Vascularized organ chip capable of collecting multi-cell co-culture or organoid metabolites in situ for multiple times

Abstract

The invention belongs to the technical field of biomedical engineering and microfluidics, and particularly relates to a vascularized organ chip capable of collecting multi-cell co-culture or organoid metabolites in situ for multiple times. The invention provides a vascularized organ chip. The vascularized organ chip sequentially comprises a runner layer, a cell culture layer, a porous membrane layer and a metabolite collection layer, the runner layer is provided with a culture solution runner, and the culture solution runner is communicated with the outside through a culture solution hole; the cell culture layer is provided with a plurality of stem cell culture chambers; each cell culture chamber is communicated with the outside through the hydrogel channel and the cell ball injection hole; the area of the porous membrane layer at least covers the bottoms of all the cell culture chambers; the metabolite collecting layer is provided with a plurality of collecting chambers, and at least one collecting chamber is arranged below each cell culture chamber; the vascularization organ chip is further provided with a plurality of liquid taking channels, and each collecting cavity is communicated with the outside through the corresponding liquid taking channel. The vascularized organ chip provided by the invention can realize in-situ collection and dynamic analysis of metabolites.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering and microfluidics, and specifically relates to a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ. Background Technology

[0002] Metabolites play a crucial role in drug mechanisms of action, toxicity assessment, and the identification of disease biomarkers. However, traditional models are inadequate in simulating these metabolic processes, leading to significant discrepancies between drug screening results and actual clinical outcomes. While three-dimensional culture techniques overcome the challenges of cell growth in three-dimensional space, limitations in material selection mean that these models still cannot fully replicate the complexity of the in vivo microenvironment, particularly in accurately simulating the generation, accumulation, and exchange of metabolites.

[0003] Organ-on-a-chip (O-chip) is a microfluidic cell culture system designed to mimic the physiological functions of human organs through microfabrication techniques and complex microfluidic environments. Through precise design, this chip can accurately control fluid flow and the cell growth environment, such as the distribution of oxygen, nutrients, and metabolic waste. It also enables dynamic monitoring and analysis of metabolites, providing crucial biological data for drug development, disease models, and personalized medicine.

[0004] Establishing tumor tissue models on organ-on-a-chip is of great significance for tumor biology research, drug screening, and toxicity assessment. However, creating a model that includes key components such as tumor cells, blood vessels, and stromal cells, and accurately simulates the complex responses within the tumor microenvironment, remains a major challenge. In particular, the metabolic characteristics of tumors play a crucial role in the tumor microenvironment, and metabolic changes in tumor cells under extreme conditions such as hypoxia and acidity are critical to drug response, drug resistance, and tumor development. Therefore, constructing models that can accurately simulate the tumor microenvironment and metabolic changes has become a cutting-edge direction in cancer research and treatment.

[0005] The limitations of traditional organ-on-a-chip structures allow tumor cell spheres to grow at arbitrary locations within the culture chamber. This not only increases the difficulty of image acquisition but also restricts the collection and analysis of metabolites during experiments. Due to the lack of precise spatial localization, the interactions between cells and the distribution of metabolites within the tumor microenvironment may not uniformly or accurately reflect the real situation, thus affecting the reliability and reproducibility of experimental results.

[0006] Furthermore, traditional cell culture suffers from difficulties in collecting metabolites. By employing organ-on-a-chip technology, which enables in-situ measurement of metabolites, researchers can capture changes in metabolites multiple times and continuously in a real microenvironment. Compared to traditional models, this technology can accurately track the generation, transformation, and accumulation of metabolites during drug screening, reducing the discrepancy between experimental results and clinical outcomes, thereby improving the accuracy and success rate of drug screening.

[0007] In summary, traditional organ-on-a-chip structures have many limitations in simulating the tumor microenvironment and metabolic processes, particularly in the interaction between tumor spheres and vascularized networks, cell migration, and the dynamic collection and analysis of metabolites. There is an urgent need for a vascularized organ-on-a-chip capable of repeatedly collecting metabolites from multi-cell co-cultures or organoids in situ to address these issues. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention provides an organ-on-a-chip with partitioning characteristics, easy fabrication, and the ability to clearly observe the growth and migration of cell spheres in a vascularized network, as well as the ability to dynamically collect and analyze metabolites. The invention also provides its preparation and usage methods, enabling large-scale culture on a small scale and high-throughput experimental testing. It allows for the separate culture of cell spheres / organoids and the observation of their growth status. Furthermore, it allows for multiple in-situ collections of metabolites generated by cells growing in the culture chambers within the chip.

[0009] The first aspect of the present invention provides a vascularized organ-on-a-chip, wherein the vascularized organ-on-a-chip sequentially comprises a flow channel layer, a cell culture layer, a porous membrane layer, and a metabolite collection layer.

[0010] The flow channel layer has a culture medium flow channel on the side near the cell culture layer, and the culture medium flow channel is connected to the outside through culture medium holes.

[0011] The cell culture layer has several cell culture chambers that penetrate the cell culture layer; the vascularized organ-on-a-chip has several hydrogel channels and cell ball injection holes, and the hydrogel channels and cell ball injection holes respectively connect each cell culture chamber to the outside.

[0012] The porous membrane layer covers at least the bottom of all cell culture chambers.

[0013] The metabolite collection layer has several collection chambers on the side near the porous membrane layer. Each cell culture chamber has at least one collection chamber below it. At least a portion of each collection chamber overlaps with the area of ​​its corresponding cell culture chamber. The vascularized organ-on-a-chip also has several liquid extraction channels. Each collection chamber is connected to the outside through a liquid extraction channel.

[0014] The second aspect of the present invention provides a method for fabricating a vascularized chip, comprising the following steps: 1) preparing a mask for each layer of microfluidic structure pattern; 2) preparing a positive mold for each layer of microfluidic structure using soft photolithography; 3) casting PDMS into the positive mold for each layer of microfluidic structure, curing and demolding to obtain each layer having a microfluidic structure; 4) perforating the channel layer and the cell culture layer; 5) sequentially bonding each layer to obtain the vascularized organ chip.

[0015] A third aspect of the present invention provides a method for vascularized co-culture of cells or organoids, comprising culturing cells or organoids using a vascularized organ microarray as described in the first aspect above.

[0016] The vascularized co-culture method comprises the following steps:

[0017] S1) The cell stock solution is mixed with thrombin to obtain a bio-hydrogel, wherein the cell stock solution includes endothelial cells and lung fibroblasts;

[0018] S2) Insert the spacer into the cell ball injection well, continuously inject the bio-hydrogel from the channel layer hydrogel injection well until the bio-hydrogel flows out from the channel layer hydrogel discharge well, and then culture.

[0019] S3) Continuously inject laminin from the injection hole (15) until the laminin flows out from the outlet hole (16), remove the occupant, and culture;

[0020] S4) Inject culture medium through injection hole (15) and incubate;

[0021] S5) Inject cell spheres or organoids through the cell sphere injection well and culture them.

[0022] The fourth aspect of the present invention provides a method for collecting metabolites from vascularized co-culture of cells or organoids, comprising using the vascularized co-culture method as described in the third aspect above, and collecting metabolites from a sampling well.

[0023] The fifth aspect of this invention provides the application of the vascularized organ-on-a-chip of the first aspect, or the preparation method of the second aspect, or the vascularized co-culture method of the third aspect, or the collection method of the fourth aspect in drug development, and / or drug testing, and / or drug screening, and / or drug metabolism research.

