Vasculation immune co-culture organoid chip for simulating tumor microenvironment and organoid culture method

Abstract

The invention discloses a vascularized immune co-culture organoid chip for simulating a tumor microenvironment. The vascularized immune co-culture organoid chip comprises a chip main body and a bottom plate, a flow channel and a liquid storage tank are arranged in the chip main body; the runners comprise a first runner, a second runner, a third runner and a fourth runner; a compartment is arranged in the middle of the third flow channel; the first flow channel is connected with a first liquid storage tank which is used for injecting a first hydrogel suspension obtained by mixing fibroblasts and hydrogel into the first flow channel; the second flow channel is connected with a second liquid storage tank for injecting a culture solution into the second flow channel; a compartment is arranged in the middle of the third flow channel; the third flow channel is connected with a third liquid storage tank communicated with the compartment, and the compartment is connected with a compartment liquid storage tank; the compartment liquid storage tank is used for injecting a second hydrogel suspension obtained by mixing tumor organs and hydrogel into the compartment, and the third liquid storage tank is used for injecting hydrogel into the third flow channel; and the fourth flow channel is connected with a fourth liquid storage tank for injecting vascular endothelial cell and immune cell suspension into the fourth flow channel. The invention further discloses a vascularized immune co-culture organoid culture method for simulating the tumor microenvironment.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically relates to a vascularized immune co-culture organoid chip simulating a tumor microenvironment and an organoid culture method. Background Art

[0002] Tumor organoids are an emerging in vitro model in recent years, which have many advantages over traditional in vitro models such as two-dimensional cell lines, animal models and 3D tumor cell spheres. Although tumor organoids are the closest in vitro model to the human body, there are still quite a few shortcomings. For example, the immune system is a system that protects the human body from pathogens and cancer cells. The immune cells in it play an important role in identifying and killing cancer cells, but tumor organoids lack immune cells. In addition, in the human body, tumor tissue has a tumor microenvironment, which includes immune cells, blood vessels, cancer cells, normal cells, fibroblasts, etc. The growth, metastasis, angiogenesis and other processes of tumor cells are closely related to it. Usually, the development of malignant tumors is accompanied by abnormal angiogenesis, which shows that blood vessels play an extremely critical role in the development of malignant tumors. Therefore, the mechanism behind this abnormal angiogenesis has been widely studied.

[0003] In summary, it is particularly important to construct an in vitro model of vascularized immune co-cultured organoids with a tumor microenvironment. In the prior art, a common solution is to use a microfluidic chip, adopt a closed space structure, and use a limiting structure (such as microcolumns and microwalls) to divide the space to form a flow channel. The middle flow channel connects the organoid, and the flow channel on one side is inoculated with endothelial cells to form blood vessels, and the flow channel on the other side flows the culture medium. The existing technical solutions have the following deficiencies:

[0004] (1) The middle channel is not divided, and one chip can only be inoculated with one type of organoid;

[0005] (2) There are only three flow channels and a lack of fibroblasts, which cannot truly simulate the human tumor microenvironment. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a vascularized immune co-culture organoid chip and an organoid culture method that simulates the tumor microenvironment, which can construct an in vitro model containing co-culture of tumor organoids, immune cells, fibroblasts and vascular endothelial cells to more realistically simulate the human tumor microenvironment.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention first proposes a vascularized immune co-culture organoid chip that simulates a tumor microenvironment, comprising a chip body and a bottom plate; a flow channel and a liquid reservoir are provided in the chip body;

[0009] The flow channel includes a first flow channel, a second flow channel, a third flow channel and a fourth flow channel; the second flow channel is located between the first flow channel and the third flow channel, and the third flow channel is located between the second flow channel and the fourth flow channel; a compartment is provided in the middle of the third flow channel;

[0010] The first flow channel is connected to a first liquid reservoir, and the first liquid reservoir is used to inject a first hydrogel suspension obtained by mixing fibroblasts and hydrogel into the first flow channel during the vascularized immune co-culture process;

[0011] The second flow channel is connected to a second liquid reservoir, and the second liquid reservoir is used to inject culture fluid into the second flow channel during the vascularized immune co-culture process;

[0012] A compartment is provided in the middle of the third flow channel; a third liquid reservoir connected to the compartment is connected to the third flow channel, and the compartment is connected to the compartment liquid reservoir; the compartment liquid reservoir is used to inject a second hydrogel suspension obtained by mixing tumor organoids and hydrogel into the compartment during the vascularized immune co-culture process, and the third liquid reservoir is used to inject hydrogel into the third flow channel during the vascularized immune co-culture process;

[0013] The fourth flow channel is connected to a fourth liquid reservoir, and the fourth liquid reservoir is used to inject vascular endothelial cells and immune cell suspensions into the fourth flow channel during the vascularized immune co-culture process.

