Vascularized tumor organ chip and application thereof

Abstract

The invention relates to the technical field of organoid culture, in particular to a vascularized tumor organoid chip, which is characterized in that a structure that a previous culture solution flow channel and a cell culture flow channel are separated by a micro-column is designed into a structure that the previous culture solution flow channel and the cell culture flow channel are dynamically separated by an acupuncture needle, so that the physical limitation of the traditional micro-column on the growth of blood vessels is avoided; the contact area of vascular cells is obviously increased, and the growth of a vascular network is promoted; meanwhile, free switching of two liquid feeding modes of a micro-injection pump and gravitational potential energy can be realized under the action of a needle inlet runner and a liquid storage tank; and dynamic detection can be realized in the process of co-culturing blood vessels and tumor organs.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and more specifically, it relates to a vascularized tumor organoid chip and its application. Background Art

[0002] Angiogenesis plays an important role in the growth, invasion, and metastasis of tumors, and there is a complex interaction between angiogenesis and tumors. Therefore, constructing an in vitro model with a vascular structure can maximize the simulation of the tumor microenvironment, thereby better studying the biological characteristics of tumors and anti-angiogenic drugs. Currently, the tumor vasculature is mainly simulated by co-culturing tumor cell lines and vascular endothelial cells, and most of its main forms are 2D cultures. This method is difficult to simulate the interaction between real tumor and vascular cells, and the conventional static culture is prone to concentration gradients due to poor gas-liquid mass transfer ability, which in turn affects the function of co-cultured cells. Therefore, it can no longer meet the needs of scientific research and clinical practice. As a cutting-edge technology, organ-on-a-chip has the advantages of high throughput and small sample requirements. At the same time, it can be connected to a continuous or circulating perfusion system, which is more in line with human physiological characteristics. By co-culturing tumor cells and vascular endothelial cells in an organ-on-a-chip, the formation of tumor blood vessels in patients and their response to drugs can be simulated.

[0003] However, the current co-culture model of tumor-vascular cells (commonly HUVECs) based on organ-on-a-chip still has some limitations: (1) Since this model requires different flow channels, such as culture medium flow channels and cell culture flow channels, and the culture medium flow channel and the cell culture flow channel are often separated by a limiting structure (micro-columns), etc., this leads to poor growth of vascular cells, especially poor perfusion openings in the vascular model, which in turn affects perfusion; (2) The current dynamic culture mode of organ-on-a-chip is mainly based on external pipelines such as micro-injection pumps / peristaltic pumps for liquid supply, or perfusion is carried out by means of a shaker to form the gravitational potential energy of the culture medium. Different liquid supply methods have different advantages, but rarely combine the two; (3) In the current co-culture model, most are mixed cultures of tumor cell lines and HUVECs, which affect observation and analysis in subsequent experiments. Summary of the Invention

[0004] The purpose of the present invention is to provide a vascularized tumor organoid chip to solve the problems of poor formation of the vascular model, incompatible culture medium liquid supply methods, and mixed co-culture mode affecting observation in existing organ-on-a-chip.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A vascularized tumor organoid chip, comprising a chip body, a cell culture flow channel is arranged in the chip body, culture medium flow channels are arranged on the upper and lower sides of the cell culture flow channel, and needle insertion flow channels for inserting acupuncture needles to dynamically separate the cell culture flow channel from the culture medium flow channels are arranged on the upper and lower sides of the cell culture flow channel; the cell culture flow channel, the needle insertion flow channel and the culture medium flow channel are interconnected; one end of the culture medium flow channel is provided with a liquid storage pool, and the other end thereof is provided with a culture medium inlet on the surface of the chip body; cell inlets are arranged on the left and right ends of the cell culture flow channel on the surface of the chip body; a plurality of micro-columns are arranged in an annular array in the cell culture flow channel, a tumor organoid flow channel is formed inside the plurality of micro-columns, and a tumor organoid inlet is arranged on the surface of the chip body on the tumor organoid flow channel.

[0006] Preferably, the culture medium flow channel comprises a first flow channel, one end of the first flow channel is provided with a second flow channel communicated with the liquid storage pool, and the other end of the first flow channel is provided with a third flow channel communicated with the culture medium inlet; the first flow channel and the second flow channel are arranged at an obtuse angle; one end of the needle insertion flow channel extends to the connection part of the first flow channel and the second flow channel, and the other end thereof extends to the side surface of the chip body.

