Method for high-throughput culture of vascularized organoid

Abstract

The invention belongs to the field of tissue engineering, and discloses a method for high-throughput culture of vascularized organoid, which comprises the following steps: (1) taking vascular endothelial cells, endothelial progenitor cells or different types of stem cells as cells with angiogenesis potential, mixing with cells to be vascularized, resuspending into a culture medium, inoculating into a plurality of concave cavities in a microarray chip, and culturing for 2-3 hours at the temperature of 20-30 DEG C; forming a plurality of fusion spheres; (2) adding a three-dimensional culture medium into the concave chamber, so that the fused sphere is surrounded in a three-dimensional environment of the three-dimensional culture medium to grow; and (3) after the fused spheres are fully fused, adding an angiogenesis promoting factor into the microarray chip, and performing induced differentiation on the fused spheres so as to form vascularized organoid in each concave cavity in batches or form a vascularized organoid network in the microarray chip. The method has the characteristics of high throughput and high efficiency, and is suitable for large-scale preparation of vascularized organoid.

Description

Technical Field

[0001] The present invention belongs to the technical field related to tissue engineering and regenerative medicine, and more specifically, relates to a method for high-throughput culture of vascularized organoids. Background Art

[0002] Compared with traditional organoids, vascularized organoids can more accurately simulate the in vivo microenvironment, providing higher accuracy for drug screening, more reliable models for disease research, and new treatments for tissue repair and organ regeneration. Currently, there are five main ways to culture vascularized organoids: stem cell co-differentiation, multi-cell co-culture, stem cell and microvascular fragment co-culture, organoid transplantation and removal, and the use of a variety of engineering technologies such as 3D printing, electrospinning, microfluidics, etc. to construct organoids with vascular networks.

[0003] However, in actual operation, the cultivation of vascularized organoids faces many difficulties. First, how to generate a stable vascular network. The current methods to solve this problem are: for example, the organoids are implanted in mice and taken out after the blood vessels mature, but this method of obtaining functional vascularized organoids is complex to implement, inefficient, and time-consuming; some scientists use microfluidic technology to cultivate vascularized organoids, but some microfluidic devices have complex microchannel structures, high manufacturing process requirements, and require precise control of flow rate and pressure, which increases the difficulty of operation; or vascularized organoids are obtained by stem cell induced differentiation, but in most cases, the final differentiation is unsuccessful or the effect is poor because the growth factors required for organoid differentiation and angiogenesis are not fully understood. Secondly, the cultivation of vascularized organoids requires primary cells and expensive culture media, which are expensive. How to effectively control costs is also a problem we need to consider. Finally, the current culturing methods of vascularized organoids are generally inefficient and difficult to meet the needs of large-scale preparation, which seriously hinders their clinical application. In particular, the screening of tumor drugs requires a high-throughput method to prepare vascularized tumor organoids on a large scale, thereby accelerating the development of new drugs and meeting the clinical needs for personalized treatment. Summary of the invention

[0004] In response to the above defects or improvement needs of the prior art, the present invention provides a method for high-throughput generation of vascularized organoids, the purpose of which is to complete the culture of batch fusion spheroids based on a microarray chip having multiple array-arranged concave chambers, and further establish a three-dimensional environment formed by a three-dimensional culture matrix in each concave chamber to complete the batch culture and growth of vascularized organoids, as well as the growth of vascularized organoid networks, thereby solving the technical problem in the prior art that it is difficult to culture vascularized organoids simply, efficiently and on a large scale.

[0005] To achieve the above object, according to one aspect of the present invention, a method for high-throughput generation of vascularized organoids is provided, comprising the following steps:

[0006] (1) endothelial cells, endothelial progenitor cells or different types of stem cells are used as cells with angiogenesis potential, mixed with cells to be vascularized, resuspended in culture medium, and then inoculated into multiple concave chambers in a microarray chip to form multiple fusion spheres; wherein the multiple concave chambers are arrayed in the lower layer of the microarray chip, the upper opening of each concave chamber faces the connecting area of ​​the middle layer of the microarray chip, and the bottom is composed of multiple U-shaped culture wells; and the size of the concave chamber is 7 to 21 mm, and the interval between the concave chambers is 1 to 2.5 mm;

[0007] (2) adding a three-dimensional culture matrix into the concave chamber, so that the fusion sphere is surrounded by the three-dimensional environment of the three-dimensional culture matrix and grows;

[0008] (3) After the fusion spheres are fully fused, angiogenic factors are added to the microarray chip to induce differentiation of the fusion spheres, so as to form vascularized organoids in each of the concave chambers in batches or to form a vascularized organoid network in the microarray chip.

