In vitro tumor heart vasculature model and methods for compound testing thereof

Abstract

The application provides a tumor heart blood vessel model in vitro and a method for detecting compounds, and belongs to the technical field of biological tissue engineering.The model comprises an artificial blood vessel group and a tumor myocardial cell 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 myocardial cell microsphere group comprises at least one tumor microsphere and at least one myocardial cell microsphere, and a tumor myocardial cell microsphere flow channel for culturing and / or perfusing the at least one tumor microsphere and the at least one myocardial cell microsphere, and the tumor myocardial cell microsphere flow channel is connected with the artificial blood vessel outer flow channel.The application establishes a model combining a heart, a blood vessel and a tumor model in vitro, has the functions of blood circulation and blood vessel filtration, realizes nutrient and drug absorption, and realizes the influence of drugs on the heart and tumor metastasis and invasion.

Description

Technical Field

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

[0002] Traditional methods for studying pharmacokinetics, pharmacodynamics, and drug toxicity mainly involve animal experiments and two-dimensional cell culture. While these methods have yielded many achievements, 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] Organ-on-a-chip technology combines methods from multiple disciplines, including cell biology, engineering, and biomaterials, to construct in vitro microenvironments of cells, tissues, and organs. As drug screening models, they more accurately reflect the effects and toxicity of drugs on corresponding cells, tissues, and organs compared to traditional toxicological animal experiments and in vitro 2D cell models. However, drug screening models in related technologies cannot reflect the complexity, functional changes, and integrity of organ functions, leading to limitations in their function and application, and a relatively narrow range of effects on the body. Summary of the Invention

[0004] 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 cardiovascular model and a method for detecting compounds using the in vitro tumor cardiovascular model.

[0005] In one aspect of the present invention, an in vitro tumor-cardiovascular model includes:

[0006] Artificial blood vessel group, and

[0007] Tumor cardiomyocyte microspheres

[0008] 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.

[0009] The tumor cardiomyocyte microsphere assembly includes at least one tumor microsphere and at least one cardiomyocyte microsphere, and a tumor cardiomyocyte microsphere channel for culturing and / or perfusing the at least one tumor microsphere and at least one cardiomyocyte microsphere, wherein the tumor cardiomyocyte microsphere channel is connected to the external channel of the artificial blood vessel.

[0010] Preferably, the artificial blood vessel comprises an endothelial layer and a smooth muscle layer; and / or,

[0011] The width of the artificial blood vessel ranges from 2mm to 40mm.

[0012] Preferably, the length of the in vitro tumor-associated cardiovascular model ranges from 10 mm to 50 mm, and the width ranges from 5 mm to 40 mm.

[0013] Preferably, the diameters of the cardiomyocyte microspheres and the tumor microspheres are both in the range of 900 μm to 1300 μm.

[0014] Preferably, the in vitro tumor-cardiovascular model further includes an independent tumor cardiomyocyte microsphere assembly.

[0015] The independent tumor cardiomyocyte microsphere group includes at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere, and an independent tumor cardiomyocyte microsphere channel for independent culture and / or perfusion of the at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere.

[0016] Preferably, the at least one independent tumor microsphere and the at least one independent cardiomyocyte microsphere are arranged symmetrically with respect to the at least one tumor microsphere and the at least one cardiomyocyte microsphere.

[0017] Preferably, the artificial blood vessel assembly further includes an artificial blood vessel inlet communicating with the internal flow channel of the artificial blood vessel;

[0018] The tumor cardiomyocyte microsphere assembly also includes a tumor cardiomyocyte microsphere inlet that is connected to the flow channel of the tumor cardiomyocyte microsphere;

[0019] The independent tumor cardiomyocyte microsphere assembly also includes an independent tumor cardiomyocyte microsphere inlet connected to the flow channel of the independent tumor cardiomyocyte microsphere.

[0020] In another aspect, the present invention provides a method for detecting compounds using the aforementioned in vitro tumor-cardiovascular model, comprising the following specific steps:

[0021] Each culture medium is introduced into each channel to culture or perfuse tumor microspheres, cardiomyocyte microspheres and / or artificial blood vessels;

[0022] The test compound was introduced into the artificial blood vessel assembly;

[0023] Obtain the regulatory effects of the test compound on tumor microspheres, cardiomyocyte microspheres, and / or artificial blood vessels.