[0014] Furthermore, at least two independent compartments are provided in the third flow channel, and each of the compartments is located between the second flow channel and the fourth flow channel; the third liquid storage tank is arranged corresponding to the compartments, and the third liquid storage tank is connected to the corresponding compartments.

[0015] Furthermore, the base plate has biocompatibility and light transmittance, and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

[0016] Furthermore, the chip body is biocompatible and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

[0017] The present invention also proposes an organoid culture method using the vascularized immune co-culture organoid chip that simulates the tumor microenvironment as described above, characterized in that it comprises the following steps:

[0018] Step 1: Organoid Chip Pretreatment

[0019] The organoid chip was cleaned with alcohol and PBS buffer in turn, and then placed under ultraviolet light for irradiation and disinfection;

[0020] Step 2: Injection of fibroblasts

[0021] Mixing fibroblasts and hydrogel to obtain a first hydrogel suspension, and injecting the first hydrogel suspension into the first flow channel through the first liquid reservoir to form a hydrogel barrier between the first flow channel and the second flow channel;

[0022] Step 3: Inoculate the organ

[0023] The organoid sample is mixed with the hydrogel to obtain a second hydrogel suspension, and the second hydrogel suspension is injected into the compartment through the compartment reservoir; the organoid chip is placed in an incubator until the hydrogel solidifies, and then a sufficient amount of culture medium is added into the second flow channel through the second reservoir and into the corresponding compartment through the compartment reservoir;

[0024] Step 4: Forming a hydrogel barrier

[0025] Injecting hydrogel into the third flow channel through the third liquid reservoir to form a hydrogel barrier between the third flow channel and the fourth flow channel, and then placing the organoid chip in an incubator until the hydrogel solidifies;

[0026] Step 5: Constructing a perfusable vascular network

[0027] 51) injecting vascular endothelial cells into the fourth flow channel through the fourth liquid reservoir, and then tilting the organoid chip so that the vascular endothelial cells adhere to the hydrogel barrier;

[0028] 52) injecting a sufficient amount of culture medium into the fourth flow channel through the fourth liquid reservoir, and then driving the chip to swing, so as to provide nutrition and shear force stimulation for the fibroblasts, tumor organoids and vascular endothelial cells; until the vascular endothelial cells cover the inner surface of the fourth flow channel and sprout toward the organoids, forming a new vascular network surrounding the organoids;

[0029] Step 6: Immune cell co-culture

[0030] The immune cell suspension is injected into the fourth flow channel through the fourth liquid reservoir to observe the killing process of cancer cells by immune cells and realize organoid immune co-culture.

[0031] Furthermore, at least two independent compartments are provided in the third flow channel, and each of the compartments is located between the second flow channel and the fourth flow channel; the third liquid storage tank is provided corresponding to the compartments, and the third liquid storage tank is communicated with the corresponding compartments;

[0032] In step 2, a second hydrogel suspension containing the same or different organoids is injected into each compartment respectively.

[0033] Further, the organoids are tumor organoids, including colon cancer organoids, lung cancer organoids, gastric cancer organoids, breast cancer organoids, liver cancer organoids, ovarian cancer organoids, cervical cancer organoids, brain cancer organoids, skin cancer organoids, prostate cancer organoids, kidney cancer organoids, bladder cancer organoids, head and neck cancer organoids, melanoma organoids, esophageal cancer organoids, thyroid cancer organoids and pancreatic cancer organoids.

[0034] The beneficial effects of the present invention are:

