Vascularized tumor organoid chip and applications thereof

Abstract

This invention relates to the field of organoid culture technology, specifically to a vascularized tumor organoid chip. It redesigns the traditional micropillar-separated culture medium and cell culture channels into a dynamically separated structure using acupuncture needles. This avoids the physical limitations of traditional micropillars on blood vessel growth, significantly increases the contact area between vascular cells, and promotes the growth of the vascular network. Simultaneously, the needle insertion channel and reservoir allow for free switching between micro-infusion pump and gravitational potential energy delivery modes. Furthermore, it enables dynamic detection during the co-culture of blood vessels and tumor organoids.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and more specifically, to a vascularized tumor organoid chip and its applications. Background Technology

[0002] Angiogenesis plays a crucial role in tumor growth, invasion, and metastasis, and a complex relationship exists between angiogenesis and tumors. Therefore, constructing in vitro models with vascular structures can maximize the simulation of the tumor microenvironment, thereby enabling better research on tumor biological characteristics and anti-angiogenic drugs. Currently, tumor vascularization is mainly simulated through co-culture of tumor cell lines and vascular endothelial cells, primarily in 2D culture. This method struggles to simulate the interaction between real tumor and vascular cells, and conventional static culture, due to poor gas-liquid mass transfer, easily creates concentration gradients that affect the function of co-cultured cells, thus failing to meet research and clinical needs. Organ-on-a-chip technology, as a cutting-edge technology, offers advantages such as high throughput and small sample requirements. It can also be connected to continuous or cyclic perfusion systems, better reflecting human physiological characteristics. By co-culturing tumor cells and vascular endothelial cells on organ-on-a-chip, it is possible to simulate tumor angiogenesis and drug responses in patients.

[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 the model requires different channels, such as culture medium channels and cell culture channels, the culture medium channels and cell culture channels are often separated by limiting structures (microcolumns), which leads to poor growth of vascular cells, especially poor perfusion opening of the vascular model, which affects perfusion; (2) At present, the dynamic culture mode of organ-on-a-chip is mainly based on external pipelines such as micropumps / peristaltic pumps for liquid supply, or perfusion by using a shaker to form the gravitational potential energy of the culture medium. Different liquid supply methods have different advantages, but few combine the two; (3) At present, most co-culture models use tumor cell lines and HUVECs for mixed culture, which affects observation and analysis in subsequent experiments. Summary of the Invention

[0004] The purpose of this invention is to provide a vascularized tumor organoid microarray to solve the problems of poor vascular model formation, incompatibility of culture medium delivery methods, and the impact of mixed co-culture mode on observation in existing organoid microarrays.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A vascularized tumor organoid microarray includes a chip body with a cell culture channel inside. Culture medium channels are located on the upper and lower sides of the cell culture channel. Needle insertion channels are located on the upper and lower sides of the cell culture channel for inserting acupuncture needles to dynamically separate the cell culture channel from the culture medium channel. The cell culture channel, needle insertion channel, and culture medium channel are interconnected. A reservoir is located at one end of the culture medium channel, and a culture medium inlet is located at the other end on the surface of the chip body. Cell inlets are located on the surface of the chip body at both ends of the cell culture channel. A plurality of micropillars are arranged in a circular array within the cell culture channel, forming tumor organoid channels on the inner sides of the micropillars. Tumor organoid inlets are located on the surface of the chip body within the tumor organoid channels.

[0007] Preferably, the culture medium flow channel includes a first flow channel, one end of which is provided with a second flow channel communicating with the storage tank, and the other end of which is provided with a third flow channel communicating 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 flow channel extends to the connection between the first flow channel and the second flow channel, and the other end extends to the side surface of the chip body.

[0008] Preferably, the culture medium flow channel has a width of 500 micrometers, a height of 250 micrometers, and a length of 12,300 micrometers.