[0009] As a preferred embodiment of the present invention, after vascularized organoids are formed in batches in each of the concave chambers, the culture is continued so that the vascularized organoids in the concave chambers grow divergently above the concave chambers until they connect with the vascularized organoids in adjacent concave chambers to form a vascularized organoid network.

[0010] As a preferred embodiment of the present invention, the microarray chip is further provided with a circulating flow pump connected to the inlet and outlet of the culture medium of the microarray chip for circulating the culture medium;

[0011] After the cells resuspended in the culture medium are inoculated into a plurality of concave chambers in the microarray chip, the culture medium is circulated through the circulation flow pump to allow the cells to grow.

[0012] As a preferred embodiment of the present invention, the different types of stem cells include induced pluripotent stem cells, mesenchymal stem cells or embryonic stem cells.

[0013] As a preferred embodiment of the present invention, the cells to be vascularized include primary tissues or passaged organoid cells.

[0014] As a preferred embodiment of the present invention, the primary tissue or passaged organoid is digested into single cells and then mixed with the cells having angiogenic potential.

[0015] As a preferred embodiment of the present invention, the primary tissue is derived from healthy tissue or tumor tissue; the passaged organoid cells are derived from organoids differentiated from pluripotent stem cells, organoids of tumor tissue or organoids of healthy tissue.

[0016] As a preferred embodiment of the present invention, the angiogenesis-promoting factors include vascular endothelial growth factor, monocyte chemoattractant protein-1, sphingosine 1-phosphate and phorbol esters.

[0017] As a preferred embodiment of the present invention, the method further comprises: in step (1), when the cells to be vascularized are mixed with the cells with angiogenic potential, fibroblasts and macrophages are added thereto and mixed together.

[0018] As a preferred embodiment of the present invention, passaged organoid cells are selected as the cells to be vascularized and co-cultured with human umbilical vein endothelial cells as the cells with angiogenic potential.

[0019] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0020] (1) The method for high-throughput culture of vascularized organoids provided by the present invention is based on a microarray chip having a plurality of array-arranged concave chambers to complete the culture of batch fusion spheres, and further establishes a three-dimensional environment formed by a three-dimensional culture matrix in each concave chamber to complete the culture and growth of batch vascularized organoids, as well as the growth of vascularized organoid networks. Therefore, the method for high-throughput culture of vascularized organoids provided by the present invention not only has low equipment requirements and is easy to operate, but also can culture a large number of vascularized organoids of uniform size in a relatively short period of time, and can directly culture to form vascularized organoid networks. It has the characteristics of high throughput and high efficiency, is suitable for large-scale preparation of vascularized organoids, and lays a foundation for its clinical application.

[0021] (2) The multiple concave chamber arrays of the present invention are arranged in the lower layer of the microarray chip, the upper opening of each concave chamber faces the connecting area of ​​the middle layer of the microarray chip, and the bottom is composed of U-shaped culture holes of micrometer size; and the spacing between the concave chambers is millimeter-level. Based on the microarray chip of the present invention, it is possible to more flexibly choose to isolate and culture a single vascularized organoid or culture a vascularized organoid network according to the culture requirements, and further can regulate and control the morphology of the vascularized organoid network.

[0022] (3) The present invention preferably cultivates the vascularized organoid network in a continuous and simple manner, that is, based on the concave chambers of millimeter size and the spacing between the concave chambers, the vascularized organoids are still in the same culture environment and only need to extend the culture time, so that the vascularized organoids in the concave chambers grow divergently above the concave chambers until they connect with the vascularized organoids in the adjacent concave chambers to form a vascularized organoid network. Therefore, cultivating the vascularized organoid network by the method of the present invention can reduce pollution and is simpler and more efficient.

[0023] (4) The present invention preferably configures a circulating flow pump on the microarray core to circulate flow to the culture medium so that the culture medium helps blood vessel maturation when the fluid is sheared.

[0024] (5) The present invention preferably adds angiogenic factors after the fusion spheres are fully fused, which significantly improves the success rate of vascularization.