[0007] Preferably, the width of the culture medium flow channel is 500 microns, the height is 250 microns, and the length is 12300 microns.

[0008] Preferably, the length of the first flow channel is 4400 microns, the length of the second flow channel is 1950 microns, the length of the third flow channel is 5950 microns, and the diameter of the culture medium inlet is 1500 microns.

[0009] Preferably, the width of the needle insertion flow channel is 250 microns, the height is 250 microns, and the length is 9800 microns.

[0010] Preferably, the cross-section of the cell culture flow channel is a hexagon with a side length of 4400 microns, the height of the cell culture flow channel is 250 microns, and the diameter of the cell inlet is 1500 microns.

[0011] Preferably, the cross-section of the tumor organoid flow channel is a circle with a diameter of 2000 microns, and the diameter of the tumor organoid inlet is 1500 microns.

[0012] Another object of the present invention is to provide an application of a vascularized tumor organoid chip, using the aforementioned vascularized tumor organoid chip for constructing a vascular model, for constructing a co-culture model, or for in vitro drug screening.

[0013] Preferably, the method for constructing a vascular model comprises the following steps: (1) Insert an acupuncture needle into the needle insertion flow channel to separate the cell culture flow channel and the culture medium flow channel; (2) Inject the gel-forming solution into the cell culture flow channel through the cell inlet, and place it in an incubator at 37°C with a CO2 concentration of 5% for 30 minutes for solidification. (3) After 30 minutes, take out the vascularized tumor organoid chip, and partially or completely pull out the acupuncture needles according to the liquid supply form. If the micro-injection pump is used for liquid supply, pull out the acupuncture needles to the connection between the first flow channel and the third flow channel, inject the HUVECs culture medium into the cell culture flow channel through the cell inlet, and then place the vascularized tumor organoid chip in an incubator at 37°C with a CO2 concentration of 5% for culture. If the gravitational potential energy is used for liquid supply, add the HUVECs culture medium to the two liquid storage pools, completely pull out the acupuncture needles, so that the HUVECs culture medium flows into the cell culture flow channel through the culture medium flow channel and the needle inlet flow channel, and then place the vascularized tumor organoid chip in an incubator at 37°C with a CO2 concentration of 5% for culture.

[0014] Preferably, the method for constructing the co-culture model includes the following steps: (1) Insert acupuncture needles into the needle inlet flow channel to separate the cell culture flow channel and the culture medium flow channel. (2) Inject the gel-forming solution into the cell culture flow channel through the cell inlet, place the vascularized tumor organoid chip in an incubator at 37°C with a CO 2 concentration of 5% for 30 minutes for solidification, and then perform vascular culture. (3) After the vascular culture is completed, take out the vascularized tumor organoid chip, inject the tumor organoids into the tumor organoid flow channel, and place the vascularized tumor organoid chip in an incubator at 37°C with a CO 2 concentration of 5% for 30 minutes for solidification. (4) Take out the vascularized tumor organoid chip and perform co-culture medium liquid supply; if the micro-injection pump is used for liquid supply, replace the HUVECs culture medium in the syringe with the co-culture medium for liquid supply; if the gravitational potential energy of the culture medium is used for liquid supply, replace the HUVECs culture medium in the liquid storage pool with the co-culture medium for liquid supply; wherein, the co-culture medium is composed of the tumor organoid culture medium and the HUVECs culture medium mixed at a volume ratio of 1:1.

[0015] The present invention has the following beneficial effects: (1) Through the design of the hexagonal cell flow channel and the circular tumor organoid flow channel, the contact area of vascular cells is significantly increased, the growth of the vascular network is promoted, and better perfused blood vessels are formed; the dynamic separation technology of acupuncture needles avoids the physical limitation of traditional micro-columns on vascular growth and further optimizes the forming effect of the vascular model.

[0016] (2)The free switching between the micro-injection pump and gravitational potential energy liquid supply modes is realized, significantly improving the flexibility and controllability of the experiment; the micro-injection pump mode is suitable for drug screening experiments with precise speed control, while the gravitational potential energy mode is suitable for long-term culture, reducing equipment dependence.

[0017] (3)An in vitro model closer to the in vivo tumor microenvironment was constructed through the co-culture of patient-derived tumor organoids and vascular endothelial cells; combined with the co-culture of immune cells, the killing process of immune cells against cancer cells can be intuitively observed, providing a powerful tool for tumor immunotherapy research.