[0024] The beneficial effects of this invention are as follows:

[0025] 1) This invention provides a vascularized organ-on-a-chip that can collect metabolites of multi-cell co-culture or organoids multiple times in situ. It can observe the growth status of cell spheres / organoids in vascularized co-culture without direct contact with cells. Furthermore, the cell spheres are placed in a convenient manner, and they can be observed directly under a microscope.

[0026] 2) The vascularized organ-on-a-chip in this invention can collect the metabolites produced by cells growing in the culture chamber within the chip multiple times in situ, and can collect the metabolites of cells under the action of drugs in real time in subsequent drug research, and dynamically monitor and analyze the material composition of the metabolites.

[0027] 3) The vascularized organ-on-a-chip in this invention has four chambers, and the height of the cell culture chamber is 400~500μm, which can realize large-scale culture on a small scale and high-throughput experimental testing.

[0028] 4) The structure used in this invention can place cell spheres / organoids in a designated location, culture cell spheres / organoids separately, and clearly observe the interaction between cell spheres / organoids and blood vessels through a microscope.

[0029] 5) The structure of this invention is simple, easy to operate, the chip is easy to manufacture, and it has good biocompatibility. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the overall structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ according to the present invention.

[0031] Figure 2 This is a schematic diagram of the exploded structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites of multi-cell co-culture or organoids in situ according to the present invention.

[0032] Figure 3 This is a schematic diagram of the combined structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites of multi-cell co-culture or organoids in situ according to the present invention.

[0033] Figure 4 This is a schematic diagram of the flow channel layer structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ according to the present invention.

[0034] Figure 5 This is a schematic diagram of the cell culture layer structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ according to the present invention.

[0035] Figure 6This is a schematic diagram of the vascularized organ-on-a-chip metabolite collection layer structure that can collect metabolites from multi-cell co-culture or organoids multiple times in situ according to the present invention.

[0036] Figure 7 This is a schematic diagram of the flow channel structure of a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ according to the present invention.

[0037] Figure 8 This is an enlarged view of the rhomboid flow channel layer of a vascularized organ-on-a-chip that can repeatedly collect metabolites from multi-cell co-culture or organoids in situ according to the present invention.

[0038] Figure 9 This is a schematic diagram illustrating the use of a vascularized organ-on-a-chip that allows for repeated in-situ collection of metabolites from multi-cell co-culture or organoids, according to the present invention.

[0039] Figure label.

[0040] 1-Flow channel layer

[0041] 111-First flow channel layer capillary burst valve orifice

[0042] 112-First flow channel layer hydrogel injection hole

[0043] 113-First cell ball injection hole

[0044] 114 - Liquid intake hole of the first flow channel layer

[0045] 115 - First flow channel layer hydrogel injection hole

[0046] 116 - First flow channel layer through hole

[0047] 121-Second flow channel layer capillary burst valve orifice

[0048] 122-Second flow channel layer hydrogel injection hole

[0049] 123 - Second cell ball injection hole

[0050] 124 - Liquid intake hole of the second flow channel layer

[0051] 125 - Second flow channel layer hydrogel injection hole

[0052] 126 - Second flow channel layer through hole

[0053] 131-Third flow channel layer capillary burst valve orifice

[0054] 132-Third flow channel layer hydrogel injection hole

[0055] 133-Third Cell Ball Injection Hole

[0056] 134 - Liquid intake hole of the third flow channel layer

[0057] 135 - Third flow channel layer hydrogel injection hole

[0058] 136 - Third flow channel layer through hole

[0059] 141-Fourth flow channel layer capillary burst valve orifice

[0060] 142-Fourth flow channel layer hydrogel injection hole

[0061] 143-Fourth cell ball injection well

[0062] 144-Fourth flow channel layer liquid sampling hole

[0063] 145-Fourth flow channel layer hydrogel injection hole

[0064] 146 - Fourth flow channel layer through hole

[0065] 15-Injection Hole

[0066] 16-Liquid outlet hole

[0067] 17-Culture medium flow channel

[0068] 171-Serpentine Flow Channel

[0069] 1711-Rhombus Flow Channel

[0070] 17111 - Rhombus outer edge

[0071] 17112-Rhomboid Microcolumn

[0072] 1712-Bent Flow Channel

[0073] 172-Straight flow channel

[0074] 2-Cell Culture Layer

[0075] 21-First Cell Culture Chamber

[0076] 211-First culture chamber capillary burst valve orifice

[0077] 212-First culture chamber hydrogel injection well

[0078] 213-First Culture Chamber Capillary Rupture Valve

[0079] 214 - First culture chamber liquid sampling port

[0080] 215-First culture chamber hydrogel injection well

[0081] 216-First culture chamber through hole

[0082] 22-Second cell culture chamber

[0083] 221-Second culture chamber capillary burst valve orifice

[0084] 222-Second culture chamber hydrogel injection well

[0085] 223-Second Culture Chamber Capillary Rupture Valve

[0086] 224 - Second culture chamber liquid sampling hole

[0087] 225 - Second culture chamber hydrogel injection well

[0088] 226-Second culture chamber through hole

[0089] 23-Third cell culture chamber

[0090] 231-Third culture chamber capillary burst valve orifice

[0091] 232-Third culture chamber hydrogel injection well

[0092] 233-Third Culture Chamber Capillary Rupture Valve

[0093] 234 - Liquid sampling port of the third culture chamber

[0094] 235 - Third culture chamber hydrogel injection well

[0095] 236-Third culture chamber through hole

[0096] 24 - Fourth Cell Culture Chamber

[0097] 241-Fourth Culture Chamber Capillary Burst Valve Orifice

[0098] 242-Fourth Culture Chamber Hydrogel Injection Hole

[0099] 243-Fourth Culture Chamber Capillary Rupture Valve

[0100] 244-Fourth culture chamber liquid sampling hole

[0101] 245-Fourth culture chamber hydrogel injection well

[0102] 246-Fourth Culture Chamber Through Hole

[0103] 3-Porous membrane layer

[0104] 4-Metabolite Collection Layer

[0105] 41-First Collection Chamber

[0106] 411-First Collection Chamber Liquid Extraction Hole

[0107] 412-First collection chamber through hole

[0108] 413-First Collection Chamber Flow Channel

[0109] 42-Second Collection Chamber

[0110] 421-Second Collection Chamber Liquid Extraction Hole

[0111] 422-Second collection chamber through hole

[0112] 423-Second Collection Chamber Flow Channel

[0113] 43-Third Collection Chamber

[0114] 431-Liquid collection port of the third collection chamber

[0115] 432-Third collection chamber through hole

[0116] 433-Third Collection Chamber Flow Channel

[0117] 44-Fourth Collection Chamber

[0118] 441-Fourth Collection Chamber Liquid Extraction Hole

[0119] 442-Fourth Collection Chamber Through Hole

[0120] 443-Fourth Collection Chamber Flow Channel Detailed Implementation

[0121] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0122] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0123] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0124] like Figures 1-3 As shown, the present invention first provides a vascularized organ-on-a-chip, which sequentially includes a flow channel layer 1, a cell culture layer 2, a porous membrane layer 3, and a metabolite collection layer 4.

[0125] The flow channel layer 1 has a culture medium flow channel 17 on the side near the cell culture layer. The culture medium flow channel 17 is connected to the outside through a culture medium hole and is used to inject culture medium into the cell culture chamber.