[0025] (6) The high-throughput culturing method of vascularized organoids provided by the present invention not only has the characteristics of high throughput and high efficiency, but also its standardized chip design and culture conditions provide a standardized culture environment for vascularized organoids, reduce experimental operation errors, and improve technical repeatability.

[0026] In summary, the method of the present invention achieves efficient, stable and reproducible preparation of vascularized organoids by utilizing high-throughput chips, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structure of a patterned microarray chip according to an example of the present invention.

[0028] Figure 2 Flowchart illustrating the high-throughput culture of vascularized organoids of the present invention.

[0029] Figure 3 These are the bright field images and fluorescence test images of the natural sedimentation state of the cells mixed and inoculated into the chip 15 minutes after Example 1 of the present invention. The left image is the bright field image, and the right image is the fluorescence test image.

[0030] Figure 4 These are the bright field images and fluorescence test images of the state of cells fusion forming spheres in Example 1 of the present invention, the left image is the bright field image, and the right image is the fluorescence test image.

[0031] Figure 5 These are the bright field images and fluorescence test images of the colon cancer organoids induced to differentiate into vascularized colon cancer organoids in the chip in Example 1 of the present invention. From left to right, they are the bright field image at 1 mm size, the fluorescence test image at 1 mm size, the bright field image at 200 μm size, and the fluorescence test image at 200 μm size.

[0032] Figure 6 This is a display of immunofluorescence staining of the colon cancer organoids induced to differentiate into vascularized colon cancer organoids in the chip in Example 1 of the present invention, from left to right are the phalloidin staining image at a size of 100 μm, the calcein staining image, the colon cancer organoid specific marker image, and the fluorescence image of the three stainings and the superposition of the cell nucleus.

[0033] Throughout the drawings, the same reference numerals are used to refer to the same structures, wherein:

[0034] 1-arrayed microwells; 2-arrayed patterns. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] The patterned microarray chip involved in the present invention is a device provided in the prior art or in this subject, wherein the device is provided with a patterned microarray culture area and can also be connected to a matching automatic cell culture system.

[0037] The following is an example of a patterned microarray chip, the structure of the patterned microarray chip is as follows: Figure 1 As shown, it includes an arrayed micropore 1 on the upper layer, an arrayed pattern 2 bonded to the lower layer of the patterned micropore array, and other conventional components of the chip; and the micropores in the patterned micropore array correspond one-to-one to each pattern of the arrayed pattern 2, so as to obtain an isolated space (concave chamber) with an array arrangement. For example, if the bottom of the concave chamber is arc-shaped, a U-shaped chamber is formed, which is conducive to the divergent growth of microvessels. Specifically, the lower layer of the microarray chip is a concave chamber, the middle layer is a connecting area, and the upper opening of the concave chamber faces the connecting area; the size of the concave chamber is 7 to 21 mm, and the minimum spacing between the concave chambers can be 1 mm, all of which are millimeter-level, and the bottom is composed of U-shaped culture holes of micron-level size (for example, a diameter of 400 to 800 μm); for example, a micropore diameter of 7 mm, a spacing of 2.5 mm between micropores, and a patterned microarray chip with a diameter of 600 μm of the U-shaped culture hole are prepared by the present invention.

[0038] According to the patterned microarray chip described above, its size is the preferred size of the above scheme. The specific preparation method is as follows, which specifically includes: first, peeling off the upper and lower layers of 1.5 mm polymethyl methacrylate for standby use; using a laser engraving machine to emboss a patterned microwell array on the polymethyl methacrylate; preparing another piece of polymethyl methacrylate, peeling off one side of the film, sticking a special double-sided tape, and then directly using a laser engraving machine to engrave the arrayed pattern at the bottom of the culture chamber; peeling off the double-sided tape layer of the engraved patterned microwell array and the arrayed pattern at the bottom of the chamber to bond them to form a patterned microwell array; An isolated chamber chip with an array arrangement; the bonded chip is placed in a hot press with the upper and lower pressing surfaces at 60°C for 20 minutes; the chip after hot pressing is allowed to return to room temperature, placed in pure water for ultrasonic treatment for 24 hours, and blown dry with a nitrogen gun; a polypropylene glycol and ethylene oxide addition polymer is used to drop polypropylene glycol and ethylene oxide addition polymer on top of each microwell in the patterned microwell array, and the microwell is completely covered and then sucked out, and it is waited for air drying to complete the modification, and is placed in a transparent plastic bag and sealed for later use; before conducting the experiment, the sealed chip is placed in a UV disinfection cabinet or clean bench for 30 minutes for subsequent experiments.