[0018] (4)Drug sensitivity testing based on patient-derived tumor organoids can more accurately predict clinical efficacy; by comparing the drug sensitivity results of the in vitro model with the actual clinical results of patients, the high accuracy rate (86.7%) of the model was verified, providing an experimental basis for individualized precision treatment.

[0019] (5)The observation conditions of tumors and blood vessels were optimized to facilitate the real-time monitoring of the proliferation, invasion of tumor cells and the dynamic changes of the vascular network; at the same time, it supports high-throughput and uniform culture of tumor organoids, improving the reliability and repeatability of experimental data. Description of the Drawings

[0020] Figure 1 is the layout diagram of each flow channel of the vascularized tumor organoid chip in the present invention; Figure 2 is Figure 1 the partial enlarged view of Figure 3 is the physical diagram of the vascularized tumor organoid chip in the present invention; Figure 4 is the vascular fluorescence diagram in the vascular model constructed in the present invention; Figure 5 is the fluorescence diagram of the co-culture model constructed by using the micro-injection pump to supply liquid in the present invention.

[0021] In the figure, 1 is the chip body; 11 is the cell culture flow channel; 12 is the culture medium flow channel; 121 is the first flow channel; 122 is the second flow channel; 123 is the third flow channel; 13 is the needle inlet flow channel; 14 is the liquid storage pool; 15 is the culture medium inlet; 16 is the cell inlet; 17 is the micro-column; 18 is the tumor organoid flow channel; 19 is the tumor organoid inlet. Detailed Embodiments

[0022] The present invention will be further described below in conjunction with embodiments, but the scope claimed by the present invention is not limited to the scope described in the embodiments.

[0023] Embodiment 1: Design of the vascularized tumor organoid chip A vascularized tumor organoid chip, comprising a chip body 1. Inside the chip body 1, there is a cell culture flow channel 11. On the upper and lower sides of the cell culture flow channel 11, there are culture medium flow channels 12. On the upper and lower sides of the cell culture flow channel 11, there are needle insertion flow channels 13 for inserting acupuncture needles to dynamically separate the cell culture flow channel 11 from the culture medium flow channel 12; the cell culture flow channel 11, the needle insertion flow channel 13 and the culture medium flow channel 12 are interconnected; one end of the culture medium flow channel 12 is provided with a liquid storage pool 14, and the other end is provided with a culture medium inlet 15 on the surface of the chip body 1; both the left and right ends of the cell culture flow channel 11 are provided with cell inlets 16 on the surface of the chip body 1; inside the cell culture flow channel 11, there are several micro-columns 17 arranged in a circular array. Inside the several micro-columns 17, a tumor organoid flow channel 18 is formed. On the tumor organoid flow channel 18, there is a tumor organoid inlet 19 on the surface of the chip body 1.

[0024] Further, the culture medium flow channel 12 includes a first flow channel 121. One end of the first flow channel 121 is provided with a second flow channel 122 communicating with the liquid storage pool 14, and the other end of the first flow channel 121 is provided with a third flow channel 123 communicating with the culture medium inlet 15; the first flow channel 121 and the second flow channel 122 are arranged at an obtuse angle; one end of the needle insertion flow channel 13 extends to the connection of the first flow channel 121 and the second flow channel 122, and the other end extends to the side surface of the chip body 1.

[0025] Further, the width of the culture medium flow channel 12 is 500 microns, the height is 250 microns, and the length is 12300 microns.

[0026] Further, the length of the first flow channel 121 is 4400 microns, the length of the second flow channel 122 is 1950 microns, the length of the third flow channel 123 is 5950 microns, and the diameter of the culture medium inlet 15 is 1500 microns.

[0027] Further, the width of the needle insertion flow channel 13 is 250 microns, the height is 250 microns, and the length is 9800 microns.

[0028] Further, the cross-section of the cell culture flow channel 11 is a hexagon with a side length of 4400 microns, the height of the cell culture flow channel 11 is 250 microns, and the diameter of the cell inlet 16 is 1500 microns.

[0029] Further, the cross-section of the tumor organoid flow channel 18 is a circle with a diameter of 2000 microns, and the diameter of the tumor organoid inlet 19 is 1500 microns.