[0126] Furthermore, such as Figure 7 As shown, the culture medium flow channel 17 includes a serpentine flow channel 171, which is composed of several alternately connected curved flow channels 1712 and rhomboid flow channels 1711. The vertical projection of the rhomboid flow channel 1711 is located within the cell culture chamber. The rhomboid flow channel 1711 includes a rhomboid outer edge 17111 and several separate, unconnected rhomboid micropillars 17112. The rhomboid outer edge 17111 is formed by several connected rhomboids, and the gap between the rhomboid outer edge 17111 and the rhomboid micropillars 17112 forms a passage for the flow of culture medium. The curved flow channel 1712 is located outside the cell culture chamber. The design of the rhomboid flow channel 1711 is to restrict the flow of the bio-hydrogel from the cell culture layer 2 to the flow channel layer 1 when it is injected. The rhomboid flow channel 1711 causes the hydrogel to form a gel-liquid interface in the z-axis direction due to surface tension when injected, which prevents it from breaking through the gel-liquid interface formed between the micropillars in the rhomboid flow channel 1711 to reach the rhomboid flow channel 1711 in the flow channel layer 1. This makes the bio-hydrogel more inclined to fill the cell culture chamber and not enter the rhomboid flow channel 1711 upwards.

[0127] Furthermore, such as Figure 8As shown, the shortest distance M between the outer edge 17111 of the rhombus and the rhombus micropillar 17112 is 20~25 μm, for example, it can be 20~21 μm, 21~22 μm, 22~23 μm, 23~24 μm, 24~25 μm; the shortest distance N between two adjacent separated rhombus micropillars 17112 is 35~40 μm, for example, it can be 35~36 μm, 36~37 μm, 37~38 μm, 38~39 μm, 39~40 μm; the width of the curved flow channel 1712 is 0.1~0.15 mm, for example, it can be 0.1~0.11 mm, 0.11~0.12 mm, 0.12~0.13 mm, 0.13~0.14 mm, 0.14~0.15 mm.

[0128] The culture medium well is located in the flow channel layer 1. The culture medium well includes an injection well 15 and an outlet well 16, both of which penetrate the flow channel layer 1. The injection well 15 and the outlet well 16 are located at opposite ends of the culture medium flow channel 17. The vertical projections of the injection well 15 and the outlet well 16 are both located outside the cell culture chamber. When the culture medium is injected through the injection well 15, it can flow through the culture medium flow channel 17 and enter the cell culture chamber, and finally flow out through the outlet well 16 to provide nutrients and mechanical stimulation required for cell growth.

[0129] Furthermore, the culture medium flow channel 17 also includes two straight flow channels 172, which are located at both ends of the serpentine flow channel 171, and the injection hole 15 and the outlet hole 16 are located at the two free ends of the straight flow channel 172.

[0130] The cell culture layer 2 is provided with a plurality of cell culture chambers that penetrate the cell culture layer 2 for culturing cells or organoids. The number of cell culture chambers can be, for example, three or more, such as three, four, five, six or more. The cell culture chambers penetrate from one side of the cell culture layer 2 to the other side to form cavities that can accommodate hydrogel to form a vascular network and accommodate cells. The cavities can be prepared by punching holes in the cell culture layer 2 using a punch of the desired shape.

[0131] The cell culture chamber is rectangular or truncated rectangular. A truncated rectangle is a complete rectangle with some parts (e.g., one or more corners) cut off. The length of the cell culture chamber can be 7-8 mm, for example, 7-7.5 mm or 7.5-8 mm, and the width can be 1.8-2.0 mm, for example, 1.8-1.9 mm or 1.9-2.0 mm.

[0132] The vascularized organ-on-a-chip is provided with several hydrogel channels, and each cell culture chamber is connected to the outside through a hydrogel channel. Further, the hydrogel channel includes a hydrogel injection channel and a hydrogel discharge channel. The hydrogel injection channel is used to inject hydrogel into the cell culture chamber from the outside to form a vascular network. The hydrogel discharge channel is used to allow the hydrogel to flow out of the cell culture chamber after it is filled. The hydrogel injection channel, cell culture chamber, and hydrogel discharge channel form a hydrogel pathway connecting to the outside, thereby enabling biological hydrogel to enter through the hydrogel injection channel, fill the cell culture chamber, and flow out through the hydrogel discharge channel, forming a vascular network within the cell culture chamber after cultivation. Even further, the hydrogel injection channel on the vascularized organ-on-a-chip is composed of hydrogel injection holes in the flow channel layer 1 and hydrogel injection holes in the culture chamber on the cell culture layer 2. The hydrogel discharge channel on the vascularized organ-on-a-chip is composed of hydrogel discharge holes in the flow channel layer 1 and hydrogel discharge holes in the culture chamber on the cell culture layer 2.

[0133] The vascularized organ-on-a-chip is provided with a plurality of cell ball injection holes, and each cell culture chamber is also connected to the outside through the cell ball injection holes. The cell ball injection holes are used to inject cell balls from the outside into the cell culture chamber for culture. The cell ball injection holes are located between the serpentine channels 171, and the serpentine channels 171 are at least diamond-shaped channels 1711 on both sides of the cell ball injection holes.

[0134] The porous membrane layer 3 covers at least the bottom of all cell culture chambers, meaning that there is a porous membrane at the bottom of at least all cell culture chambers. The porous membrane can also be extended. The pore size of the porous membrane allows metabolites to pass through but does not allow cells to pass through. Therefore, when cells or organoids produce metabolites through culture, the metabolites can permeate through the porous membrane layer 3 into the collection chamber of the metabolite collection layer 4.

[0135] The metabolite collection layer 4 has several collection chambers on the side near the porous membrane layer, and each cell culture chamber has at least one collection chamber below it for collecting metabolites produced by cell or organoid culture in the cell culture chamber.

[0136] The collection chamber can be rectangular, with a width greater than that of the corresponding cell culture chamber. The length of the collection chamber can be 6.4~7.4 mm, for example, 6.4~6.6 mm, 6.6~6.8 mm, 6.8~7.0 mm, 7.0~7.2 mm, or 7.2~7.4 mm, and the width can be 3~4 mm, for example, 3~3.5 mm or 3.5~4 mm.

[0137] The vascularized organ-on-a-chip is also provided with several liquid collection channels. Each collection chamber is connected to the outside through the liquid collection channel for taking out metabolic products in the collection chamber. Furthermore, the liquid collection channel on the vascularized organ-on-a-chip is composed of liquid collection holes on the flow channel layer 1, liquid collection holes on the culture chamber on the cell culture layer 2, and liquid collection holes on the collection chamber on the metabolic product collection layer 4.

[0138] The vascularized organ-on-a-chip also has a capillary burst valve channel. The cell culture chamber is connected to the outside through the capillary burst valve channel. When the hydrogel is injected into the cell culture chamber, the pressure in the cell culture chamber is released to ensure that the hydrogel can smoothly fill the cell culture chamber. Furthermore, the capillary burst valve channel is composed of a flow channel layer burst valve hole on the flow channel layer 1 and a culture chamber capillary burst valve hole on the cell culture layer 2.

[0139] The vascularized organ-on-a-chip also has a chip channel, which is composed of flow channel through-holes on the flow channel layer 1, culture chamber through-holes on the cell culture layer 2, and collection chamber through-holes on the metabolite collection layer 4.

[0140] Furthermore, the flow channel layer 1, cell culture layer 2, porous membrane layer 3, and metabolite collection layer 4 are described respectively.