[0024] Preferably, obtaining the regulatory results of the test compound on tumor microspheres, cardiomyocyte microspheres, and / or artificial blood vessels includes:

[0025] To obtain cell viability and / or cell migration results of tumor microspheres cultured via artificial blood vessels, and the peristaltic frequency of cardiomyocyte microspheres; and / or,

[0026] Obtain the morphology of the artificial blood vessel;

[0027] Based on the cell activity and / or cell migration results of the tumor microspheres, test compounds that match the tumor were screened.

[0028] The effects of the test compound on the cardiomyocyte microspheres and / or the morphology of the artificial blood vessel are obtained based on the peristaltic frequency of the cardiomyocyte microspheres and / or the artificial blood vessel.

[0029] Preferably, obtaining the regulatory results of the test compound on tumor microspheres and cardiomyocyte microspheres further includes:

[0030] To obtain cell viability and / or cell migration results of independent tumor microspheres, and peristaltic frequency of independent cardiomyocyte microspheres;

[0031] Based on the cell activity and / or cell migration results of the tumor microspheres and the independent tumor microspheres, and the peristaltic frequency of the cardiomyocyte microspheres and the independent cardiomyocyte microspheres, the regulatory effect of the test compound on the tumor microspheres and cardiomyocyte microspheres after absorption by artificial blood vessels can be obtained.

[0032] This invention is based on a multi-organ design, establishing an in vitro model combining heart, blood vessel, and tumor models. This model possesses functions such as blood flow and vascular filtration, enabling the creation of an environment conducive to nutrient and drug absorption, drug effects on the heart, and tumor metastasis and invasion. Using this biological model as a drug screening model allows for accurate, efficient, and convenient testing and research of pharmacokinetic and pharmacodynamic parameters related to tumor drugs, and more importantly, enables efficient testing of the cardiovascular toxicity of antitumor drugs. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the overall structure of an in vitro tumor-cardiovascular model according to an embodiment of the present invention;

[0034] Figure 2 This is a schematic diagram of the structure of the culture layer according to an embodiment of the present invention;

[0035] Figure 3 This is a schematic diagram of the structure of a sealing layer according to an embodiment of the present invention;

[0036] Figure 4 This is a schematic diagram of the connection layer according to an embodiment of the present invention;

[0037] Figure 5 This is a schematic diagram of the structure of a cultivation system according to an embodiment of the present invention;

[0038] Figure 6 This is a graph showing the change in the beating frequency of myocardial cell microspheres as a function of the number of days of drug action according to an embodiment of the present invention;

[0039] 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.

[0040] 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

[0041] 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.

[0042] like Figures 1 to 5 As shown, one aspect of the present invention provides an in vitro tumor-cardiovascular model, comprising: an artificial blood vessel group and a tumor cardiomyocyte microsphere group, wherein the artificial blood vessel group includes an artificial blood vessel, an artificial blood vessel internal flow channel for independent culture and / or perfusion of the interior of the artificial blood vessel, and an artificial blood vessel external flow channel for culture and / or perfusion of the exterior of the artificial blood vessel; the tumor cardiomyocyte microsphere group includes at least one tumor microsphere and at least one cardiomyocyte microsphere, and a tumor cardiomyocyte microsphere flow channel for culture and / or perfusion of at least one tumor microsphere and at least one cardiomyocyte microsphere, the tumor cardiomyocyte microsphere flow channel being connected to the artificial blood vessel external flow channel, i.e., tumor microspheres and cardiomyocyte microspheres with vascular function can be formed based on this model.

[0043] The in vitro tumor cardiovascular model of this embodiment can realize the absorption of drugs and nutrients through blood vessels, and has the functions of blood circulation and vascular filtration. 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In some other preferred embodiments, the width of the artificial blood vessel ranges from 2 mm to 40 mm.

[0048] As a further preferred option, the width of the artificial blood vessel ranges from 2mm to 7mm to simulate human veins.

[0049] 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.

[0050] Furthermore, in some preferred embodiments, the length of the in vitro tumor-associated cardiovascular model ranges from 10 mm to 50 mm, and the width ranges from 5 mm to 40 mm.

[0051] The model in this embodiment is relatively small, which improves the convenience of experimental operation.

[0052] Furthermore, in some preferred embodiments, at least one cardiomyocyte microsphere and at least one tumor microsphere are both spherical structures, and the diameters of the cardiomyocyte microspheres and tumor microspheres are both in the range of 900 μm to 1300 μm.