[0035] The vascularized immune co-culture organoid chip of the present invention simulates the tumor microenvironment. By setting four flow channels including a first flow channel, a second flow channel, a third flow channel and a fourth flow channel, during the organoid culture process, after pre-treatment of the organoid chip, a first hydrogel suspension obtained by mixing fibroblasts and hydrogel is first injected into the first flow channel, thereby introducing fibroblasts into the human tumor microenvironment. The main function of fibroblasts is to produce and maintain the tumor microenvironment, so they can maintain the tumor microenvironment, interact with endothelial cells to enhance angiogenesis, and affect drug efficacy and therapeutic response; the second hydrogel suspension obtained by mixing the organoid sample with the hydrogel is injected into the second flow channel through the compartment reservoir to achieve organoid inoculation; the hydrogel is injected into the second flow channel through the third reservoir to form a hydrogel barrier; and then the vascular endothelial cells are injected into the fourth flow channel, and nutrition is provided by a sufficient amount of culture medium until the vascular endothelial cells cover the inner surface of the fourth flow channel, and sprout to the organoid to form a new vascular network surrounding the organoid; finally, the immune cell suspension is injected into the fourth flow channel, and the killing process of immune cells on cancer cells can be visually observed, and organoid immune co-culture is achieved. In summary, the vascularized immune co-culture organoid chip that simulates the tumor microenvironment of the present invention can construct an in vitro model comprising tumor organoids, immune cells, fibroblasts and vascular endothelial cells co-cultured to more realistically simulate the human tumor microenvironment.

[0036] In the present invention, at least two independent compartments are provided in the third flow channel. Thus, during the organoid culture process, different second hydrogel suspensions obtained by mixing different organoid samples with hydrogels can be injected into different compartments. Thus, at least two organoids can be inoculated during one organoid culture process, thereby improving efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0038] Figure 1 This is a schematic diagram of the structure of an embodiment of a vascularized immune co-culture organoid chip that simulates a tumor microenvironment according to the present invention;

[0039] Figure 2 is a schematic diagram of the structure of the flow channel;

[0040] Figure 3 It is a structural schematic diagram of the liquid storage tank;

[0041] Figure 4 The present invention is a flow chart of the vascularized immune co-culture organoid culture method for simulating the tumor microenvironment.

[0042] Description of reference numerals:

[0043] 101-chip body; 1011-first flow channel; 1011a-first liquid reservoir; 1012-second flow channel; 1012a-second liquid reservoir; 1013-third flow channel; 1013a-third liquid reservoir; 1014-fourth flow channel; 1014a-fourth liquid reservoir; 1015-first compartment; 1015a-first compartment liquid reservoir; 1016-second compartment; 1016a-second compartment liquid reservoir;

[0044] 102-base plate. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0046] like Figure 1 As shown, the vascularized immune co-culture organoid chip simulating the tumor microenvironment in this embodiment includes a chip body 101 and a base plate 102. Specifically, a flow channel and a liquid reservoir are provided in the chip body 101.

[0047] like Figure 2 As shown, in this embodiment, the flow channel includes a first flow channel 1011, a second flow channel 1012, a third flow channel 1013 and a fourth flow channel 1014. Specifically, the second flow channel 1012 is located between the first flow channel 1011 and the third flow channel 1013, and the third flow channel 1013 is located between the second flow channel 1012 and the fourth flow channel 1014; a compartment is provided in the middle of the third flow channel 1013.

[0048] like Figure 3As shown, in this embodiment, the reservoir includes a first reservoir 1011a, a second reservoir 1012a, a third reservoir 1013a, a fourth reservoir 1014a and a compartment reservoir. Specifically, the first flow channel 1011 is connected to the first reservoir 1011a, and the first reservoir 1011a is used to inject the first hydrogel suspension obtained by mixing fibroblasts and hydrogel into the first flow channel 1011 during the vascularized immune co-culture process. In this embodiment, the first reservoir 1011a is set to two, and the two first reservoirs 1011a are respectively connected to the two ends of the first stay 1011. The second flow channel 1012 is connected to the second reservoir 1012a, and the second reservoir 1012a is used to inject culture fluid into the second flow channel 1012 during the vascularized immune co-culture process. Specifically, the second reservoir 1012a of this embodiment is set to two, and the two second reservoirs 1012a are respectively connected to the two ends of the second flow channel 1012. In this embodiment, a compartment is provided in the middle of the third flow channel 1013, and a third liquid reservoir 1013a connected to the compartment is connected to the third flow channel 1013, and the compartment is connected to the compartment liquid reservoir. Specifically, the compartment liquid reservoir is used to inject a second hydrogel suspension obtained by mixing tumor organoids and hydrogel into the compartment during the vascularized immune co-culture process, and the third liquid reservoir 1013a is used to inject hydrogel into the third flow channel 1013 during the vascularized immune co-culture process. In this embodiment, a fourth liquid reservoir 1014a is connected to the fourth flow channel 1014, and the fourth liquid reservoir 1014a is used to inject vascular endothelial cells and immune cell suspensions into the fourth flow channel 1014 during the vascularized immune co-culture process. Specifically, the fourth liquid reservoir 1014a is set to two and respectively connected to the two ends of the fourth flow channel 1014.