[0039] Furthermore, the patterned microarray chip is equipped with a circulating flow pump, and can also be used in conjunction with a fully automated culture system. The fully automated culture system includes a control panel, a temperature control system, a carbon dioxide valve, and a UV sterilization system, and the patterned microarray chip, the temperature control system, the carbon dioxide valve, and the UV sterilization system are connected through the control panel.

[0040] The method for high-throughput generation of vascularized organoids exemplified in the present invention is based solely on the above-mentioned patterned microarray chip for culture, that is, it is possible to achieve large-scale and efficient production of vascularized organoids of uniform size without using more complex devices. Figure 2 , the cultivation steps are as follows:

[0041] (1) Preparing a patterned microarray chip according to the preferred preparation method described above, and providing an automatic cell culture system in the chip;

[0042] (2) mixing the cells to be vascularized with cells with angiogenic potential such as endothelial cells, endothelial progenitor cells or different types of stem cells, resuspending them in a culture medium, and then inoculating them into a microarray chip and evenly distributing them in each concave chamber of the microarray chip to form a plurality of fused spheres of uniformly sized cell populations in the microarray chip;

[0043] (3) After the culture medium is aspirated from the microarray chip, a three-dimensional culture matrix (including matrix gel and hydrogel that can allow other cells to grow, etc.) is added into each concave chamber, so that each fusion sphere is surrounded by the three-dimensional environment of the three-dimensional culture matrix and fully grows;

[0044] (4) After the fusion spheres are fully fused (1-2 days), multiple angiogenic factors are added to the chip to induce differentiation into batches of vascularized organoids or vascularized organoid networks in the same chip environment.

[0045] As a preferred embodiment of the present invention, a three-dimensional culture matrix is ​​added to each isolation chamber so that each fusion sphere is surrounded by the three-dimensional environment of the three-dimensional culture matrix and fully grows into a single vascularized organoid. The culture is then continued so that the vascularized organoids in the concave chamber grow radiatively above the concave chamber until they connect with the vascularized organoids in adjacent concave chambers to form a vascularized organoid network.

[0046] As a preferred embodiment of the present invention, the microarray chip is also equipped with a circulating flow pump, which is connected to the inlet and outlet of the culture medium of the microarray chip and is used to circulate the culture medium; after the cells resuspended in the culture medium are inoculated into multiple concave chambers in the microarray chip, the culture medium is circulated to the culture medium through the circulating flow pump for cell growth.

[0047] As preferred embodiments of the present invention, different types of stem cells include induced pluripotent stem cells, mesenchymal stem cells or embryonic stem cells.

[0048] As a preferred embodiment of the present invention, fibroblasts and macrophages may be added to the microarray core in the culture medium before forming the mixed inoculation to promote the formation of vascularization.

[0049] As a preferred embodiment of the present invention, the cells to be vascularized include primary tissues or passaged organoid cells. The cells to be vascularized also include common cell lines such as immortalized tumor cells.

[0050] As a preferred method of the present invention, primary tissues or passaged organoids are digested into single cells for use.

[0051] As a preferred embodiment of the present invention, the primary tissue is derived from healthy tissue or tumor tissue, and is not limited to species (including human tissue or other animal tissues, etc.); the passaged organoid cells are derived from organoids differentiated from pluripotent stem cells, organoids of tumor tissue or organoids of healthy tissue, and are not limited to species (including human tissue or other animal tissues, etc.).

[0052] As a preferred embodiment of the present invention, cells with angiogenic potential include human umbilical vein endothelial cells (HUVEC), endothelial progenitor cells (EPCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) or embryonic stem cells (ESCs).

[0053] As a preferred embodiment of the present invention, the fused spheres are differentiated into vascularized organoids under the induction of multiple angiogenic factors, wherein the angiogenic factors are specifically vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), sphingosine 1-phosphate (S1P) and phorbol 12-myristate 13-acetate (PMA).

[0054] As a preferred embodiment of the present invention, human umbilical vein endothelial cells and passaged organoid cells are selected for co-culture.

[0055] The following is a further explanation of the method for high-throughput culture of vascularized organoids of the present invention through specific examples.