[0053] In this embodiment, the peristalsis frequency of the myocardial cell microspheres is 18 times / minute, and the tumor microspheres have functional characteristics, specifically, the center is dark in color, the edges are light and the outline is clear.

[0054] It should be noted that this embodiment does not specifically limit the type of at least one tumor microsphere. 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.

[0055] Specifically, such as Figure 1 and Figure 2 As shown, the tumor cardiomyocyte microsphere flow channel includes, in the direction of fluid flow, a tumor cardiomyocyte microsphere culture inlet B2, a tumor cardiomyocyte 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 cardiomyocyte microsphere outlet flow channel B1-1, and a tumor cardiomyocyte microsphere culture outlet B1.

[0056] Furthermore, such as Figures 1 to 3 As shown, the internal flow channel of the artificial blood vessel includes 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 in the direction of fluid flow. 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.

[0057] It should be noted that this embodiment does not specifically limit 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.

[0058] For example, to facilitate the installation of artificial blood vessels, such as Figure 1 and Figure 2 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.

[0059] Based on the specific structures of tumor cardiomyocyte microspheres and artificial blood vessels, the principles of culture or perfusion of artificial blood vessels, tumor microspheres, and cardiomyocyte microspheres are as follows: (Combined with...) Figure 5 As shown, a first culture medium and a second culture medium are introduced into the tumor cardiomyocyte microsphere channel and the artificial blood vessel channel, respectively, for culturing or perfusing tumor microspheres, cardiomyocyte 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 and cardiomyocyte microspheres in the first group of culture chambers, thus obtaining tumor microspheres and cardiomyocyte microspheres with vascular function. This can be used to screen tumor drugs and also to obtain results on the effects of drugs on the cardiovascular system.

[0060] In some other preferred embodiments, in order to provide a control over tumor microspheres and cardiomyocyte microspheres with vascular function, the in vitro tumor cardiovascular model further includes an independent tumor cardiomyocyte microsphere group, which includes at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere, and an independent tumor cardiomyocyte microsphere channel for independently culturing and / or perfusing at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere.

[0061] Specifically, such as Figure 2 As shown, the independent tumor cardiomyocyte microsphere flow channel includes an independent culture inlet B5, an independent inlet channel B5-1, a second culture chamber B7, an independent outlet channel B6-1, and an independent culture outlet B6, arranged sequentially on the culture layer B in the direction of fluid flow.

[0062] 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 tumor microspheres and cardiomyocyte microspheres.

[0063] 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.

[0064] Furthermore, when the channels in the tumor cardiomyocyte microsphere flow channel and the independent tumor cardiomyocyte microsphere flow channel 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.

[0065] 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 B, a third fluid conversion hole B8 is formed between the two flow channels.

[0066] 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 cardiomyocyte microsphere group further includes a tumor cardiomyocyte microsphere inlet communicating with the flow channel of the tumor cardiomyocyte microsphere; the independent tumor cardiomyocyte microsphere group further includes an independent tumor cardiomyocyte microsphere inlet communicating with the flow channel of the independent tumor cardiomyocyte microsphere, so that the corresponding culture medium can be introduced through each inlet. Of course, an outlet should also be provided to draw out the culture medium from each flow channel.

[0067] For example, such as Figures 1 to 4 As 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 cardiomyocyte microsphere inlet A2, a tumor cardiomyocyte microsphere outlet A1, and independent cardiomyocyte microsphere inlets A5 and A6. Specifically, the artificial blood vessel inlet A3 is connected to the blood vessel 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 outlet C4 via the artificial blood vessel culture inlet B4 on the culture layer B. Furthermore, the tumor cardiomyocyte microsphere inlet A2 is connected to the tumor cardiomyocyte microsphere culture inlet B2, and the tumor cardiomyocyte microsphere outlet A1 is connected to the tumor cardiomyocyte microsphere culture outlet B1. Additionally, the independent tumor cardiomyocyte microsphere inlet A5 is connected to the independent culture inlet B5, and the independent tumor cardiomyocyte microsphere outlet A6 is connected to the independent culture outlet B6.

[0068] 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 ease of obtaining results on the effects of different drugs on tumor microspheres and cardiomyocyte microspheres.