[0009] Preferably, the length of the first flow channel is 4400 micrometers, the length of the second flow channel is 1950 micrometers, the length of the third flow channel is 5950 micrometers, and the diameter of the culture medium inlet is 1500 micrometers.

[0010] Preferably, the width of the needle inlet channel is 250 micrometers, the height is 250 micrometers, and the length is 9800 micrometers.

[0011] Preferably, the cell culture channel has a hexagonal cross-section with a side length of 4400 micrometers, a height of 250 micrometers, and a cell inlet diameter of 1500 micrometers.

[0012] Preferably, the cross-section of the tumor organoid flow channel is a circle with a diameter of 2000 micrometers, and the diameter of the tumor organoid inlet is 1500 micrometers.

[0013] Another objective of this invention is to provide an application of vascularized tumor organoid chips, which are used to construct vascular models, construct co-culture models, or screen drugs in vitro.

[0014] Preferably, the method for constructing a vascular model includes the following steps:

[0015] (1) Use acupuncture needles to insert into the needle channel to separate the cell culture channel and the culture medium channel;

[0016] (2) Inject the gel-forming solution into the cell culture channel through the cell inlet and place it in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify;

[0017] (3) After 30 minutes, remove the vascularized tumor organoid chip and, depending on the administration method, partially or completely remove the acupuncture needles.

[0018] If a microinfusion pump is used for infusion, the acupuncture needle is pulled out to the connection between the first and third flow channels, and the HUVECs culture medium is injected into the cell culture channel through the cell inlet. Then, the vascularized tumor organoid chip is placed in an incubator at 37°C with a CO2 concentration of 5% for culture.

[0019] If gravitational potential energy is used for liquid supply, add HUVECs culture medium to the two reservoirs, completely remove the acupuncture needles, and allow the HUVECs culture medium to be injected into the cell culture channel through the culture medium channel and the needle insertion channel. Then, place the vascularized tumor organoid chip in an incubator at 37°C with a CO2 concentration of 5% for culture.

[0020] Preferably, the method for constructing a co-cultivation model includes the following steps:

[0021] (1) Use acupuncture needles to insert into the needle channel to separate the cell culture channel and the culture medium channel;

[0022] (2) The gel generation solution was injected into the cell culture channel through the cell inlet, and the vascularized tumor organoid chip was placed in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify, and then the blood vessels were cultured.

[0023] (3) After the vascular culture is completed, the vascularized tumor organoid chip is removed and the tumor organoid is injected into the tumor organoid channel. The vascularized tumor organoid chip is then placed in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify.

[0024] (4) Remove the vascularized tumor organoid chip and administer co-culture medium; if the microinfusion pump is used, replace the HUVECs culture medium in the syringe with the co-culture medium; if the culture medium is administered by its own gravitational potential energy, replace the HUVECs culture medium in the reservoir with the co-culture medium; wherein, the co-culture medium is composed of tumor organoid culture medium and HUVECs culture medium mixed at a volume ratio of 1:1.

[0025] The present invention has the following beneficial effects:

[0026] (1) The design of hexagonal cell channels and circular tumor organoid channels significantly increases the contact area of ​​vascular cells, promotes the growth of vascular networks, and forms better perfusion vessels; the dynamic separation technology of acupuncture needles avoids the physical limitation of traditional microcolumns on vascular growth and further optimizes the forming effect of vascular models.

[0027] (2) It enables free switching between two liquid delivery modes: micro-pump and gravitational potential energy, which significantly improves the flexibility and controllability of the experiment. The micro-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.

[0028] (3) By co-culturing tumor organoids derived from patients with vascular endothelial cells, an in vitro model that is closer to the tumor microenvironment in vivo was constructed; combined with the co-culture of immune cells, the killing process of immune cells on cancer cells can be observed directly, providing a powerful tool for tumor immunotherapy research.

[0029] (4) Drug sensitivity testing based on tumor organoids from patients 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 of the model (86.7%) was verified, providing experimental basis for individualized precision treatment.