[0049] In the preferred implementation of this embodiment, in order to achieve the technical purpose of culturing multiple organoids at the same time, at least two independent compartments are provided in the third flow channel 1013 of this embodiment, and the compartments are used to connect the organoids, and each compartment is located between the second flow channel 1012 and the fourth flow channel 1014. The third liquid reservoir 1013a is arranged corresponding to the compartment, and the third liquid reservoir 1013a is connected to the corresponding compartment. Specifically, in this embodiment, there are two compartments, and the two compartments are the first compartment 1015 and the second compartment 1016. The third liquid reservoir 1013a of this embodiment is also set to two, and the two third liquid reservoirs 1013a are connected to the first compartment 1015 and the second compartment 1016 respectively. The compartment liquid reservoir of this embodiment is also set to two, the first liquid reservoir 1015a is connected to the first compartment 1015 and is located directly above the first compartment 1015, and the second liquid reservoir 1016a is connected to the second compartment 1016 and is located directly above the second compartment 1016. Thus, in this embodiment, two types of organoids can be inoculated simultaneously through the first compartment 1015 and the second compartment 1016 .

[0050] Specifically, the bottom plate 102 needs to be biocompatible and light-transmissive so as to facilitate observation of the organoid culture process and the killing process of the immune cells against the cancer cells. Thus, the bottom plate 102 can be made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

[0051] Similarly, the chip body 101 should also be biocompatible and made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin. The manufacturing method of the chip body 101 includes one-piece injection molding, mechanical milling and 3D printing, etc., which will not be repeated.

[0052] like Figure 4 As shown, this embodiment also proposes a vascularized immune co-culture organoid culture method that simulates the tumor microenvironment, comprising the following steps:

[0053] Step 1: Organoid Chip Pretreatment

[0054] The organoid chip is cleaned with alcohol and PBS buffer in turn, and then the organoid chip is placed under ultraviolet light for irradiation and disinfection. Specifically, the specific method for pre-treatment of the organoid chip is as follows: add 75% concentration alcohol to all reservoirs of the organoid chip, and tilt it repeatedly to ensure that the alcohol flows through the internal flow channel; after one minute of treatment, the remaining alcohol is sucked out. Then add PBS buffer to the reservoir for cleaning, and after one minute of treatment, the remaining buffer is sucked out. After absorbing the liquid in the chip as much as possible, place it under ultraviolet irradiation for 30 minutes to complete the pre-treatment of the organoid immune co-culture chip.

[0055] Step 2: Injection of fibroblasts

[0056] The fibroblasts and the hydrogel are mixed to obtain a first hydrogel suspension. 4 μl of the first hydrogel suspension is aspirated using a pipette and injected into the first flow channel 1011 through the first liquid reservoir 1011a using a pipette to form a hydrogel barrier between the first flow channel 1011 and the second flow channel 1012 .

[0057] Step 3: Inoculate the organ

[0058] The organoid sample is mixed with the hydrogel to obtain a second hydrogel suspension, so that each microliter of the second hydrogel suspension contains 2000-4000 cells. The second hydrogel suspension is sucked with a pipette, and the second hydrogel suspension is injected into the compartment through the compartment reservoir, and the amount of the second hydrogel suspension added to the compartment is 25 microliters. The organoid chip is placed in an incubator, and the hydrogel is solidified at an ambient temperature of 37°C, and then a sufficient amount of culture medium is added to the second flow channel 1012 through the second reservoir 1012a, and a sufficient amount of culture medium is added to the corresponding compartment through the compartment reservoir.

[0059] Specifically, at least two independent compartments are provided in the third flow channel 1013, and each compartment is located between the second flow channel 1012 and the fourth flow channel 1014; the third liquid reservoir 1013a is arranged corresponding to the compartment, and the third liquid reservoir 1013a is connected to the corresponding compartment. When culturing organoids, the same second hydrogel suspension can be injected into each compartment respectively, or different second hydrogel suspensions of organoids can be injected into each compartment respectively. Specifically, the organoids are tumor organoids, including colon cancer organoids, lung cancer organoids, gastric cancer organoids, breast cancer organoids, liver cancer organoids, ovarian cancer organoids, cervical cancer organoids, brain cancer organoids, skin cancer organoids, prostate cancer organoids, kidney cancer organoids, bladder cancer organoids, head and neck cancer organoids, melanoma organoids, esophageal cancer organoids, thyroid cancer organoids and pancreatic cancer organoids.