[0141] In a specific embodiment of the present invention, such as Figure 4 As shown, the flow channel layer 1 is provided with a culture medium flow channel 17, a liquid injection hole 15, a liquid outlet hole 16, and several flow channel layer pore groups. The number of flow channel layer pore groups can be three or more, for example, three, four, five, six, or more. Each flow channel layer pore group includes a flow channel layer hydrogel injection hole, a cell ball injection hole, a flow channel layer hydrogel outlet hole, a flow channel layer liquid collection hole, a flow channel layer capillary burst valve hole, and a flow channel layer through hole; the culture medium flow channel 17, the liquid injection hole 15, and the liquid outlet hole 16 are as described above.

[0142] The hydrogel injection holes, cell ball injection holes, hydrogel discharge holes, liquid extraction holes, capillary burst valve holes, and through holes of the flow channel layer all penetrate the flow channel layer 1.

[0143] The shapes of the hydrogel injection holes, cell ball injection holes, hydrogel outlet holes, liquid extraction holes, capillary burst valve holes, through holes, injection holes 15, and outlet holes 16 in the flow channel layer are not limited, and can be circular, square, triangular, or polygonal. When the shape of the hole is circular, the diameter of the hole can be 1~1.5 mm, for example, 1~1.1 mm, 1.1~1.2 mm, 1.2~1.3 mm, 1.3~1.4 mm, or 1.4~1.5 mm.

[0144] The vertical projection of the cell ball injection hole is located within the cell culture chamber. With this configuration, cells to be cultured can be injected into the vascular network already formed within the cell culture chamber through the cell ball injection hole. Preferably, the vertical projection of the cell ball injection hole is located at the center of the cell culture chamber.

[0145] The thickness of the flow channel layer 1 can be 0.5~0.6 mm, for example, 0.5~0.55 mm or 0.55~0.6 mm; the length of the flow channel layer 1 can be 55~60 mm, for example, 55~57 mm, 57~59 mm or 59~60 mm; the width of the flow channel layer 1 can be 24~26 mm, for example, 24~25 mm or 25~26 mm; and the height of the culture medium flow channel 17 can be 100~200 μm, for example, 100~120 μm, 120~140 μm, 140~160 μm, 160~180 μm or 180~200 μm.

[0146] In a specific embodiment of the present invention, such as Figure 5 As shown, the cell culture layer 2 includes several cell culture chambers and cell culture layer pore groups. The number of cell culture chambers and cell culture layer pore groups are equal, that is, each cell culture chamber corresponds to a group of cell culture layer pore groups. The number can be more than three, for example, three, four, five, six, or more. Each group of cell culture layer pore groups includes a culture chamber hydrogel injection hole, a culture chamber hydrogel discharge hole, a culture chamber liquid extraction hole, a culture chamber capillary burst valve, a culture chamber capillary burst valve hole, and a culture chamber through hole; the cell culture chambers are as described above.

[0147] The liquid extraction hole and the through hole of the culture chamber penetrate the cell culture layer 2.

[0148] The hydrogel injection holes, hydrogel outlet holes, capillary burst valves, and capillary burst valve holes in the cell culture layer 2 do not penetrate the culture layer. The heights of the hydrogel injection holes, hydrogel outlet holes, capillary burst valves, and capillary burst valve holes can all be 1~1.5 mm, for example, 1~1.1 mm, 1.1~1.2 mm, 1.2~1.3 mm, 1.3~1.4 mm, or 1.4~1.5 mm. The height refers to the shortest distance from the side of the cell culture layer 2 closest to the flow channel layer to the bottom of the hole.

[0149] Both the hydrogel injection port and the hydrogel discharge port of the culture chamber are connected to the cell culture chamber.

[0150] The lateral width of the hydrogel injection hole in the culture chamber decreases from the direction away from the cell culture chamber to the direction closer to the cell culture chamber. For example, the end away from the cell culture chamber is round and the end closer to the cell culture chamber is pointed. The hydrogel injection hole in the culture chamber is generally teardrop-shaped. When the bio-hydrogel is injected, the bio-hydrogel flows from the hydrogel injection hole in the flow channel layer to the hydrogel injection hole in the culture chamber and enters the cell culture chamber.

[0151] The lateral width of the hydrogel inlet pore in the culture chamber decreases from the direction away from the cell culture chamber to the direction closer to the cell culture chamber. For example, the end away from the cell culture chamber is round, and the end closer to the cell culture chamber is pointed. The hydrogel inlet pore in the culture chamber is generally teardrop-shaped. When the bio-hydrogel fills the cell culture chamber, it flows out to the outside from the hydrogel inlet pore in the culture chamber to the hydrogel inlet pore in the flow channel layer.

[0152] When the hydrogel injection hole and hydrogel discharge hole in the culture chamber are teardrop-shaped as described above, their maximum lateral width can be 1~1.5 mm, for example, 1~1.1 mm, 1.1~1.2 mm, 1.2~1.3 mm, 1.3~1.4 mm, or 1.4~1.5 mm.

[0153] The capillary burst valve of the culture chamber is connected to the hydrogel injection port of the culture chamber and the cell culture chamber, and is also connected to the outside through the capillary burst valve port of the culture chamber and the capillary burst valve port of the flow channel layer. It is used to release the pressure generated during the injection of the bio-hydrogel. The valve orifice width of the capillary burst valve of the culture chamber is greater than the shortest distance N between two adjacent rhomboid micropillars 17112, thereby preventing the bio-hydrogel from flowing into the culture medium flow channel 17 when filling the cell culture chamber. Preferably, the valve orifice width of the capillary burst valve of the culture chamber can be 65 μm to 100 μm, for example, 65 to 70 μm, 70 to 75 μm, 75 to 80 μm, 80 to 85 μm, 85 to 90 μm, 90 to 95 μm, or 95 to 100 μm; the pipe width of the capillary burst valve of the culture chamber can be 0.25 to 0.35 mm, for example, 0.25 to 0.26 mm. The diameters are 0.26~0.27 mm, 0.27~0.28 mm, 0.28~0.29 mm, 0.29~0.30 mm, 0.30~0.31 mm, 0.31~0.32 mm, 0.32~0.33 mm, 0.33~0.34 mm, and 0.34~0.35 mm. The pipe width of the capillary rupture valve in the culture chamber refers to the overall width of the liquid channel within the rupture valve, typically referring to the inner diameter of the channel excluding the valve orifice. The valve orifice width of the capillary rupture valve in the culture chamber refers to the inner diameter of the narrowest point in the liquid channel within the rupture valve.

[0154] The shape of the culture chamber liquid intake hole, the culture chamber capillary burst valve hole, and the culture chamber through hole is not limited, and can be circular, square, triangular, or polygonal. When the shape of the hole is circular, the diameter of the hole can be 1~1.5 mm, for example, 1~1.1 mm, 1.1~1.2 mm, 1.2~1.3 mm, 1.3~1.4 mm, or 1.4~1.5 mm.

[0155] The position of the capillary burst valve orifice in the culture chamber corresponds to the position of the capillary burst valve orifice in the flow channel layer. They are identical in shape and size. Thus, the capillary burst valve orifice in the flow channel layer and the capillary burst valve orifice in the culture chamber together form a capillary burst valve channel from the surface of the vascularized organ-on-a-chip to the cell culture layer 2, and connects the outside world with the vascularized organ-on-a-chip in the cell culture chamber. This channel is used to release the pressure in the cell culture chamber when the hydrogel is injected into the cell culture chamber, ensuring that the hydrogel can smoothly fill the cell culture chamber.