[0030] (5) The observation conditions for tumors and blood vessels have been optimized, which facilitates real-time monitoring of tumor cell proliferation, invasion and dynamic changes in vascular networks; at the same time, it supports high-throughput and uniform tumor organoid culture, which improves the reliability and reproducibility of experimental data. Attached Figure Description

[0031] Figure 1 This is a flow channel layout diagram of the vascularized tumor organoid chip in this invention;

[0032] Figure 2 yes Figure 1 A magnified view of a portion of the image;

[0033] Figure 3 This is a physical image of the vascularized tumor organoid chip of this invention;

[0034] Figure 4 This is a fluorescence image of blood vessels in the blood vessel model constructed in this invention;

[0035] Figure 5 This is a fluorescence image of the co-culture model constructed using a micro-injection pump for liquid delivery according to the present invention.

[0036] In the figure, 1 is the chip body; 11 is the cell culture channel; 12 is the culture medium channel; 121 is the first channel; 122 is the second channel; 123 is the third channel; 13 is the needle insertion channel; 14 is the reservoir; 15 is the culture medium inlet; 16 is the cell inlet; 17 is the microcolumn; 18 is the tumor organoid channel; and 19 is the tumor organoid inlet. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0038] Example 1: Design of vascularized tumor organoid microarrays

[0039] A vascularized tumor organoid microarray includes a chip body 1, a cell culture channel 11 within the chip body 1, culture medium channels 12 on the upper and lower sides of the cell culture channel 11, and needle insertion channels 13 on the upper and lower sides of the cell culture channel 11 for inserting acupuncture needles to dynamically separate the cell culture channel 11 from the culture medium channels 12; the cell culture channel 11, needle insertion channels 13, and culture medium channels 12 are interconnected; a reservoir 14 is provided at one end of the culture medium channel 12, and a culture medium inlet 15 is provided at the other end on the surface of the chip body 1; cell inlets 16 are provided at both ends of the cell culture channel 11 on the surface of the chip body 1; a plurality of micropillars 17 arranged in a ring array are provided within the cell culture channel 11, and tumor organoid channels 18 are formed inside the plurality of micropillars 17, with tumor organoid inlets 19 on the surface of the chip body 1 on the tumor organoid channels 18.

[0040] Furthermore, the culture medium flow channel 12 includes a first flow channel 121, one end of which is provided with a second flow channel 122 communicating with the storage tank 14, and the other end of which 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 flow channel 13 extends to the connection between 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.

[0041] Furthermore, the culture medium flow channel 12 has a width of 500 micrometers, a height of 250 micrometers, and a length of 12,300 micrometers.

[0042] Furthermore, the length of the first flow channel 121 is 4400 micrometers, the length of the second flow channel 122 is 1950 micrometers, the length of the third flow channel 123 is 5950 micrometers, and the diameter of the culture medium inlet 15 is 1500 micrometers.

[0043] Furthermore, the needle inlet channel 13 has a width of 250 micrometers, a height of 250 micrometers, and a length of 9800 micrometers.

[0044] Furthermore, the cell culture channel 11 has a cross-section of a hexagon with a side length of 4400 micrometers, a height of 250 micrometers, and a diameter of 1500 micrometers for the cell inlet 16.

[0045] Furthermore, the cross-section of the tumor organoid flow channel 18 is a circle with a diameter of 2000 micrometers, and the diameter of the tumor organoid inlet 19 is 1500 micrometers.

[0046] Specifically, the chip body 1 needs to be used in conjunction with a substrate (not shown in the attached drawings). The chip body 1 and the substrate together constitute a vascularized tumor organoid chip. The chip body 1 and the substrate are made of transparent resin material, and the connection method between them is a conventional connection method in the art (refer to prior art CN116875460A and CN119242442A). Since this does not involve any innovation of the present invention, it will not be described in detail here. The dimensions of the chip body 1 are approximately 25mm (length) * 25mm (width).