[0030] Specifically, the chip body 1 needs to be used in cooperation with a bottom plate (not shown in the drawings). The chip body 1 and the bottom plate form a vascularized tumor organoid chip. The chip body 1 and the bottom plate are made of a transparent resin material, and the connection method between the two is a conventional connection method in the art (reference can be made to the prior arts CN116875460A and CN119242442A). Since it does not involve the innovative points of the present invention, it will not be described in detail herein. The size of the chip body 1 is approximately 25 mm (length) * 25 mm (width).

[0031] Two culture medium flow channels 12 are respectively located at the top and bottom of the organ chip. The right end of each culture medium flow channel 12 is connected to a culture medium inlet 15 on the surface of the chip, and the left end is connected to a storage pool 14. The width of the culture medium flow channel 12 is 500 microns, the height is 250 microns, and the length is 12300 microns, which includes three parts: the first flow channel 121, the second flow channel 122, and the third flow channel 123. The first flow channel 121 is in contact with the needle insertion flow channel 13, with a length of 4400 microns, and one end thereof communicates with the second flow channel 122 at an angle of 150 degrees; the second flow channel 122 is about 1950 microns long, and the other end thereof communicates with the storage pool 14; the other end of the first flow channel 121 communicates with the third flow channel 123 at an angle of 150 degrees, and the third flow channel 123 is about 5950 microns long; the opening of the third flow channel 123 is the culture medium inlet 15 with a circular cross-section on the surface of the organ chip with a diameter of 1500 microns.

[0032] Two needle insertion flow channels 13 communicate with the culture medium flow channel 12 and the cell culture flow channel 11 (HUVECs are used as a representative in this embodiment). The width of the needle insertion flow channel 13 is 250 microns, the height is 250 microns, and the length is 9800 microns. The left end thereof extends to the connection between the first flow channel 121 and the second flow channel 122 in the culture medium flow channel 12, and the right end opening is located on the side surface of the organ chip.

[0033] One cell culture flow channel 11 is a flow channel with a hexagonal cross-section (including a tumor organoid flow channel 18 in the middle); the side length of the hexagonal flow channel is 4400 microns, the height is 250 microns, and its upper and lower ends respectively communicate with the upper and lower two culture medium flow channels 12, and the left and right ends are connected to two cell inlets 16 on the surface of the chip. The cross-section of the cell inlet 16 is a circle with a diameter of 1500 microns.

[0034] One tumor organoid flow channel 18 is separated by micro-columns 17 in the cell culture flow channel 11. Its cross-section is a circle with a diameter of 2000 microns, and there is a tumor organoid inlet 19 communicating with the outside at its top. The cross-section of the tumor organoid inlet 19 is a circle with a diameter of 1500 microns.

[0035] Two storage pools 14 for accommodating the culture medium are respectively connected to the two culture medium flow channels 12. The height is 6000 microns, and the cross-section is a circle with a diameter of 7500 microns.

[0036] Example 2: Construction of a blood vessel model A method for constructing a blood vessel model, comprising the following steps: (1) Take out the HUVECs to be resuscitated from liquid nitrogen, quickly place them in a 37°C water bath, take them out after they melt to small ice crystals, add 1 ml of HUVECs culture medium, mix well and transfer to a 15 ml centrifuge tube, centrifuge at 300 g for 3 mins, discard the supernatant, wash with PBS, resuspend, and centrifuge at 300 g for 3 mins.

[0037] (2) Resuspend the HUVECs in the culture medium, plate them on a culture dish, and place them in an incubator at 37°C with CO 2 ...

[0038] (3) Replace the HUVECs culture medium once every 2 - 3 days. When the cell density reaches about 70% - 80% under the microscope, aspirate the culture medium, add trypsin digestive solution, and place it in an incubator at 37°C with CO 2 ... for 3 mins.

[0039] (4) After taking it out, add HUVECs culture medium, gently pipette, wait for the HUVECs to detach, transfer to a 15 ml centrifuge tube, and centrifuge at 300 g for 3 mins.

[0040] (5) Discard the supernatant, resuspend with HUVECs culture medium to make the HUVECs density 1400 m / ml, and place it on ice to obtain a HUVECs suspension.

[0041] (6) Prepare a mixed solution containing prothrombin and aprotinin with the HUVECs suspension to make a solution with a prothrombin concentration of 2 UI / ml and aprotinin of 10 UI / ml to obtain a pretreated HUVECs culture medium.

[0042] (7) Prepare a fibrinogen stock solution with a fibrinogen concentration of 6 mg / ml with the HUVECs culture medium.