[0156] The circular end of the hydrogel injection hole in the culture chamber corresponds to the position of the hydrogel injection hole in the flow channel layer. They are the same in shape and size. Thus, the hydrogel injection hole in the flow channel layer and the hydrogel injection hole in the culture chamber together form a hydrogel injection channel for the vascularized organ chip from the surface of the vascularized organ chip to the cell culture layer 2, and connects the outside world with the cell culture chamber. This channel is used for the injection of bio-hydrogels that form a vascular network.

[0157] The circular end of the hydrogel injection hole in the culture chamber corresponds to the position of the hydrogel injection hole in the flow channel layer. They are the same in shape and size. Thus, the hydrogel injection holes in the flow channel layer and the hydrogel injection holes in the culture chamber together form a hydrogel injection channel for the vascularized organ chip from the surface of the vascularized organ chip to the cell culture layer 2, and connects the outside world with the cell culture chamber. This channel is used to form the outflow of bio-hydrogel that forms a vascular network.

[0158] The thickness of the cell culture layer 2 can be 0.4~0.5 mm, for example 0.4~0.45 mm, 0.45~0.5 mm; the length can be 55~60 mm, for example 55~57 mm, 57~59 mm, 59~60 mm; and the width can be 24~26 mm, for example 24~25 mm, 25~26 mm.

[0159] In a specific embodiment of the present invention, such as Figure 2As shown, the porous membrane layer 3 has selective permeability, meaning it allows metabolites to pass through but not cells or organoids. For example, it can be selected from polycarbonate membranes, polydimethylsiloxane membranes, polyethylene terephthalate membranes, or other polymer membranes. In one embodiment, the porous membrane is a polycarbonate membrane. The porous membrane layer 3 can be a continuous or discontinuous porous membrane. It at least covers the area defined by all cell culture chambers, meaning that a porous membrane is present at the bottom of all cell culture chambers, but not at the bottom surface of the liquid collection holes or the bottom surface of the through holes. The porous membrane layer 3 is attached between the cell culture layer 2 and the metabolite collection layer 4.

[0160] Preferably, when the porous membrane layer 3 is a continuous porous membrane, the length of the porous membrane layer 3 can be 25~35 mm, for example, 25~30 mm, 30~35 mm; the width can be 8~10 mm, for example, 8~9 mm, 9~10 mm; the pore size of the porous membrane can be 5.0~20.0 μm, for example, 5.0~10.0 μm, 10.0~15.0 μm, 15.0~20.0 μm; the thickness can be 30~40 μm, for example, 30~35 μm, 35~40 μm; and the porosity can be 10%~15%, for example, 10%~12%, 12%~14%, 14%~15%.

[0161] In a specific embodiment of the present invention, such as Figure 6 As shown, the metabolite collection layer 4 includes several collection chambers and sets of metabolite collection channels. The number of collection chambers is equal to the number of sets of metabolite collection channels, that is, each collection chamber corresponds to a set of sets of metabolite collection channels. The number can be three or more, for example, three, four, five, six, or more. The number of sets of metabolite collection channels is equal to the number of sets of channels in the flow channel layer. Each set of metabolite collection channels includes a collection chamber liquid intake hole, a collection chamber through hole, and a collection chamber flow channel. Each collection chamber liquid intake hole and collection chamber through hole is connected to the collection chamber through the collection chamber flow channel. The collection chambers are as described above.

[0162] The position of the liquid collection hole in the collection chamber corresponds to the position of the liquid collection hole in the flow channel layer and the liquid collection hole in the culture chamber. They are the same in shape and size. Thus, the liquid collection hole in the flow channel layer, the liquid collection hole in the culture chamber, and the liquid collection hole in the collection chamber together form a liquid collection channel from the surface of the vascularized organ-on-a-chip to the metabolite collection layer 4, and connect the outside world with the vascularized organ-on-a-chip in the collection chamber, for the purpose of extracting metabolites.

[0163] The position of the collection chamber through hole corresponds to the position of the flow channel layer through hole and the culture chamber through hole, and they have the same shape and size. Thus, the flow channel layer through hole, the culture chamber through hole, and the collection chamber through hole together constitute the chip channel of the vascularized organ chip from the surface of the vascularized organ chip to the metabolic product collection layer 4, and connect the outside world with the collection chamber.

[0164] The height of the liquid collection hole, the through hole, and the flow channel of the collection chamber can be 0.2~0.5 mm, for example, 0.2~0.3 mm, 0.3~0.4 mm, or 0.4~0.5 mm.

[0165] The width of the flow channel in the collection chamber can be 0.1~0.2 mm, for example, 0.1~0.12 mm, 0.12~0.14 mm, 0.14~0.16 mm, 0.16~0.18 mm, or 0.18~0.2 mm.

[0166] The diameter of the liquid collection hole and the through hole of the collection chamber can be 1~1.5 mm, for example, 1~1.1 mm, 1.1~1.2 mm, 1.2~1.3 mm, 1.3~1.4 mm, or 1.4~1.5 mm.

[0167] The thickness of the metabolite collection layer 4 can be 0.6~0.8 mm, for example 0.6~0.7 mm, 0.7~0.8 mm, the length can be 55~60 mm, for example 55~57 mm, 57~59 mm, 59~60 mm, and the width can be 24~26 mm, for example 24~25 mm, 25~26 mm.

[0168] The materials of the flow channel layer 1, cell culture layer 2, and metabolite collection layer 4 are high molecular weight polymers, including but not limited to polydimethylsiloxane. These polymers are softer than glass, allowing for better cell fusion. The polymers are transparent.

[0169] The flow channel layer 1 and the cell culture layer 2 are connected by bonding to form a perfusion space. The cell culture layer 2 and the metabolite collection layer 4 are connected by bonding. The porous membrane layer 3 is attached between the cell culture layer 2 and the metabolite collection layer 4. Specifically, before the cell culture layer 2 and the metabolite collection layer 4 are fixed, the porous membrane layer 3 is first attached to the bottom surface of the cell culture layer 2. Then, the cell culture layer 2 with the porous membrane layer 3 attached is sealed to the metabolite collection layer 4 by bonding, thereby fixing the flow channel layer 1, the cell culture layer 2, the porous membrane layer 3, and the metabolite collection layer 4 relatively. A cell culture chamber for cell culture is isolated above the porous membrane layer 3, and a collection chamber for collecting metabolites after cell culture is isolated below the porous membrane. During cell culture, metabolites generated in the cell culture chamber permeate into the collection chamber through the porous membrane. The bonding methods include, but are not limited to, hot-press bonding, solvent-assisted bonding, adhesive bonding, plasma-activated bonding, surface-modified grafting bonding, copolymer interface layer bonding, nanoimprint bonding, ultrasonic bonding, and enzyme-catalyzed bonding.

[0170] The present invention also provides a method for preparing the above-mentioned vascularized organ chip, which adopts soft photolithography and may specifically include the following steps: 1) preparing a mask for the microchannel structure pattern of each layer; 2) preparing a positive mold for each layer of microchannel structure using soft photolithography; 3) casting a polymer into the positive mold of each layer of microchannel structure, curing and demolding to obtain each layer with microchannel structure; 4) perforating the channel layer 1 and the cell culture layer 2; 5) sequentially bonding each layer to obtain the vascularized organ chip.