[0047] Two culture medium channels 12 are located at the top and bottom of the organ-on-a-chip, respectively. The right end of each channel 12 connects to a culture medium inlet 15 on the chip surface, and the left end connects to a reservoir 14. Each culture medium channel 12 is 500 micrometers wide, 250 micrometers high, and 12,300 micrometers long, comprising three parts: a first channel 121, a second channel 122, and a third channel 123. The first channel 121, which is in contact with the needle insertion channel 13, is 4,400 micrometers long, and one end of it connects to the second channel 122 at a 150-degree angle. The second channel 122 is approximately 1,950 micrometers long, and its other end connects to the reservoir 14. The other end of the first channel 121 connects to the third channel 123 at a 150-degree angle, and the third channel 123 is approximately 5,950 micrometers long. The opening of the third channel 123 is a circular culture medium inlet 15 with a cross-sectional diameter of 1,500 micrometers on the organ-on-a-chip surface.

[0048] Two needle insertion channels 13 are connected to the culture medium channel 12 and the cell culture channel 11 (using HUVECs as a representative in this embodiment). They are 250 micrometers wide, 250 micrometers high, and 9800 micrometers long. Their left end extends to the connection between the first channel 121 and the second channel 122 in the culture medium channel 12, and their right end opening is located on the side surface of the organ-on-a-chip.

[0049] A cell culture channel 11 is a channel with a hexagonal cross-section (including a tumor organoid channel 18 in the middle); the hexagonal channel has a side length of 4400 micrometers and a height of 250 micrometers. Its upper and lower ends are connected to the upper and lower culture medium channels 12 respectively, and its left and right ends are connected to two cell inlets 16 located on the chip surface. The cell inlets 16 have a cross-section of 1500 micrometers.

[0050] A tumor organoid flow channel 18 is located within the cell culture flow channel 11 and is separated by micropillars 17. Its cross-section is a circle with a diameter of 2000 micrometers. At its top, there is a tumor organoid inlet 19 that communicates with the outside. The cross-section of the tumor organoid inlet 19 is a circle with a diameter of 1500 micrometers.

[0051] Two reservoirs 14 for holding culture medium are connected to two culture medium channels 12, respectively. They are 6000 micrometers high and have a circular cross-section with a diameter of 7500 micrometers.

[0052] Example 2: Constructing a vascular model

[0053] A method for constructing a vascular model includes the following steps:

[0054] (1) Take out the HUVECs to be resuscitated from liquid nitrogen, quickly place them in a 37°C water bath, wait for them to melt into small ice crystals, then take them out, add 1ml of HUVECs culture medium, mix well, transfer to a 15ml centrifuge tube, centrifuge at 300g for 3mins, remove the supernatant, wash with PBS, resuspend, and centrifuge at 300g for 3mins.

[0055] (2) HUVECs were resuspended in culture medium, plated in petri dishes, and placed in an incubator at 37°C and CO2.

[0056] (3) Change the HUVECs culture medium every 2-3 days. When the cell density reaches about 70%-80% under a microscope, remove the culture medium, add trypsin digestion solution, and place in a 37℃ CO2 incubator for 3 minutes.

[0057] (4) After taking it out, add HUVECs culture medium, gently blow it until HUVECs fall off, transfer it to a 15ml centrifuge tube, centrifuge at 300g for 3mins.

[0058] (5) Remove the supernatant, resuspend the HUVECs in HUVECs culture medium to make the HUVECs density 1400 m / ml, and place it on ice to obtain HUVECs suspension.

[0059] (6) Prepare a mixed solution containing prothrombin and aprotinin using HUVECs suspension, so that the concentration of prothrombin is 2 UI / ml and the concentration of aprotinin is 10 UI / ml, to obtain the pretreated HUVECs culture medium.