[0060] In this embodiment, there are two compartments, which are the first compartment 1015 and the second compartment 1016. In this embodiment, there are also two third liquid reservoirs 1013a, which are respectively connected to the first compartment 1015 and the second compartment 1016. In this embodiment, there are also two compartment liquid reservoirs, the first compartment liquid reservoir 1015a is connected to the first compartment 1015 and is located directly above the first compartment 1015, and the second compartment liquid reservoir 1016a is connected to the second compartment 1016 and is located directly above the second compartment 1016. In this way, in this embodiment, two types of organoids can be inoculated simultaneously through the first compartment 1015 and the second compartment 1016. Thus, when preparing the second hydrogel suspension, two different organoid samples can be mixed with the hydrogel respectively to prepare two different second hydrogel suspensions, and then the two different second hydrogel suspensions are added to the first compartment 1015 and the second compartment 1016 respectively for inoculation, thereby achieving the technical purpose of culturing two organoids at the same time.

[0061] Step 4: Forming a hydrogel barrier

[0062] The hydrogel is sucked by a pipette, and the hydrogel is injected into the third flow channel 1013 through the third liquid reservoir 1013a. After a hydrogel barrier is formed between the third flow channel 1013 and the fourth flow channel 1014, the organoid chip is placed in an incubator until the hydrogel solidifies. Specifically, in this embodiment, 0.5-20 microliters of hydrogel are injected through two third liquid reservoirs 1013a respectively. After injecting the hydrogel, it should be observed whether the hydrogel barrier is formed, whether there is any overflow from the flow channel, etc.; if the hydrogel barrier has been formed and the hydrogel has not overflowed, the organoid chip is placed in an incubator at an ambient temperature of 37°C to solidify the hydrogel.

[0063] Step 5: Constructing a perfusable vascular network

[0064] 51) Prepare human umbilical vein endothelial cells, use a pipette to draw 3-100 μl of human umbilical vein endothelial cells, inject the endothelial cells into the fourth flow channel 1014 through the fourth liquid reservoir 1014a, and then tilt the organoid chip 90 degrees for 1-6 hours. The endothelial cells adhere to the hydrogel barrier between the third flow channel and the fourth flow channel to form a thin cell layer, so that the endothelial cells adhere to the hydrogel barrier.

[0065] 52) A sufficient amount of culture medium is injected into the fourth flow channel 1014 through the fourth liquid reservoir 1014a, and then the organoid chip is placed on a shaking platform, and the chip is driven to swing to drive the fluid flow, providing nutrition and shear force stimulation for fibroblasts, tumor organoids and vascular endothelial cells. After 3-7 days, the vascular endothelial cells cover the inner surface of the fourth flow channel 1014 and sprout toward the organoid, forming a new vascular network surrounding the organoid.

[0066] Step 6: Immune cell co-culture

[0067] Prepare immune cell suspension, use a pipette to absorb 1-50 μl of immune cells, inject the immune cell suspension into the fourth flow channel 1014 through the fourth liquid reservoir 1014a, and then place the organoid chip on a live cell imager to observe the killing process of immune cells on cancer cells to achieve organoid immune co-culture.

[0068] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A vascularized immune co-culture organoid chip simulating a tumor microenvironment, comprising a chip body (101) and a base plate (102); characterized in that: The chip body (101) is provided with a flow channel and a liquid storage tank; The flow channel comprises a first flow channel (1011), a second flow channel (1012), a third flow channel (1013) and a fourth flow channel (1014); the second flow channel (1012) is located between the first flow channel (1011) and the third flow channel (1013), and the third flow channel (1013) is located between the second flow channel (1012) and the fourth flow channel (1014); a compartment is provided in the middle of the third flow channel (1013); The first flow channel (1011) is connected to a first liquid reservoir (1011a), and the first liquid reservoir (1011a) is used to inject a first hydrogel suspension obtained by mixing fibroblasts and hydrogel into the first flow channel (1011) during the vascularized immune co-culture process; The second flow channel (1012) is connected to a second liquid reservoir (1012a), and the second liquid reservoir (1012a) is used to inject culture fluid into the second flow channel (1012) during the vascularized immune co-culture process; A compartment is provided in the middle of the third flow channel (1013); the third flow channel (1013) is connected to a third liquid reservoir (1013a) in communication with the compartment, and the compartment is connected to a compartment liquid reservoir; the compartment liquid reservoir is used to inject a second hydrogel suspension obtained by mixing tumor organoids and hydrogel into the compartment during the vascularized immune co-culture process, and the third liquid reservoir (1013a) is used to inject hydrogel into the third flow channel (1013) during the vascularized immune co-culture process; The fourth flow channel (1014) is connected to a fourth liquid reservoir (1014a), and the fourth liquid reservoir (1014a) is used to inject vascular endothelial cells and immune cell suspension into the fourth flow channel (1014) during the vascularized immune co-culture process.