[0171] In a specific implementation, to increase the bonding strength between each layer, the chip obtained after bonding can be heated for at least 5 minutes in step 5).

[0172] The present invention also provides a method for vascularized co-culture of cells or organoids, including culturing cells or organoids using the above-mentioned vascularized organ microarray. Vascularized co-culture refers to constructing a multi-cell co-culture system containing a functional vascular network in vitro to simulate the microenvironment of organs or tissues in vivo, and to study intercellular interactions, material exchange (such as oxygen, nutrients, and metabolic waste), and vascular-related physiological or pathological processes.

[0173] like Figure 9 As shown, the vascularization co-culture method specifically includes the following steps:

[0174] S1) The cell stock solution is mixed with thrombin to obtain a biohydrogel, wherein the cell stock solution includes endothelial cells and / or fibroblasts;

[0175] S2) Insert the placebo into the cell ball injection hole, and continuously inject the bio-hydrogel from the channel layer hydrogel injection hole until the bio-hydrogel fills the cell culture chamber, flows out from the channel layer hydrogel outlet hole, and is cultured to form a vascular network.

[0176] S3) Continuously inject laminin through injection hole 15 until the laminin flows out through outlet hole 16, remove the occupant, and culture.

[0177] S4) Inject culture medium through injection hole 15 and incubate;

[0178] S5) Inject cell spheres or organoids through the cell sphere injection well and culture them.

[0179] In step S1), a cell suspension is prepared using human umbilical vein endothelial cells of the 7th generation or less, human lung fibroblasts, and endothelial-specific culture medium. The cell suspension is mixed with fibrinogen to obtain the cell stock solution.

[0180] In step S1), the volume ratio of cell stock solution to thrombin can be 8~12:1, for example, 8~10:1, 10~12:1. In one embodiment, the volume ratio of cell stock solution to thrombin is 10:1.

[0181] In step S1), the endothelial cell concentration in the bio-hydrogel can be 7 × 10⁻⁶. 6 ~9×10 6 Cells / mL, for example, can be 7 × 10⁻⁶ 6 ~8×10 6 cells / mL, 8×10 6 ~9×10 6 per mL.

[0182] In step S1), the concentration of fibroblasts in the bio-hydrogel can be 6 × 10⁻⁶. 6 ~8×10 6 Cells / mL, for example, can be 7 × 10⁻⁶ 6 ~8×10 6 cells / mL, 8×10 6 ~9×10 6 per mL.

[0183] In step S1), the volume of the cell stock solution can be 10-15 μL per cell culture chamber, for example, 10-11 μL, 11-12 μL, 12-13 μL, 13-14 μL, or 14-15 μL.

[0184] In step S2), the placeholder only needs to be able to be inserted into the cell sphere injection well so that a cavity is formed at the placeholder when the bio-hydrogel fills the cell culture chamber. This cavity is then used to accommodate subsequently injected cell spheres or organoids after the bio-hydrogel has been cultured and formed a vascular network. The placeholder can be, for example, a pipette tip.

[0185] In step S2), the bio-hydrogel flows out of the hydrogel injection hole of the flow channel layer, which ensures that the bio-hydrogel fills the entire cell culture chamber and does not enter the culture medium flow channel 17 of the flow channel layer.

[0186] In step S2), the injection volume of the bio-hydrogel can be 10~15 μL, for example, 10~11 μL, 11~12 μL, 12~13 μL, 13~14 μL, or 14~15 μL.

[0187] In step S2), the culture temperature is 36.5~37℃, the culture time is 10~15 minutes, and the culture environment is a CO2-containing environment, for example, the CO2 content can be 5.0~5.1%.

[0188] In step S3), the amount of laminin used can be 10~15μL, for example, 10~11μL, 11~12μL, 12~13μL, 13~14μL, or 14~15μL.

[0189] In step S4), the flow rate of the culture medium can be 1~1.5 μL / min, for example, 1~1.1 μL / min, 1.1~1.2 μL / min, 1.2~1.3 μL / min, 1.3~1.4 μL / min, or 1.4~1.5 μL / min. The culture medium can be perfused from the injection hole 15 to the culture medium channel 17 by a microfluidic pump such as a micropump, peristaltic pump, or syringe pump to maintain a constant flow rate, so as to provide stable fluid stimulation to the cell culture chamber.

[0190] The present invention also provides a method for collecting metabolites from vascularized co-culture of cells or organoids, comprising using the above-described vascularized co-culture method and collecting metabolites from the sampling well.

[0191] The present invention also provides the application of the above-mentioned vascularized organ-on-a-chip, the above-mentioned preparation method, the above-mentioned vascularized co-culture method, or the above-mentioned collection method in drug development, and / or drug testing, and / or drug screening, and / or drug metabolism research.

[0192] The present invention will be described below through specific embodiments. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Third Edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; the series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATIN STRUCTURE AND FUNCTION*, Third Edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN... MOLECULARBIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc. Unless otherwise specified, the instruments, reagents, and materials used in the examples can be obtained through conventional means.

[0193] Example 1: Vascularized organ-on-a-chip

[0194] This embodiment provides a vascularized organ-on-a-chip, see [link to documentation]. Figures 1-7 .

[0195] like Figures 1-3 As shown, the vascularized organ-on-a-chip sequentially includes a flow channel layer 1, a cell culture layer 2, a porous membrane layer 3, and a metabolic product collection layer 4.

[0196] The thickness of the flow channel layer 1 can be 0.5~0.6 mm, the length of the flow channel layer 1 can be 55~60 mm, and the width of the flow channel layer 1 can be 24~26 mm.

[0197] The cell culture layer 2 has a thickness of 0.4-0.5 mm, a length of 55-60 mm, and a width of 24-26 mm.

[0198] The porous membrane layer 3 is a polydimethylsiloxane membrane. The length of the porous membrane layer 3 is 25~35 mm, the width is 8~10 mm, the pore size is 5.0~20.0 μm, the thickness is 30~40 μm, and the porosity is 10%~15%. The porous membrane layer 3 covers the area defined by all cell culture chambers, but does not include the area defined by the liquid extraction holes and through holes of the culture chambers.

[0199] The thickness of the metabolite collection layer 4 is 0.6~0.8 mm, the length can be 55~60 mm, and the width is 24~26 mm.

[0200] The flow channel layer 1, cell culture layer 2, and metabolite collection layer 4 are all made of polydimethylsiloxane. The flow channel layer 1 and cell culture layer 2 are bonded together. The porous membrane layer 3 is attached between the cell culture layer 2 and the metabolite collection layer 4, which are also bonded together.

[0201] like Figure 4 As shown, the flow channel layer 1 includes a culture medium flow channel 17, an injection port 15, an outlet port 16, and four sets of flow channel layer pore groups. These four sets of flow channel layer pore groups are: a first flow channel layer pore group, a second flow channel layer pore group, a third flow channel layer pore group, and a fourth flow channel layer pore group. The first flow channel layer pore group includes a first flow channel layer hydrogel injection port 112, a first cell spheroid injection port 113, a first flow channel layer hydrogel outlet port 115, a first flow channel layer liquid collection port 114, a first flow channel layer capillary burst valve port 111, and a first flow channel layer through-hole 116. The second flow channel layer pore group includes a second flow channel layer hydrogel injection port 122, a second cell spheroid injection port 123, and a second flow channel layer hydrogel injection port 124. The third flow channel layer includes a hydrogel injection hole 132, a cell ball injection hole 133, a hydrogel injection outlet hole 135, a liquid extraction hole 134, a capillary rupture valve hole 131, and a through hole 136; the fourth flow channel layer includes a hydrogel injection hole 142, a cell ball injection hole 143, a hydrogel injection outlet hole 145, a liquid extraction hole 144, a capillary rupture valve hole 141, and a through hole 146.