[0060] (7) Prepare a fibrin mother solution containing a fibrinogen concentration of 6 mg / ml using HUVECs culture medium.

[0061] (8) Insert two acupuncture needles into the needle 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.

[0062] (9) Mix equal volumes of the pretreated HUVECs culture medium obtained in step (6) and the fibrin mother liquor obtained in step (7), and blow them 5 times to obtain the gel generating solution; quickly inject the gel generating solution into the cell inlet 16 located at one end of the reservoir 14.

[0063] (10) Carefully place the organ-on-a-chip in a 37°C, CO2 incubator and cure for 30 minutes until the solution containing HUVECs is cured.

[0064] (11) Remove the organ chip from the incubator and carefully pull out the acupuncture needle from the needle inlet channel 13. Depending on the method of fluid administration, if it is a micro-infusion pump, pull out the acupuncture needle to the connection between the first channel 121 and the third channel 123; if it is a culture medium using its own gravitational potential energy for fluid administration, pull out the acupuncture needle completely.

[0065] (12) Microinfusion pump administration: Draw 2 ml of HUVECs culture medium into a syringe, remove the needle, connect a silicone tubing, and insert the other end of the silicone tubing into the culture medium inlet 15. Place the syringe in the microinfusion pump, set the administration rate to 10 μL / h, and place the organ-on-a-chip in a 37°C, CO2 incubator. A complete and continuous vascular network can be formed after 7 days (see...). Figure 4 ).

[0066] (13) Gravitational potential energy supply of the culture medium: 200 μL of HUVECs culture medium was added to two reservoirs 14. Since the acupuncture needles were completely removed, the needle insertion channel 13 was connected to the reservoir 14, and the opening of the needle insertion channel 13 was open to the outside. With the addition of HUVECs culture medium, a height difference was formed between the reservoir 14 and the opening of the needle insertion channel 13, enabling gravity potential energy supply. The organ-on-a-chip was then placed in a 37°C, CO2 incubator. After 7 days, a complete and continuous vascular network was formed (see...). Figure 4 The results were compared and quantitatively analyzed with those of previous culture medium channels and cell culture channels separated by microcolumns (control group), and the results are shown in Table 1.

[0067] Table 1 Vascular models under different infusion methods

[0068]

[0069] Note: "+" represents the baseline value of the control group, and "++" represents an increase of 0-50%.

[0070] As shown in Table 1, the organ-on-a-chip of this invention, through its hexagonal cell flow channel and circular tumor organoid flow channel design, significantly increases the contact area of ​​vascular cells, promotes the growth of vascular networks, and forms better perfusion vessels. The dynamic segmentation technology of acupuncture needles avoids the physical limitations on vascular growth imposed by traditional microcolumns, further optimizing the formation effect of the vascular model. Furthermore, the organ-on-a-chip of this invention allows for free switching between two infusion modes: microinfusion pump and gravitational potential energy, significantly improving the flexibility and controllability of the experiment. The microinfusion pump mode is suitable for drug screening experiments requiring precise rate control, while the gravitational potential energy mode is suitable for long-term culture, reducing equipment dependence.

[0071] Furthermore, in this embodiment, the angiogenesis of the vascular model is not limited to the HUVECs cell line, but also includes other vascular endothelial cell lines, primary vascular cells, iPS cells, etc.

[0072] Example 3: Preparation of Tumor Organoids

[0073] A method for preparing tumor organoids includes the following steps:

[0074] (1) Fresh tumor tissue was collected. The specimens were obtained by surgery and biopsy. The tissue transport medium was placed on ice and transported. The tissue transport medium was Advanced DMEM / F12 basal medium with 1% (V / V%) Glutamax, 10 mmol / L HEPES, and 2% (V / V%) penicillin / streptomycin solution added.

[0075] (2) Use high-temperature sterilized scissors to cut off excess tissue such as muscle and fat, and wash the tumor tissue 1-2 times with pre-cooled physiological saline. Cut the tumor tissue strips into pieces less than 1 mm, and wash them several times in centrifuge tubes with pre-cooled physiological saline.