[0043] (8) Insert two acupuncture needles into the inlet channel 13 from the side of the organ chip, with the distal end reaching the connection between the first channel 121 and the second channel 122 of the culture medium channel 12.

[0044] (9) Mix an equal volume of the pretreated HUVECs culture medium obtained in step (6) and the fibrinogen stock solution obtained in step (7), pipette 5 times to obtain a gel-forming solution; quickly inject the gel-forming solution from the cell inlet 16 at one end of the reservoir 14.

[0045] (10) Carefully place the organ chip in an incubator at 37°C with CO 2 ... for 30 mins to solidify, and wait for the solution containing HUVECs to solidify.

[0046] (11) Take out the organ-on-a-chip from the incubator, carefully pull out the acupuncture needle from the injection flow channel 13. According to the form of liquid supply, if it is liquid supply by a micro-injection pump, pull out the acupuncture needle to the connection between the first flow channel 121 and the second flow channel 122; if it is liquid supply by the gravitational potential energy of the culture medium itself, pull out the acupuncture needle completely.

[0047] (12) For liquid supply by a micro-injection pump, suck 2 ml of HUVECs culture medium into a syringe, remove the needle, connect a silicone hose, and insert the other end of the silicone hose into the culture medium inlet 15. Place the syringe in the micro-injection pump, set the liquid supply speed at 10 ul / h, and place the organ-on-a-chip in an incubator at 37 °C with CO 2 . A complete and continuous vascular network can be formed after 7 days (see Figure 4 ).

[0048] (13) For liquid supply by the gravitational potential energy of the culture medium itself, add 200 ul of HUVECs culture medium to the two liquid storage pools 14. Since the acupuncture needle is completely pulled out, the injection flow channel 13 is connected to the liquid storage pool 14 and is open to the outside at the opening of the injection flow channel 13. A height difference is formed between the liquid storage pool 14 with added HUVECs culture medium and the opening of the injection flow channel 13, enabling liquid supply by gravitational potential energy. Place the organ-on-a-chip in an incubator at 37 °C with CO 2 . A complete and continuous vascular network can be formed after 7 days (see Figure 4 ). A comparison and quantitative analysis were carried out with the culture results (control group) where the previous culture medium flow channel and cell culture flow channel were separated by micro-columns, and the results are shown in Table 1.

[0049] Table 1 Vascular models under different liquid supply forms Group Average vessel length Average vessel diameter Vessel density Control group + + + Microinfusion pump fluid administration group ++ ++ ++ Gravitational potential energy fluid administration group ++ ++ ++ Note: “+” is the basic value of the control group, and “++” indicates an increase within 0 - 50%.

[0050] As can be seen from Table 1, the organ-on-a-chip of the present invention, through the design of hexagonal cell flow channels and circular tumor organoid flow channels, significantly increases the contact area of vascular cells, promotes the growth of the vascular network, and forms better perfused blood vessels; the dynamic separation technology of the acupuncture needle avoids the physical limitation of traditional micro-columns on vascular growth and further optimizes the forming effect of the vascular model. In addition, the organ-on-a-chip of the present invention realizes the free switching between two liquid supply modes of a micro-injection pump and gravitational potential energy, significantly improving the flexibility and controllability of the experiment; the micro-injection pump mode is suitable for drug screening experiments with precise speed control, and the gravitational potential energy mode is suitable for long-term culture, reducing equipment dependence.

[0051] In addition, the angiogenesis of the blood vessel model in this embodiment is not limited to the HUVECs cell line, but also includes other vascular endothelial cell lines, primary source vascular cells, IPS-derived cells, etc.

[0052] Example 3: Preparation of tumor organoids A method for preparing tumor organoids, comprising the following steps: (1) Collect fresh tumor tissues. The specimen acquisition methods include surgery and biopsy, and they are transported on ice using a tissue transport solution. The tissue transport solution is Advanced DMEM / F12 basal medium, supplemented with 1% (V / V%) Glutamax, 10 mmol / L HEPES, and 2% (V / V%) penicillin / streptomycin solution.

[0053] (2) Use sterilized scissors to cut off excess tissues such as muscle and fat, and wash the tumor tissues 1-2 times with pre-cooled normal saline. Cut the tumor tissue strips into pieces within 1 mm, and wash them several times with pre-cooled normal saline in a centrifuge tube.