[0056] Unless otherwise specified, the reagents used in the present invention are commercially available and of commercial grade and used as received. Standard experimental reagents and methods are used for the experimental tests involved in the present invention. The scientific and technical terms and abbreviations used in the present invention have the meanings commonly understood by those skilled in the art.

[0057] Example 1: Cultivation of vascularized colon cancer (CRC) organoids:

[0058] (1) Chip manufacturing method Referring to the above preparation scheme, the chip size is the specific size of the above example, which will not be described in detail here.

[0059] (2) Colon cancer organoids grown for 5 to 7 days were digested with TrypLE enzyme to single cells and resuspended in PBS solution with digested HUVECs in a certain proportion;

[0060] (3) The mixed cells were inoculated onto the chip and allowed to settle naturally to the middle of the chip for about 15 min. Figure 3 As shown, at this time, the chip is tilted and the PBS solution is slowly aspirated with a pipette tip, and complete co-culture medium is added and placed in a cell culture incubator.

[0061] (3) Wait for the cells to form fusion spheres (such as Figure 4 As shown), all the culture medium is slowly aspirated, 30uL of the three-dimensional culture matrix is ​​dripped into each chip chamber and placed in a cell culture incubator for solidification for 30 minutes. After solidification is complete, fresh complete culture medium is added to continue culturing.

[0062] (5) After the cells are fully combined, add the following four growth factors to the culture medium: VEGF, MCP1, S1P, and PMA. After about 2 days of induction, vascularized organoids will form.

[0063] like Figure 5 Shown are the bright field images and fluorescence test images of colon cancer organoids induced to differentiate into vascularized colon cancer organoids in the chip. From left to right are the bright field image at 1 mm size, the fluorescence test image at 1 mm size, the bright field image at 200 μm size, and the fluorescence test image at 200 μm size. It can be seen that our chip can batch generate a large number of uniform microvascularized organoids.

[0064] like Figure 6 Shown is an immunofluorescence staining display of colon cancer organoids induced to differentiate into vascularized colon cancer organoids in a chip. From left to right are the phalloidin staining image at a size of 100 μm, the calcein staining image, the colon cancer organoid specific marker image, and the fluorescence image of the three stainings and the superposition of cell nuclei, showing the appearance of microvascularized structures in the colon organoids.

[0065] Example 2: Formation of vascularized intestinal organoids:

[0066] (1) Chip manufacturing method Referring to the above preparation scheme, the chip size is the specific size of the above example, which will not be described in detail here.

[0067] (2) The fresh small intestinal tissue was briefly rinsed and cut into 1-2 cm fragments, and washed with PBS added with 5% double antibody for 12-15 times until there was no obvious turbidity in the supernatant; after discarding the supernatant, 2 mM EDTA solution was added and digested on a seesaw shaker for 15 min. PBS solution containing serum was added to terminate the digestion and the tissue was vigorously blown to remove large tissue blocks and other impurities through a cell sieve. At this time, small intestinal crypt stem cells were obtained.

[0068] (3) Intestinal stem cells and HUVECs were mixed and inoculated into the chip. After forming fused spheres, the culture medium was aspirated and a three-dimensional culture matrix mixture was added to bury the spheres. After solidification at 37°C for 30 min, co-culture medium was added and the spheres were cultured in an automated culture system for 1 to 2 days.

[0069] (4) Add the following four growth factors to the culture medium: VEGF, MCP1, S1P, and PMA. After about 2 days of induction, vascularized organoids will form.

[0070] Example 3: Formation of vascularized lung organoids:

[0071] (1) Chip manufacturing method Referring to the above preparation scheme, the chip size is the specific size of the above example, which will not be described in detail here.

[0072] (2) Embryonic stem cells and lung organoid single cells were inoculated onto the chip as in Example 1. After sphering, 30 ul of three-dimensional culture matrix diluent was added, and the cells were placed at 37° C. to solidify for 30 min, and complete co-culture medium was added.

[0073] (3) Adding pro-angiogenic factors promotes the differentiation of embryonic stem cells and ultimately obtains vascularized lung organoids.

[0074] Example 4: Formation of vascularized breast cancer spheroids

[0075] (1) The chip manufacturing method is similar to the above-mentioned preferred preparation scheme and will not be elaborated here.

[0076] (2) Breast cancer cells MCF7, fibroblasts and human umbilical vein endothelial cells HUVEC were digested separately and then mixed and inoculated into the chip. Complete culture medium was added and placed in a cell culture incubator.