2. The vascularized immune co-culture organoid chip simulating a tumor microenvironment according to claim 1, characterized in that: At least two independent compartments are provided in the third flow channel (1013), and each of the compartments is located between the second flow channel (1012) and the fourth flow channel (1014); the third liquid storage tank (1013a) is arranged corresponding to the compartments, and the third liquid storage tank (1013a) is connected to the corresponding compartments.

3. The vascularized immune co-culture organoid chip simulating a tumor microenvironment according to claim 1, characterized in that: The base plate (102) has biocompatibility and light transmittance, and is made of one of glass, polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

4. The vascularized immune co-culture organoid chip simulating a tumor microenvironment according to claim 1, characterized in that: The chip body (101) is biocompatible and is made of one of polydimethylsiloxane, polypropylene, polystyrene, polycarbonate, cycloolefin copolymer, polymethyl methacrylate, hydrogel and transparent resin.

5. An organoid culture method using a vascularized immune co-culture organoid chip that simulates a tumor microenvironment as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: Organoid Chip Pretreatment The organoid chip was cleaned with alcohol and PBS buffer in turn, and then placed under ultraviolet light for irradiation and disinfection; Step 2: Injection of fibroblasts Mixing fibroblasts and hydrogel to obtain a first hydrogel suspension, and injecting the first hydrogel suspension into the first flow channel (1011) through the first liquid reservoir (1011a), so that a hydrogel barrier is formed between the first flow channel (1011) and the second flow channel (1012); Step 3: Inoculate the organ The organoid sample is mixed with the hydrogel to obtain a second hydrogel suspension, and the second hydrogel suspension is injected into the compartment through the compartment reservoir; the organoid chip is placed in an incubator until the hydrogel solidifies, and then a sufficient amount of culture medium is added into the second flow channel (1012) through the second reservoir (1012a) and into the corresponding compartment through the compartment reservoir; Step 4: Forming a hydrogel barrier Injecting hydrogel into the third flow channel (1013) through the third liquid reservoir (1013a) to form a hydrogel barrier between the third flow channel (1013) and the fourth flow channel (1014), and then placing the organoid chip in an incubator until the hydrogel solidifies; Step 5: Constructing a perfusable vascular network 51) injecting vascular endothelial cells into the fourth flow channel (1014) through the fourth liquid reservoir (1014a), and then tilting the organoid chip so that the vascular endothelial cells adhere to the hydrogel barrier; 52) injecting a sufficient amount of culture medium into the fourth flow channel (1014) through the fourth liquid reservoir (1014a), and then driving the chip to swing, so as to provide nutrition and shear force stimulation to the fibroblasts, tumor organoids and vascular endothelial cells; until the vascular endothelial cells cover the inner surface of the fourth flow channel (1014) and sprout toward the organoids, forming a new vascular network surrounding the organoids; Step 6: Immune cell co-culture An immune cell suspension is injected into the fourth flow channel (1014) through the fourth liquid storage tank (1014a), and the killing process of the immune cells on the cancer cells is observed to achieve organoid immune co-culture.

6. The method for culturing vascularized immune co-culture organoids simulating a tumor microenvironment according to claim 5, characterized in that: At least two independent compartments are provided in the third flow channel (1013), and each of the compartments is located between the second flow channel (1012) and the fourth flow channel (1014); the third liquid storage tank (1013a) is arranged corresponding to the compartments, and the third liquid storage tank (1013a) is connected to the corresponding compartments; In step 2, a second hydrogel suspension containing the same or different organoids is injected into each compartment respectively.

7. The method for culturing vascularized immune co-culture organoids simulating a tumor microenvironment according to claim 5, characterized in that: The organoids are tumor organoids, including colon cancer organoids, lung cancer organoids, gastric cancer organoids, breast cancer organoids, liver cancer organoids, ovarian cancer organoids, cervical cancer organoids, brain cancer organoids, skin cancer organoids, prostate cancer organoids, kidney cancer organoids, bladder cancer organoids, head and neck cancer organoids, melanoma organoids, esophageal cancer organoids, thyroid cancer organoids, and pancreatic cancer organoids.

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