[0069] In another aspect, the present invention provides a method for detecting compounds using the in vitro tumor-cardiovascular model described above, comprising the following specific steps:

[0070] First, introduce culture media into each flow channel to culture or perfuse tumor microspheres, cardiomyocyte microspheres, and / or artificial blood vessels;

[0071] Second, introduce the test compound into the artificial blood vessel group;

[0072] Third, obtain the regulatory results of the test compound on tumor microspheres, cardiomyocyte microspheres and / or artificial blood vessels.

[0073] It should be understood that when using the models described above to culture or perfuse tumor microspheres or cardiomyocyte microspheres, it is also necessary to connect the model to the culture tubing, such as... Figure 5As shown, the culture tubing includes a first culture flask 1, a second culture flask 2, a third culture flask 3, and corresponding first, second, and third culture media contained in 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 cardiomyocyte 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 cardiomyocyte microsphere channel. The pumps on each culture tubing introduce the culture media from each culture flask into each channel.

[0074] In some preferred embodiments, the detection method specifically includes:

[0075] First, a first culture medium is introduced into the tumor cardiomyocyte microsphere group to culture or perfuse the tumor microspheres and cardiomyocyte microspheres;

[0076] 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.

[0077] Third, the tumor microspheres and cardiomyocyte microspheres in the tumor cardiomyocyte microsphere group were analyzed to obtain the cell activity and / or cell migration results of the tumor microspheres cultured in artificial blood vessels, the peristalsis frequency of the cardiomyocyte microspheres; and / or, the morphology of the artificial blood vessels was analyzed to obtain the morphological changes of the artificial blood vessels.

[0078] Based on the cell activity and / or cell migration results of tumor microspheres, to screen for test compounds that match tumors;

[0079] Based on the peristaltic frequency of cardiomyocyte microspheres and the morphological changes of artificial blood vessels, the effects of the above-mentioned test compounds on cardiovascular toxicity were obtained.

[0080] It should be noted that, to ensure the tumor microspheres possess the preset functional characteristics and the cardiomyocyte microspheres possess the preset peristaltic frequency, a first activity analysis is performed on the artificial blood vessels, tumor microspheres, and cardiomyocyte microspheres cultured in the second culture medium without the test compound. This ensures that the vascular endothelial cells are tightly packed and directional, the peristaltic frequency of the cardiomyocyte microspheres is 18 times / minute, and the tumor microspheres exhibit good functional characteristics. Subsequently, a second activity analysis is performed on the tumor microspheres, cardiomyocyte microspheres, and artificial blood vessels cultured in the second culture medium with the test compound added, to obtain the results of the test compound's effects on tumors, the heart, and blood vessels.

[0081] 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. The drug acts on the tumor microspheres and cardiomyocyte 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 peristaltic frequency of cardiomyocyte microspheres increases with the increase of drug concentration, and the vascular morphology also changes after drug administration.

[0082] In some other preferred embodiments, the detection method further includes:

[0083] First, a third culture medium is introduced into the independent tumor cardiomyocyte microsphere group to culture or perfuse the independent tumor microspheres and independent cardiomyocyte microspheres;

[0084] Second, the independent tumor microspheres and independent cardiomyocyte microspheres in the independent tumor cardiomyocyte microsphere group were analyzed to obtain the cell activity and cell migration results of the independent tumor microspheres and the peristaltic frequency of the independent cardiomyocyte microspheres.

[0085] Based on the cell activity and / or cell migration results of tumor microspheres and independent tumor microspheres, as well as the peristaltic frequency of cardiomyocyte microspheres and independent cardiomyocyte microspheres, the regulatory effect of the test compound on tumor microspheres and cardiomyocyte microspheres after absorption through artificial blood vessels can be obtained.

[0086] It should be understood that, since no drug exchange occurred in this embodiment, no drug acted on the independent tumor microspheres and independent cardiomyocyte microspheres. The independent tumor microspheres proliferated normally, and the peristaltic frequency of the independent cardiomyocyte microspheres did not change. This formed a control group with the tumor microspheres and cardiomyocyte microspheres group described above, in order to obtain the regulatory results of the test compound on the tumor and the heart.

[0087] This invention simulates the interaction between human blood vessels, tumors, and the heart in vitro, establishing a tumor-cardiovascular model. This model can more realistically reflect the effects of the test compounds in the human body, enabling the absorption of drugs and nutrients through blood vessels. By analyzing tumor microspheres, cardiomyocyte microspheres, and artificial blood vessels, it can be used to screen tumor drugs and simultaneously assess the impact of tumor drugs on cardiovascular toxicity.