[0054] (3) After washing several times and allowing it to stand, discard the supernatant, and place the tissue precipitate in a tissue digestion solution for digestion. The tissue digestion solution consists of Advanced DMEM / F12 basal medium, 2.5 mg / mL type IV collagenase, 0.1 mg / mL deoxyribonuclease 1, and 2% (V / V%) penicillin / streptomycin solution. Place it in a 37°C incubator and shake for digestion for 50 minutes.

[0055] (4) Add an equal volume of pre-cooled digestion termination solution to terminate digestion. The digestion termination solution consists of complete cell medium (Advanced DMEM / F12 basal medium + 10% fetal bovine serum), 10 μmol / L cell detachment and apoptosis protector Y27632, and 1% (V / V%) penicillin / streptomycin solution. The cell suspension is filtered through a 100-μm filter, centrifuged at 300 g for 10 minutes in a 4°C centrifuge. Discard the supernatant, add pre-cooled normal saline and wash twice, and the finally collected precipitate is the tumor cells.

[0056] (5) Resuspend the tumor cells in a 1:2 mixture of pre-cooled tumor organoid medium and Matrigel, adjust the cell concentration to 2500 - 4000 cells / 50 μL, and inoculate them into a cell culture plate.

[0057] (6) Place it in a cell culture incubator at 37°C and 5% carbon dioxide for solidification for 30 minutes.

[0058] (7) Add tumor organoid medium and change the medium every 2 - 3 days.

[0059] Example 4: Construction of a co-culture model A method for constructing a co-culture model, based on Example 2 and Example 3, further includes the following steps: (1) Aspirate the organoid culture medium in the culture plate, pipette the gel droplets with TryPLE to disrupt the gel droplets, and place them in a cell culture incubator at 37°C and 5% carbon dioxide for 20 min.

[0060] (2) After taking out, pipette, transfer to a 15 ml centrifuge tube, centrifuge at 300 g in a 4°C centrifuge for 3 mins, discard the supernatant, wash with PBS, centrifuge at 300 g in a 4°C centrifuge for 3 mins. Discard the supernatant and resuspend in Matrigel.

[0061] (3) Aspirate 10 μl of the organoid suspension, carefully inject it into the tumor organoids from the tumor organoid inlet 19 at the top of the tumor organoid flow channel 18, and place it in an incubator at 37°C and 5% CO 2 for 30 mins.

[0062] (4) If the micro-injection pump is used for liquid supply, replace the HUVECs culture medium in the syringe with the co-culture medium for liquid supply; if the liquid supply is by the gravitational potential energy of the culture medium itself, replace the HUVECs culture medium in the reservoir 14 with the co-culture medium for liquid supply; wherein, the co-culture medium is composed of the tumor organoid culture medium and the HUVECs culture medium mixed in a volume ratio of 1:1. After co-culturing for 7 days, it can be seen that the organoids and vascular endothelial cells can be co-cultured well (see Figure 5 ).

[0063] From Figure 5 it can be known that the present invention optimizes the observation conditions of tumors and blood vessels, facilitating the real-time monitoring of the proliferation, invasion of tumor cells and the dynamic changes of the vascular network; at the same time, it supports high-throughput and uniform tumor organoid culture, improving the reliability and repeatability of experimental data.

[0064] In addition, the tumor formation in the co-culture model in this example is not limited to tumor organoids, but also includes tumor cell lines, primary tumor cell tissues, etc.

[0065] Example 5: Drug sensitivity test of anti-tumor drugs A method for drug sensitivity test of anti-tumor drugs, including the following steps: (1) Construct a vascular model according to the technical solution of Example 2 in the present invention.

[0066] (2) Select patients who clinically receive FOLFOX + bevacizumab drug treatment from the tumor organoid biobank (selecting colon cancer organoids).

[0067] (3) Take out the corresponding cryopreserved patient-derived organoids from liquid nitrogen, quickly place them in a 37°C water bath, take them out after they melt to small ice crystals, add 1 ml of organoid culture medium, mix well and transfer them to a 15 ml centrifuge tube, centrifuge at 300 g for 3 minutes, discard the supernatant, wash with PBS, resuspend, and centrifuge at 300 g for 3 minutes.

[0068] (4) Resuspend the tumor cells in a pre-cooled mixture of tumor organoid medium and Matrigel at a ratio of 1:2, adjust the cell concentration to 2500 - 4000 cells / 50 μL, and seed them in a cell culture plate.