[0076] (3) After washing several times, allow the mixture 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 culture medium, 2.5 mg / mL type IV collagenase, 0.1 mg / mL deoxyribonuclease 1, and 2% (V / V%) penicillin / streptomycin solution. Incubate at 37°C with shaking for 50 minutes.

[0077] (4) Add an equal volume of pre-cooled digestion termination solution to terminate digestion. The digestion termination solution consists of complete cell culture medium (basal medium Advanced DMEM / F12 + 10% fetal bovine serum), 10 µmol / L cell exfoliation and apoptosis protection agent Y27632, and 1% (V / V%) penicillin / streptomycin solution. The cell suspension is filtered through a 100 µm filter and centrifuged at 300 g for 10 minutes at 4 °C. The supernatant is discarded, and the cells are washed twice with pre-cooled physiological saline. The final precipitate is the tumor cells.

[0078] (5) Resuspend the tumor cells in a pre-cooled mixture of tumor organoid culture medium and matrix gel at a ratio of 1:2, adjust the cell concentration to 2500-4000 cells / 50μL, and seed them in cell culture plates.

[0079] (6) Place in a cell culture incubator at 37°C and 5% carbon dioxide for 30 minutes to solidify.

[0080] (7) Add tumor organoid culture medium and change the medium every 2-3 days.

[0081] Example 4: Constructing a co-cultivation model

[0082] A method for constructing a co-cultivation model, based on Examples 2 and 3, further includes the following steps:

[0083] (1) Remove the organoid culture medium from the culture plate, use TryPLE to blow the droplets to break them up, and place them in a cell culture incubator at 37°C and 5% carbon dioxide for 20 min.

[0084] (2) After taking it out, blow it into a 15ml centrifuge tube, centrifuge at 300g for 3min at 4℃, discard the supernatant, wash with PBS, centrifuge at 300g for 3min at 4℃. Discard the supernatant and resuspend it in a matrix gel.

[0085] (3) Take 10 μL of organoid suspension and carefully inject it into the tumor organoid through the tumor organoid inlet 19 at the top of the tumor organoid channel 18, and place it in an incubator at 37°C and 5% CO2 for 30 mins.

[0086] (4) If using a microinfusion pump, replace the HUVECs culture medium in the syringe with co-culture medium; if using the culture medium's own gravitational potential energy for dispensing, replace the HUVECs culture medium in reservoir 14 with co-culture medium; wherein, the co-culture medium is composed of tumor organoid culture medium and HUVECs culture medium mixed at a volume ratio of 1:1. After 7 days of co-culture, organoids and vascular endothelial cells can be observed to co-culture well (see...). Figure 5 ).

[0087] from Figure 5 It is evident that this invention optimizes the observation conditions for tumors and blood vessels, facilitating real-time monitoring of tumor cell proliferation, invasion, and dynamic changes in vascular networks; it also supports high-throughput and uniform tumor organoid culture, improving the reliability and reproducibility of experimental data.

[0088] Furthermore, the tumor formation in the co-culture model in this embodiment is not limited to tumor organoids, but also includes tumor cell lines, primary tumor cell tissues, etc.

[0089] Example 5: Drug susceptibility testing of antitumor drugs

[0090] A method for drug sensitivity testing of an antitumor drug, comprising the following steps:

[0091] (1) A vascular model is constructed according to the technical solution of Embodiment 2 of the present invention.

[0092] (2) Select patients who received FOLFOX + bevacizumab treatment from the tumor organoid biobank (selecting colon cancer organoids).

[0093] (3) Take out the corresponding cryopreserved patient organoids from liquid nitrogen, quickly place them in a 37°C water bath, wait for them to melt into small ice crystals, then take them out, add 1ml of organoid culture medium, mix well, transfer to a 15ml centrifuge tube, centrifuge at 300g for 3mins, remove the supernatant, wash with PBS, resuspend, and centrifuge at 300g for 3mins.