[0088] The in vitro tumor cardiovascular model and its specific applications will be further illustrated below with reference to specific embodiments:

[0089] Example 1

[0090] This example uses an in vitro tumor cardiovascular model and a method for detecting compounds as an illustration, including the following steps:

[0091] S1. Obtain models with artificial blood vessel groups, tumor microsphere groups, and independent tumor microsphere groups.

[0092] S2. Use Huvec endothelial cells to culture artificial blood vessels until the artificial blood vessels exhibit functional characteristics.

[0093] S3. Using human pluripotent stem cells, we induce them to differentiate into human cardiomyocytes. We then use these cardiomyocytes to create cardiomyocyte microspheres and culture them until they exhibit functional characteristics, with the myocardium exhibiting a regular beat of 18 times per minute.

[0094] 3D tumor microspheres were fabricated using human lung cancer cells NCI-H23 and cultured until the NCI-H23 tumor microspheres exhibited functional characteristics.

[0095] 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.

[0096] S5. Under aseptic conditions, place two cultured cardiomyocyte microspheres and three NCI-H23 tumor microspheres sequentially into the first and second culture chambers.

[0097] S6. The connecting layer and the culture layer are sealed together using non-biotoxic double-sided adhesive to form a closed artificial blood vessel flow channel, a closed tumor cardiomyocyte microsphere flow channel, and a closed independent tumor cardiomyocyte microsphere flow channel.

[0098] 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.

[0099] like Figure 5 As shown, the first culture medium in the first culture flask 1 is connected to the tumor cardiomyocyte microsphere channel via pump M1, the second culture medium in the second culture flask 2 is connected to the intravascular channel via pump M2, and the third culture medium in the third culture flask 3 is connected to the independent tumor cardiomyocyte microsphere channel via pump M3.

[0100] 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; continuously perfuse culture for 24 h, 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, cardiomyocyte microspheres, and tumor microspheres in the model to ensure that the artificial blood vessels, cardiomyocyte microspheres, and tumor microspheres have good functional characteristics, the vascular endothelium is tightly arranged, and there is a certain directionality after continuous perfusion culture; the tumor microspheres are dark in the middle, light at the edges and have clear outlines, and the peristaltic frequency of the cardiomyocyte microspheres is 18 times / minute.

[0101] Furthermore, in a sterile environment, the second culture medium corresponding to the artificial blood vessel was replaced with a culture medium containing 5-fluorouracil (5-FU), 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 organ tissue.

[0102] Furthermore, the obtained tumor microspheres were analyzed using a high-content system. During the aforementioned continuous perfusion culture process, cardiomyocyte microspheres (corresponding to...) were analyzed every 24 hours in two sub-culture chambers of culture chamber B12 in the drug testing area. Figure 6 Cardiac cell microspheres (corresponding to 5-FU-1) in the middle and independent control areas of culture chamber B7 in the second group of culture chambers B7. Figure 6 The amplitude of the jumping frequency was analyzed using 5-FU-2. The graph of the analysis data is shown in the figure. Figure 6 The study found that 5-FU had no significant effect on the beating frequency of cardiomyocyte microspheres in the two sub-culture chambers of the drug test area in the first culture chamber (B12). It did have a damaging effect on the beating of the cardiomyocyte microspheres, but this effect was not significant. In the second culture chamber (B7 control group), 5-FU significantly affected the beating frequency of cardiomyocyte microspheres at both 24 and 48 hours, reducing the amplitude of the beating. The cardiomyocyte microspheres stopped beating on the seventh day and could not recover after washing. This experimental phenomenon is consistent with the effects of 5-FU on human myocardium, indicating that 5-FU can affect cardiomyocyte microsphere tissue after absorption, filtration, and shielding through artificial blood vessels.