[0069] (5) Incubate in a cell culture incubator at 37°C and 5% carbon dioxide for 30 minutes for solidification.

[0070] (6) Add tumor organoid medium and change the medium every 2 - 3 days.

[0071] (7) Observe until the growth density of the organoids is good and the size is normal, then passage them, and construct a co-culture model from patient-derived according to the technical solution of Example 4 in the present invention.

[0072] (8) Add FOLFOX + bevacizumab to the culture medium, where the FOLFOX regimen uses a concentration ratio of (5-FU:leucovorin:oxaliplatin, 25:5:1), the concentration of 5-FU is 10 μM, and the concentrations of bevacizumab are 0.04, 0.2, and 1 μM respectively. 3 Perform drug response tests at these three concentration gradients, and set up negative control groups and positive control groups.

[0073] (9) Take pictures 6 days after drug administration, and use ImageJ software to calculate the vascular density and average tumor organoid diameter at each concentration.

[0074] (10) Define the positive control group as 0% vascular density, and define the negative control (solvent control) as 100% vascular density. The calculation formula is: percentage of vascular density = (vascular density value - average positive control value) / (average negative control value - average positive control value) * 100%.

[0075] (11) Define the positive control group as 0% organoid diameter, and define the negative control (solvent control) as 100% organoid diameter. The calculation formula is: percentage of organoid diameter graph = (average organoid diameter value - average positive control value) / (average negative control value - average positive control value) * 100%.

[0076] (12)The efficacy determination criteria for the in vitro model were established based on the changes in the vascular network density and the changes in the diameter of the organoids in the organ-on-a-chip in vitro model. When the concentration of bevacizumab was 1 μm, if the vascular network density was less than 50% of the negative control group, it was determined that the vascular network was sensitive to the drug; otherwise, it was drug-resistant. If the average diameter of the organoids was less than 50% of the negative control group, it was determined that the colorectal cancer organoids were sensitive to the drug; otherwise, it was drug-resistant (see Table 2).

[0077] Table 2 Drug screening data of vascularized tumor organ-on-a-chip from different sources Source Relative vessel density Relative organoid diameter Model prediction P001 45% 46% Sensitive P002 52% 58% Drug-resistant P003 72% 68% Drug-resistant P004 23% 37% Sensitive P005 69% 72% Drug-resistant P006 12% 26% Sensitive P007 42% 35% Sensitive P008 73% 65% Drug-resistant P009 56% 71% Drug-resistant P010 32% 45% Sensitive P011 21% 16% Sensitive P012 29% 41% Sensitive P013 71% 82% Drug-resistant P014 13% 24% Sensitive P015 55% 63% Drug-resistant (13)The relevant clinical information before and after receiving FOLFOX + bevacizumab treatment was analyzed, including imaging data and colorectal cancer-related tumor markers such as CEA, CA199, and CA242. According to RECIST, the CR and PR stages were defined as clinical efficacy, and the SD and PD stages were defined as clinical drug resistance.

[0078] (14)The results of the drug sensitivity experiments in the organ-on-a-chip from patient sources were compared with the actual clinical results of the patients (see Table 3).

[0079] Table 3 Comparison between the new vascularized tumor organ-on-a-chip model and clinical results Clinically sensitive Clinically drug-resistant Total Predicted sensitive 7 1 8 Predicted drug-resistant 1 6 7 Total 8 7 15 As can be seen from Table 3, the present invention conducts drug sensitivity tests based on patient-derived tumor organoids, which can more accurately predict clinical efficacy. By comparing the drug sensitivity results of the in vitro model with the actual clinical results of the patients, the high accuracy of the model is verified, providing an experimental basis for individualized precision treatment. The accuracy rate is specifically (7 + 6) / (8 + 7)*100% = 86.7%.

[0080] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A vascularized tumor organoid chip, characterized in that: The chip body comprises a cell culture channel, the cell culture channel has culture fluid channels on the upper and lower sides, the cell culture channel has needle insertion channels on the upper and lower sides for inserting acupuncture needles to dynamically separate the cell culture channel and the culture fluid channel; the cell culture channel, the needle insertion channel and the culture fluid channel are interconnected; a liquid reservoir is provided at one end of the culture fluid channel, and a culture fluid inlet is provided at the other end thereof, which is located on the surface of the chip body; cell inlets are provided on the surface of the chip body at both left and right ends of the cell culture channel; a plurality of microcolumns arranged in a circular array are provided in the cell culture channel, a tumor organoid channel is formed on the inner side of the plurality of microcolumns, and a tumor organoid inlet is provided on the tumor organoid channel, which is located on the surface of the chip body.