[0094] (4) Resuspend the tumor cells in a pre-cooled mixture of tumor organoid culture medium and matrix gel at a ratio of 1:2, adjust the cell concentration to 2500-4000 cells / 50μL, and seed them in cell culture plates.

[0095] (5) Place in a cell culture incubator at 37°C and 5% carbon dioxide for 30 min to solidify.

[0096] (6) Add tumor organoid culture medium and change the medium every 2-3 days.

[0097] (7) After observing that the organoids have good growth density and normal size, they are passaged and a co-culture model derived from patients is constructed according to the technical solution of Example 4 of this invention.

[0098] (8) FOLFOX + bevacizumab was added to the culture medium. The FOLFOX regimen used a concentration ratio of (5-FU:leucovorin:oxaliplatin, 25:5:1), with a 5-FU concentration of 10 μm and bevacizumab concentrations of 0.04, 0.2, and 1 μm, respectively. 3 Drug response tests were conducted using a concentration gradient, with negative and positive control groups set up.

[0099] (9) Take photos 6 days after administration and use ImageJ software to calculate the vascular density and average tumor organoid diameter at each concentration.

[0100] (10) The positive control group is defined as 0% vascular density, and the negative control (solvent control) is defined as 100% vascular density. The calculation formula is: vascular density percentage = (vascular density value - average positive control value) / (average negative control value - average positive control value) * 100%.

[0101] (11) The positive control group is defined as 0% organoid diameter, and the negative control (solvent control) is defined as 100% organoid diameter. The calculation formula is: organoid diameter percentage = (average organoid diameter value - average positive control value) / (average negative control value - average positive control value) * 100%.

[0102] (12) Based on the changes in vascular network density and organoid diameter in the organ-on-a-chip in vitro model, the in vitro model efficacy judgment criteria were formulated. When the bevacizumab concentration was 1 μm, the vascular network density was less than 50% of the negative control group, indicating that the vascular network was sensitive to the drug, and vice versa. The average diameter of the organoid was less than 50% of the negative control group, indicating that the colorectal cancer organoid was sensitive to the drug, and vice versa (see Table 2).

[0103] Table 2. Drug screening data from organoid microarrays of vascularized tumors from different sources.

[0104]

[0105] (13) Analyze relevant clinical information before and after receiving FOLFOX + bevacizumab treatment, including imaging data and colorectal cancer-related tumor markers such as CEA, CA199 and CA242. Based on the recist stage, CR and PR stages are defined as clinically effective, and SD and PD stages are defined as clinically resistant.

[0106] (14) Compare the drug sensitivity test results of the organ-on-a-chip from the patient with the actual clinical results of the patient (see Table 3).

[0107] Table 3 Comparison of novel vascularized tumor organoid microarray models and clinical outcomes

[0108]

[0109] As shown in Table 3, this invention, based on drug sensitivity testing using patient-derived tumor organoids, can more accurately predict clinical efficacy. By comparing the in vitro model drug sensitivity results with actual clinical outcomes in patients, the high accuracy of the model was verified, providing experimental evidence for personalized precision treatment. The specific accuracy rate is (7+6) / (8+7)*100%=86.7%.