[0202] like Figure 5As shown, the cell culture layer 2 includes four cell culture chambers and four cell culture layer pore groups, with each cell culture chamber corresponding to one cell culture layer pore group. The four cell culture chambers are designated as the first cell culture chamber 21, the second cell culture chamber 22, the third cell culture chamber 23, and the fourth cell culture chamber 24. The four cell culture layer pore groups are designated as the first cell culture layer pore group, the second cell culture layer pore group, the third cell culture layer pore group, and the fourth cell culture layer pore group. The first cell culture layer pore group includes a first culture chamber hydrogel injection hole 212, a first culture chamber hydrogel outlet hole 215, a first culture chamber liquid extraction hole 214, a first culture chamber capillary burst valve 213, a first culture chamber capillary burst valve hole 211, and a first culture chamber through hole 216. The second cell culture layer pore group includes a second culture chamber water... The third cell culture layer pore group includes a gel injection hole 222, a hydrogel injection hole 225 in the second culture chamber, a liquid extraction hole 224 in the second culture chamber, a capillary burst valve 223 in the second culture chamber, a capillary burst valve hole 221 in the second culture chamber, and a through hole 226 in the second culture chamber; the fourth cell culture layer pore group includes a hydrogel injection hole 232 in the third culture chamber, a hydrogel injection hole 235 in the third culture chamber, a liquid extraction hole 234 in the third culture chamber, a capillary burst valve 233 in the third culture chamber, a capillary burst valve hole 231 in the third culture chamber, and a through hole 236 in the third culture chamber; the fourth cell culture layer pore group includes a hydrogel injection hole 242 in the fourth culture chamber, a hydrogel injection hole 245 in the fourth culture chamber, a liquid extraction hole 244 in the fourth culture chamber, a capillary burst valve 243 in the fourth culture chamber, a capillary burst valve hole 241 in the fourth culture chamber, and a through hole 246 in the fourth culture chamber.

[0203] like Figure 6As shown, the metabolite collection layer 4 includes four collection chambers and four sets of metabolite collection channels, with each collection chamber corresponding to one set of metabolite collection channels. The four collection chambers are a first collection chamber 41, a second collection chamber 42, a third collection chamber 43, and a fourth collection chamber 44. The four sets of metabolite collection channels are a first set of metabolite collection channels, a second set of metabolite collection channels, a third set of metabolite collection channels, and a fourth set of metabolite collection channels. The first set of metabolite collection channels includes a first collection chamber liquid intake hole 411, a first collection chamber through hole 412, and a first collection chamber flow channel 413. The first collection chamber liquid intake hole 411 and the first collection chamber through hole 412 are both connected to the first collection chamber 41 through the first collection chamber flow channel 413. The second set of metabolite collection channels includes a second collection chamber liquid intake hole. The third metabolite collection channel group includes a third collection chamber liquid extraction hole 431, a third collection chamber through hole 432, and a third collection chamber flow channel 433. The third collection chamber liquid extraction hole 431 and the third collection chamber through hole 432 are respectively connected to the third collection chamber 43 through the third collection chamber flow channel 423. The fourth metabolite collection channel group includes a fourth collection chamber liquid extraction hole 441, a fourth collection chamber through hole 442, and a fourth collection chamber flow channel 443. The fourth collection chamber liquid extraction hole 441 and the fourth collection chamber through hole 442 are respectively connected to the fourth collection chamber 44 through the fourth collection chamber flow channel 443.

[0204] like Figure 7 As shown, the culture medium flow channel 17 includes straight flow channels 172 located at both ends and a serpentine flow channel 171 located between the two straight flow channels 172. The serpentine flow channel 171 includes eight diamond-shaped flow channels 1711 and a curved flow channel 1712 that connects the eight diamond-shaped flow channels 1711 in sequence.

[0205] The vertical projection of each rhomboid channel 1711 is located within the cell culture chamber. For example, the vertical projections of the first and second rhomboid channels are located within the first cell culture chamber 21, the vertical projections of the third and fourth rhomboid channels are located within the second cell culture chamber 22, the vertical projections of the fifth and sixth rhomboid channels are located within the third cell culture chamber 23, and the vertical projections of the seventh and eighth rhomboid channels are located within the fourth cell culture chamber 24.

[0206] like Figure 8As shown, the rhomboid flow channel 1711 includes a rhomboid outer edge 17111 and several non-connected rhomboid micropillars 17112. The rhomboid outer edge 17111 is formed by connecting several rhomboids. The gap between the rhomboid outer edge 17111 and the rhomboid micropillars 17112 forms a channel for the flow of culture medium. The shortest distance M between the rhomboid outer edge 17111 and the rhomboid micropillars 17112 is 20~30 μm, and the shortest distance N between two adjacent rhomboid micropillars 17112 is 35~45 μm. The vertical projection of the curved flow channel 1712 is basically located outside the cell culture chamber, and the width of the curved flow channel 1712 is 0.1~0.2 mm. The height of the culture medium flow channel 17 can be 100~200 μm.

[0207] The injection hole 15 and the outlet hole 16 are located at the free ends of the two straight flow channels 172, respectively, and the vertical projections of the injection hole 15 and the outlet hole 16 are both located outside the cell culture chamber.

[0208] Injection hole 15, outlet hole 16, all flow channel layer hydrogel injection holes, cell ball injection holes, flow channel layer hydrogel injection holes, flow channel layer liquid extraction holes, flow channel layer capillary burst valve holes, and flow channel layer through holes all penetrate the flow channel layer 1.

[0209] All the hydrogel injection holes, cell ball injection holes, hydrogel outlet holes, liquid extraction holes, capillary burst valve holes, through holes, injection holes 15, and outlet holes 16 in the flow channel layer 1 are circular in shape, and their diameters can be 1~1.5 mm.

[0210] All cell culture chambers penetrate cell culture layer 2, and all culture chamber liquid extraction holes and culture chamber through holes penetrate cell culture layer 2. All culture chamber hydrogel injection holes, culture chamber hydrogel discharge holes, culture chamber capillary burst valves, and culture chamber capillary burst valve holes are blind holes, that is, one end of the hole is open and the other end does not penetrate the culture layer, and their height is 1~1.5 mm.

[0211] All culture chamber liquid intake holes, culture chamber capillary burst valve holes, and culture chamber through holes are circular in shape, with a diameter of 1~1.5 mm. All culture chamber hydrogel injection holes and culture chamber hydrogel dispensing holes are teardrop-shaped. The lateral width of the culture chamber hydrogel injection holes and culture chamber hydrogel dispensing holes decreases from the direction away from the cell culture chamber to the direction closer to the cell culture chamber, with a maximum lateral width of 1~1.5 mm.

[0212] The first culture chamber capillary rupture valve 213 is connected to the connection between the first culture chamber hydrogel injection port 212 and the first cell culture chamber 21; the second culture chamber capillary rupture valve 223 is connected to the connection between the second culture chamber hydrogel injection port 222 and the second cell culture chamber 22; the third culture chamber capillary rupture valve 233 is connected to the connection between the third culture chamber hydrogel injection port 232 and the third cell culture chamber 23; and the fourth culture chamber capillary rupture valve 243 is connected to the connection between the fourth culture chamber hydrogel injection port 242 and the fourth cell culture chamber 24. The valve orifice width of all culture chamber capillary rupture valves is 65 μm to 100 μm, and the pipe width of all culture chamber capillary rupture valves is 0.3 to 0.4 mm.