2. The vascularized tumor organ-on-a-chip according to claim 1, characterized in that: The culture fluid flow channel includes a first flow channel, one end of which is provided with a second flow channel connected to a liquid storage tank, and the other end of the first flow channel is provided with a third flow channel connected to a culture fluid inlet; the first flow channel and the second flow channel are arranged at an obtuse angle; one end of the needle inlet flow channel extends to the connection between the first flow channel and the second flow channel, and the other end thereof extends to the side surface of the chip body.

3. The vascularized tumor organ-on-a-chip according to claim 2, characterized in that: The width of the culture fluid flow channel is 500 microns, the height is 250 microns, and the length is 12,300 microns.

4. The vascularized tumor organ-on-a-chip according to claim 3, characterized in that: The length of the first flow channel is 4400 microns, the length of the second flow channel is 1950 microns, the length of the third flow channel is 5950 microns, and the diameter of the culture fluid inlet is 1500 microns.

5. The vascularized tumor organ-on-a-chip according to claim 3, characterized in that: The needle inlet channel has a width of 250 microns, a height of 250 microns, and a length of 9800 microns.

6. The vascularized tumor organ-on-a-chip according to claim 3, characterized in that: The cross section of the cell culture flow channel is a hexagon with a side length of 4400 microns, the height of the cell culture flow channel is 250 microns, and the diameter of the cell inlet is 1500 microns.

7. The vascularized tumor organ-on-a-chip according to claim 3, characterized in that: The cross-section of the tumor organoid flow channel is a circle with a diameter of 2000 microns, and the diameter of the tumor organoid inlet is 1500 microns.

8. An application of a vascularized tumor organoid chip, characterized in that: The vascularized tumor organoid chip according to any one of claims 1 to 7 is used to construct a vascular model, to construct a co-culture model, or to be used for in vitro drug screening.

9. The use of the vascularized tumor organoid chip according to claim 8, characterized in that: The method for constructing a blood vessel model comprises the following steps: (1) Insert an acupuncture needle into the needle channel to separate the cell culture channel and the culture fluid channel; (2) Inject the gel-forming liquid into the cell culture channel from the cell inlet and place it in an incubator at 37°C and 5% CO2 concentration to solidify for 30 minutes; (3) After 30 minutes, the vascularized tumor organoid chip was removed and the acupuncture needle was partially or completely removed according to the form of fluid administration; If the microinjection pump is used for fluid administration, the acupuncture needle is pulled out to the connection between the first flow channel and the third flow channel, the HUVECs culture fluid is injected into the cell culture flow channel through the cell inlet, and then the vascularized tumor organoid chip is placed in an incubator at 37°C and a CO2 concentration of 5% for culture; If gravity potential energy is used for fluid feeding, add HUVECs culture fluid into the two reservoirs, pull out the acupuncture needle completely, and allow the HUVECs culture fluid to be injected into the cell culture channel through the culture fluid channel and the needle channel. Then place the vascularized tumor organoid chip in an incubator at 37°C and a CO2 concentration of 5% for culture.

10. The use of the vascularized tumor organoid chip according to claim 8, characterized in that: The method for constructing the co-culture model includes the following steps: (1) Insert an acupuncture needle into the needle channel to separate the cell culture channel and the culture fluid channel; (2) Inject the gel-forming liquid into the cell culture channel from the cell inlet, place the vascularized tumor organoid chip in an incubator at 37°C and 5% CO2 concentration for 30 minutes to solidify, and then perform vascular culture; (3) After the vascular culture is completed, the vascularized tumor organoid chip is taken out, and the tumor organoid is injected into the tumor organoid flow channel. The vascularized tumor organoid chip is placed in an incubator at 37°C and a CO2 concentration of 5% to solidify for 30 minutes; (4) Take out the vascularized tumor organoid chip and perform co-culture fluid administration; if the fluid is administered by a microinjection pump, replace the HUVECs culture fluid in the syringe with the co-culture fluid; if the fluid is administered by the culture fluid's own gravitational potential energy, replace the HUVECs culture fluid in the reservoir with the co-culture fluid; wherein the co-culture fluid is composed of a mixture of tumor organoid culture fluid and HUVECs culture fluid in a volume ratio of 1:1.

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