[0110] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A vascularized tumor organoid microarray, characterized in that, The chip includes a main body, within which a cell culture channel is provided. Culture medium channels are located on the upper and lower sides of the cell culture channel. Needle insertion channels are located on the upper and lower sides of the cell culture channel for inserting acupuncture needles, thereby dynamically separating the cell culture channel from the culture medium channel. The cell culture channel, needle insertion channel, and culture medium channel are interconnected. A reservoir is located at one end of the culture medium channel, and a culture medium inlet is located at the other end on the surface of the chip. Cell inlets are located on the surface of the chip at both ends of the cell culture channel. Several micropillars are arranged in a ring array within the cell culture channel, and tumor organoid channels are formed inside these micropillars. Tumor organoid inlets are located on the surface of the chip. The culture medium flow channel includes a first flow channel, a second flow channel connected to the storage tank at one end of the first flow channel, and a third flow channel connected to the culture medium inlet at the other end of the first flow channel; the first flow channel and the second flow channel are arranged at an obtuse angle; one end of the needle flow channel extends to the connection between the first flow channel and the second flow channel, and the other end extends to the side surface of the chip body. The culture medium flow channel is 500 micrometers wide, 250 micrometers high, and 12,300 micrometers long; The needle inlet channel has a width of 250 micrometers, a height of 250 micrometers, and a length of 9800 micrometers.

2. The vascularized tumor organoid chip according to claim 1, characterized in that, The length of the first flow channel is 4400 micrometers, the length of the second flow channel is 1950 micrometers, the length of the third flow channel is 5950 micrometers, and the diameter of the culture medium inlet is 1500 micrometers.

3. The vascularized tumor organoid chip according to claim 1, characterized in that, The cell culture channel has a hexagonal cross-section with a side length of 4400 micrometers, a height of 250 micrometers, and a cell inlet diameter of 1500 micrometers.

4. The vascularized tumor organoid chip according to claim 1, characterized in that, The cross-section of the tumor organoid flow channel is a circle with a diameter of 2000 micrometers, and the diameter of the tumor organoid inlet is 1500 micrometers.

5. An application of an organoid microarray for vascularized tumors, characterized in that, The vascularized tumor organoid chip according to any one of claims 1-4 is used to construct vascular models, to construct co-culture models, or for in vitro drug screening.

6. The application of the vascularized tumor organoid chip according to claim 5, characterized in that, The method for constructing a vascular model includes the following steps: (1) Use acupuncture needles to insert into the needle channel to separate the cell culture channel and the culture medium channel; (2) Inject the gel-forming solution into the cell culture channel through the cell inlet and place it in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify; (3) After 30 minutes, remove the vascularized tumor organoid chip and, depending on the administration method, partially or completely remove the acupuncture needles. If a microinfusion pump is used for infusion, the acupuncture needle is pulled out to the connection between the first and third channels, and the HUVECs culture medium is injected into the cell culture channel through the culture medium inlet. Then, the vascularized tumor organoid chip is placed in an incubator at 37°C with a CO2 concentration of 5% for culture. If gravitational potential energy is used for liquid supply, add HUVECs culture medium to the two reservoirs, completely remove the acupuncture needles, and inject the HUVECs culture medium into the cell culture channel through the culture medium channel and the needle insertion channel. Then, place the vascularized tumor organoid chip in an incubator at 37°C with a CO2 concentration of 5% for culture.

7. The application of the vascularized tumor organoid chip according to claim 5, characterized in that, The method for constructing a co-cultivation model includes the following steps: (1) Use acupuncture needles to insert into the needle channel to separate the cell culture channel and the culture medium channel; (2) The gel generation solution was injected into the cell culture channel through the cell inlet, and the vascularized tumor organoid chip was placed in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify, and then the blood vessels were cultured. (3) After the vascular culture is completed, the vascularized tumor organoid chip is removed and the tumor organoid is injected into the tumor organoid channel. The vascularized tumor organoid chip is then placed in an incubator at 37°C and 5% CO2 concentration for 30 mins to solidify. (4) Remove the vascularized tumor organoid chip and administer co-culture medium; if the microinfusion pump is used, replace the HUVECs culture medium in the syringe with the co-culture medium; if the culture medium is administered by its own gravitational potential energy, replace the HUVECs culture medium in the reservoir with the co-culture medium; wherein, the co-culture medium is composed of tumor organoid culture medium and HUVECs culture medium mixed at a volume ratio of 1:1.

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