[0103] Furthermore, the obtained organ tissues were further analyzed using a high-content system, such as... Figure 7 As shown, on days 1, 3, 5, 7, and 10 of the continuous perfusion culture, NCI-H23 tumor microspheres (corresponding to...) in the three sub-culture chambers of culture chamber B12 in the first group of the drug testing area were... Figure 7 NCI-H23 tumor microspheres (corresponding to 5-FU-1) and three subculture chambers of culture chamber B7 in the second group of independent control area. Figure 7 Activity analysis was performed on 5-FU-2, and the analysis data and acquired images are shown in [the image]. Figure 7 , Figure 8 Analysis of the above experimental data revealed that after 10 days of drug treatment, compared with the independent tumor microsphere activity of NCI-H23 in the control group (B7) of the second culture chamber, the activity of the three NCI-H23 tumor microspheres in the drug test area of ​​the first culture chamber (B12) decreased by approximately 25%. This experimental phenomenon is consistent with the effect of 5-FU on human lung cancer cells NCI-H23, indicating that 5-FU can have a toxic effect on NCI-H23 tumor microsphere tissue after absorption, filtration, and shielding through artificial blood vessels.

[0104] In summary, after being absorbed, filtered, and shielded by artificial blood vessels, the test drug can affect tissues such as cardiomyocyte microspheres and tumor microspheres. Furthermore, compared to the control group that did not undergo artificial blood vessel treatment, the experimental results are closer to those found in actual clinical studies. The detection method for multi-tissue function regulation using this invention can more accurately reflect the effects of the test drug in the human body.

[0105] This invention proposes an in vitro tumor-cardiovascular model and a method for compound detection using this model, offering the following advantages: Based on a multi-organ-chip design, this invention establishes an in vitro model combining the heart, blood vessels, and tumor, enabling functions such as blood flow and vascular filtration. This allows for the creation of an environment conducive to nutrient and drug absorption, drug effects on the heart, and tumor metastasis and invasion. Using this biological model as a drug screening model allows for accurate, efficient, and convenient testing and research of pharmacokinetics and pharmacodynamics related to tumor drugs, and more importantly, enables efficient testing of the cardiovascular toxicity of antitumor drugs.

[0106] 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-associated cardiovascular model, characterized in that, include: Artificial blood vessel group, and Tumor cardiomyocyte 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 cardiomyocyte microsphere assembly includes at least one tumor microsphere and at least one cardiomyocyte microsphere, and a tumor cardiomyocyte microsphere channel for culturing and / or perfusing the at least one tumor microsphere and at least one cardiomyocyte microsphere. The tumor cardiomyocyte microsphere channel is connected to the external channel of the artificial blood vessel. The test compound in the internal channel 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 channel of the artificial blood vessel to act on the tumor microsphere and cardiomyocyte microsphere.

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-associated cardiovascular model ranges from 10 mm to 50 mm, and the width ranges from 5 mm to 40 mm.

4. The model according to claim 1, characterized in that, The diameters of both the cardiomyocyte microspheres and the tumor microspheres range from 900 μm to 1300 μm.

5. The model according to claim 1, characterized in that, The in vitro tumor-cardiovascular model also includes an independent tumor cardiomyocyte microsphere assembly. The independent tumor cardiomyocyte microsphere group includes at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere, and an independent tumor cardiomyocyte microsphere channel for independent culture and / or perfusion of the at least one independent tumor microsphere and at least one independent cardiomyocyte microsphere.

6. The model according to claim 5, characterized in that, The at least one independent tumor microsphere and the at least one independent cardiomyocyte microsphere are arranged symmetrically with the at least one tumor microsphere and the at least one cardiomyocyte microsphere.

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 cardiomyocyte microsphere assembly also includes a tumor cardiomyocyte microsphere inlet that is connected to the flow channel of the tumor cardiomyocyte microsphere; The independent tumor cardiomyocyte microsphere assembly also includes an independent tumor cardiomyocyte microsphere inlet connected to the flow channel of the independent tumor cardiomyocyte microsphere.

8. A method for detecting compounds using an in vitro tumor-cardiovascular model according to any one of claims 1 to 7, characterized in that, The specific steps include the following: Each culture medium is introduced into each channel to culture or perfuse tumor microspheres, cardiomyocyte 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, cardiomyocyte microspheres, and / or artificial blood vessels.

9. The method according to claim 8, characterized in that, The process of obtaining the regulatory results of the test compound on tumor microspheres, cardiomyocyte 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 the peristaltic frequency of cardiomyocyte microspheres; and / or, Obtain the morphology of the artificial blood vessel; Based on the cell activity and / or cell migration results of the tumor microspheres, test compounds that match the tumor were screened. The effects of the test compound on the cardiomyocyte microspheres and / or the morphology of the artificial blood vessel are obtained based on the peristaltic frequency of the cardiomyocyte microspheres and / or the artificial blood vessel.

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

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