[0077] (3) After the fusion spheres are formed, the culture medium is aspirated, and a hydrogel in which cells can grow is added to bury the spheres. After solidification at 37°C, a co-culture medium is added and the spheres are cultured in an automated culture system.

[0078] (4) After 5 days of normal culture, the tumor cells will be promoted to form blood vessels under the action of fibroblasts and endothelial cells. The addition of pro-angiogenic factors can shorten the time and obtain more mature vascularized breast cancer spheroids.

[0079] Example 5: Formation of vascularized organoid networks

[0080] (1) Chip manufacturing method Referring to the above preparation scheme, the chip size is the specific size of the above example, which will not be described in detail here.

[0081] (2) Organoid single cells and HUVECs were implanted into the chip to form fusion spheres. The specific operation was as described in Example 1.

[0082] (3) After the cells form fused spheres, add the three-dimensional culture matrix mixture, and after solidification, add complete culture medium and culture normally.

[0083] (4) After the cells in the fusion sphere are fully combined, add the following four growth factors to the culture medium: VEGF, MCP1, S1P, and PMA. After about 2 days of induction, a single vascularized organoid will form in each well. After about 8 days of induction, the vascular networks of the single organoids are connected to form a large vascular organoid network.

[0084] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the present invention and its equivalent technologies, the present invention is also intended to include these changes and variations. The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and their protection scope is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are all within the protection scope of the present invention.

Claims

1. A method for high-throughput culture of vascularized organoids, characterized in that: The steps include: (1) Vascular endothelial cells, endothelial progenitor cells or different types of stem cells are used as cells with angiogenesis potential, mixed with cells to be vascularized, resuspended in culture medium, and then inoculated into multiple concave chambers in a microarray chip to form multiple fusion spheres; wherein the multiple concave chambers are arrayed in the lower layer of the microarray chip, the upper opening of each concave chamber faces the connecting area of ​​the middle layer of the microarray chip, and the bottom is composed of multiple U-shaped culture wells; and the size of the concave chamber is 7 to 21 mm, and the interval between the concave chambers is 1 to 2.5 mm. (2) adding a three-dimensional culture matrix into the concave chamber, so that the fusion sphere is surrounded by the three-dimensional environment of the three-dimensional culture matrix and grows; (3) After the fusion spheres are fully fused, angiogenic factors are added to the microarray chip to induce differentiation of the fusion spheres, so as to form vascularized organoids in each of the concave chambers in batches or to form a vascularized organoid network in the microarray chip.

2. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: After vascularized organoids are formed in batches in each of the concave chambers, the culture is continued so that the vascularized organoids in the concave chambers grow radiatively above the concave chambers until they connect with the vascularized organoids in adjacent concave chambers to form a vascularized organoid network.

3. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The microarray chip is also equipped with a circulating flow pump, which is connected to the inlet and outlet of the culture medium of the microarray chip and is used to circulate the culture medium; After the cells resuspended in the culture medium are inoculated into a plurality of concave chambers in the microarray chip, the culture medium is circulated through the circulation flow pump to allow the cells to grow.

4. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The different types of stem cells include induced pluripotent stem cells, mesenchymal stem cells or embryonic stem cells.

5. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The cells to be vascularized include primary tissues or passaged organoid cells.

6. The method for high-throughput culture of vascularized organoids according to claim 5, characterized in that: The primary tissue or passaged organoid is digested into single cells and then mixed with the cells with angiogenic potential.

7. The method for high-throughput culture of vascularized organoids according to claim 5, characterized in that: The primary tissue is derived from healthy tissue or tumor tissue; the passaged organoid cells are derived from organoids differentiated from pluripotent stem cells, organoids of tumor tissue or organoids of healthy tissue.

8. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The angiogenic factors include vascular endothelial growth factor, monocyte chemoattractant protein-1, sphingosine 1-phosphate and phorbol ester.

9. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The method further comprises: in step (1), when the cells to be vascularized are mixed with the cells with angiogenesis potential, fibroblasts and macrophages are added thereto and mixed together.

10. The method for high-throughput culture of vascularized organoids according to claim 1, characterized in that: The passaged organoid cells are selected as the cells to be vascularized and co-cultured with human umbilical vein endothelial cells as the cells with angiogenic potential.

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