after continuous perfusion culture; the tumor microspheres are darker in the center and lighter at the edges with clear outlines. The images of the tumor microspheres acquired by the high-content system are shown in [image missing]. Figure 6 .

[0103] Furthermore, in a sterile environment, the second culture medium corresponding to the artificial blood vessel was replaced with a culture medium containing doxorubicin (Dox), and the model along with the culture system was placed in a sterile incubator at 37°C for continuous perfusion culture for 10 days to obtain tumor microspheres with vascular function.

[0104] Furthermore, the obtained tumor microspheres were analyzed using a high-content system, such as... Figure 7 and Figure 8 As shown, on days 1, 3, 5, 7, and 10 of the continuous culture perfusion culture described above, NCI-H23 tumor microspheres (corresponding to...) in the five sub-culture chambers of the first culture chamber B12 in the drug testing area were... Figure 7 NCI-H23 tumor microspheres (corresponding to Dox-1) in five subculture chambers of culture chamber B7 in the second group culture chamber (B7) and the independent control area. Figure 7 Activity analysis was performed using Dox-2 (DDoX-2). The analysis data and acquired images are shown below. Figure 7 , Figure 8Analysis of the above experimental data revealed that after 10 days of drug treatment, the activity of the five NCI-H23 tumor microspheres in the drug test area of ​​culture chamber B12 in the first group decreased to 20% of the activity of the NCI-H23 tumor microspheres in the control group of culture chamber B7 in the second group. This experimental phenomenon is consistent with the effect of doxorubicin on human lung cancer cells NCI-H23, indicating that doxorubicin can have a toxic effect on NCI-H23 tumor microsphere tissue after absorption, filtration, and shielding by artificial blood vessels.

[0105] It should be noted that the same method described above can also be used to conduct experiments with docetaxel at a concentration of 40 μmol / L, and the experimental data obtained will be similar. Figure 8 This indicates that within the model of the present invention, docetaxel (corresponding to...) Figure 8 DMSO can also have a toxic effect on NCI-H23 lung cancer cells.

[0106] This invention proposes an in vitro tumor vascular model and a method for detecting compounds using the in vitro tumor vascular model, which has the following beneficial effects:

[0107] First, this invention establishes a tumor vascular model in vitro, enabling the absorption of drugs and nutrients through blood vessels, as well as the construction of a microenvironment for tumor metastasis and invasion, and possessing functions such as blood circulation and vascular filtration.

[0108] Secondly, the model of this invention can realize the co-culture of multiple organs, which can reflect the complexity, functional changes and integrity of the actual organ functions of the body;

[0109] Third, the in vitro tumor vascular model of the present invention, on the one hand, allows drugs and nutrients to act on tumors after being absorbed through blood vessels and / or to act directly on tumors, enabling more accurate, efficient, and convenient testing of anti-tumor drugs; on the other hand, it can enable accurate, efficient, and convenient testing of the effects of anti-tumor drugs on blood vessels (for example, anti-tumor drugs may affect blood vessels, leading to vasculitis, hypertension, thrombosis, and other vascular-related diseases).

[0110] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. An in vitro tumor vascular model, characterized in that, include: Artificial blood vessel group, and Tumor microspheres The artificial blood vessel group includes an artificial blood vessel, an intravascular flow channel for independently culturing and / or perfusing the interior of the artificial blood vessel, and an extravascular flow channel for culturing and / or perfusing the exterior of the artificial blood vessel; the artificial blood vessel includes an endothelial layer and a smooth muscle layer; The tumor microsphere group includes one or more tumor microspheres and tumor microsphere channels for culturing and / or perfusing the one or more tumor microspheres. The tumor microsphere channels are connected to the external channels of the artificial blood vessel. The test compound in the internal channels of the artificial blood vessel is absorbed and filtered by the endothelial layer and smooth muscle layer of the artificial blood vessel and then seeps out into the external channels of the artificial blood vessel to act on the tumor microspheres.

2. The model according to claim 1, characterized in that, The width of the artificial blood vessel ranges from 2mm to 40mm.

3. The model according to claim 1, characterized in that, The length of the in vitro tumor vascular model ranges from 10mm to 50mm, and the width ranges from 5mm to 40mm.

4. The model according to claim 1, characterized in that, The diameter of the tumor microspheres in the single or multiple tumor microspheres ranges from 900 μm to 1300 μm.

5. The model according to claim 1, characterized in that, The in vitro tumor vascular model also includes an independent tumor microsphere assembly. The independent tumor microsphere group includes one or more independent tumor microspheres and independent tumor microsphere channels for independent culture and / or perfusion of the one or more independent tumor microspheres.

6. The model according to claim 5, characterized in that, The independent tumor microspheres are arranged symmetrically with the tumor microspheres.

7. The model according to claim 5, characterized in that, The artificial blood vessel group also includes an artificial blood vessel inlet that communicates with the internal flow channel of the artificial blood vessel; The tumor microsphere assembly also includes a tumor microsphere inlet that communicates with the tumor microsphere flow channel; The independent tumor microsphere assembly also includes an independent tumor microsphere inlet that is connected to the independent tumor microsphere flow channel.

8. A method for detecting compounds using an in vitro tumor vascular model according to any one of claims 1 to 7, characterized in that, The specific steps include the following: Culture media are introduced into each flow channel to culture or perfuse tumor microspheres and / or artificial blood vessels; The test compound was introduced into the artificial blood vessel assembly; Obtain the regulatory effects of the test compound on tumor microspheres and / or artificial blood vessels.

9. The method according to claim 8, characterized in that, The process of obtaining the regulatory effects of the test compound on tumor microspheres and / or artificial blood vessels includes: To obtain cell viability and / or cell migration results of tumor microspheres cultured via artificial blood vessels; and / or, Obtain the morphology of the artificial blood vessel; Based on the cell activity and / or cell migration results of the tumor microspheres, to screen for test compounds that match the tumor; and / or, Based on the morphology of the artificial blood vessel, the regulatory effect of the test compound on the artificial blood vessel is obtained.

10. The method according to claim 9, characterized in that, The step of obtaining the regulatory effect of the test compound on tumor microspheres further includes: To obtain cell viability and / or cell migration results for independent tumor microspheres; Based on the cell activity and / or cell migration results of the independent tumor microspheres and the tumor microspheres, the regulatory effect of the test compound on the tumor microspheres after absorption through artificial blood vessels is obtained.

Images