[0213] The first culture chamber capillary rupture valve orifice 211 is located on the side of the first culture chamber capillary rupture valve 213 away from the first cell culture chamber; the second culture chamber capillary rupture valve orifice 221 is located on the side of the second culture chamber capillary rupture valve 223 away from the second cell culture chamber; the third culture chamber capillary rupture valve orifice 231 is located on the side of the third culture chamber capillary rupture valve 233 away from the third cell culture chamber; and the fourth culture chamber capillary rupture valve orifice 241 is located on the side of the fourth culture chamber capillary rupture valve 243 away from the fourth cell culture chamber.

[0214] All cell culture chambers are truncated rectangles, with a length of 7-8 mm and a width of 1.8-2.0 mm.

[0215] The first culture chamber hydrogel injection port 212 and the first culture chamber hydrogel injection port 215 are located at both ends of the first cell culture chamber 21 and are both connected to the first cell culture chamber 21; the second culture chamber hydrogel injection port 222 and the second culture chamber hydrogel injection port 225 are located at both ends of the second cell culture chamber 22 and are both connected to the second cell culture chamber 22; the third culture chamber hydrogel injection port 232 and the third culture chamber hydrogel injection port 235 are located at both ends of the third cell culture chamber 23 and are both connected to the third cell culture chamber 23; the fourth culture chamber hydrogel injection port 242 and the fourth culture chamber hydrogel injection port 245 are located at both ends of the fourth cell culture chamber 24 and are both connected to the fourth cell culture chamber 24.

[0216] The position of the first cell culture chamber 21 corresponds to the position of the first collection chamber 41; the position of the second cell culture chamber 22 corresponds to the position of the second collection chamber 42; the position of the third cell culture chamber 23 corresponds to the position of the third collection chamber 43; and the position of the fourth cell culture chamber 24 corresponds to the position of the fourth collection chamber 44.

[0217] All collection chamber sampling holes, collection chamber through holes, and collection chamber flow channels do not penetrate the metabolite collection layer 4, and their height is 0.2~0.5 mm.

[0218] The length of any of the collection chambers is 6.4 to 7.4 mm and the width is 3 to 4 mm. All collection chambers do not penetrate the metabolite collection layer 4.

[0219] All collection chamber sampling holes and collection chamber through holes are circular in shape, with a diameter of 1~1.5 mm.

[0220] The position of the capillary burst valve orifice 111 in the first flow channel layer corresponds to the position of the capillary burst valve orifice 211 in the first culture chamber, and they are the same in shape and size; the position of the capillary burst valve orifice 121 in the second flow channel layer corresponds to the position of the capillary burst valve orifice 221 in the second culture chamber, and they are the same in shape and size; the position of the capillary burst valve orifice 131 in the third flow channel layer corresponds to the position of the capillary burst valve orifice 231 in the third culture chamber, and they are the same in shape and size; the position of the capillary burst valve orifice 141 in the fourth flow channel layer corresponds to the position of the capillary burst valve orifice 241 in the fourth culture chamber, and they are the same in shape and size; thus forming four capillary burst valve channels that extend from the surface of the flow channel layer 1 into the cell culture layer 2.

[0221] The position of the first flow channel layer hydrogel injection hole 112 corresponds to the position of the first culture chamber hydrogel injection hole 212, and they are the same in shape and size; the position of the second flow channel layer hydrogel injection hole 122 corresponds to the position of the second culture chamber hydrogel injection hole 222, and they are the same in shape and size; the position of the third flow channel layer hydrogel injection hole 132 corresponds to the position of the third culture chamber hydrogel injection hole 232, and they are the same in shape and size; the position of the fourth flow channel layer hydrogel injection hole 142 corresponds to the position of the fourth culture chamber hydrogel injection hole 242, and they are the same in shape and size; thus forming four hydrogel injection channels that extend from the surface of the flow channel layer 1 into the cell culture layer 2.

[0222] The vertical projection of the first cell sphere injection hole 113 is located at the center of the first cell culture chamber 21; the vertical projection of the second cell sphere injection hole 123 is located at the center of the second cell culture chamber 22; the vertical projection of the third cell sphere injection hole 133 is located at the center of the third cell culture chamber 23; and the vertical projection of the fourth cell sphere injection hole 143 is located at the center of the fourth cell culture chamber 24.

[0223] The position of the hydrogel injection hole 115 in the first flow channel layer corresponds to the position of the hydrogel injection hole 215 in the first culture chamber, and they are the same in shape and size; the position of the hydrogel injection hole 125 in the second flow channel layer corresponds to the position of the hydrogel injection hole 225 in the second culture chamber, and they are the same in shape and size; the position of the hydrogel injection hole 135 in the third flow channel layer corresponds to the position of the hydrogel injection hole 235 in the third culture chamber, and they are the same in shape and size; the position of the hydrogel injection hole 145 in the fourth flow channel layer corresponds to the position of the hydrogel injection hole 245 in the fourth culture chamber, and they are the same in shape and size; thus forming four hydrogel injection channels that extend from the surface of the flow channel layer 1 into the cell culture layer 2.

[0224] The position of the first flow channel layer liquid collection hole 114 corresponds to the position of the first culture chamber liquid collection hole 214 and the first collection chamber liquid collection hole 411, and they are the same in shape and size; the position of the second flow channel layer liquid collection hole 124 corresponds to the position of the second culture chamber liquid collection hole 224 and the second collection chamber liquid collection hole 421, and they are the same in shape and size; the position of the third flow channel layer liquid collection hole 134 corresponds to the position of the third culture chamber liquid collection hole 234 and the third collection chamber liquid collection hole 431, and they are the same in shape and size; the position of the fourth flow channel layer liquid collection hole 144 corresponds to the position of the fourth culture chamber liquid collection hole 244 and the fourth collection chamber liquid collection hole 441, and they are the same in shape and size; thus forming four liquid collection channels that extend from the surface of the flow channel layer 1 into the metabolite collection layer 4.

[0225] The position of the first flow channel layer through-hole 116 corresponds to the position of the first culture chamber through-hole 216 and the first collection chamber through-hole 412, and they are the same in shape and size; the position of the second flow channel layer through-hole 126 corresponds to the position of the second culture chamber through-hole 226 and the second collection chamber through-hole 422, and they are the same in shape and size; the position of the third flow channel layer through-hole 136 corresponds to the position of the third culture chamber through-hole 236 and the third collection chamber through-hole 432, and they are the same in shape and size; the position of the fourth flow channel layer through-hole 146 corresponds to the position of the fourth culture chamber through-hole 246 and the fourth collection chamber through-hole 442, and they are the same in shape and size; thus forming four chip channels that extend from the surface of the flow channel layer 1 into the metabolite collection layer 4.

[0226] Example 2: Fabrication of Vascularized Organ-on-a-Chip

[0227] After the flow channel layer 1 and cell culture layer 2 are prepared using soft lithography to form silicon wafers, they are cast using polydimethylsiloxane (PDMS). Specifically, the chip layer with the microfluidic structure is first drawn into a blueprint using AutoCAD software and then processed into a mask by the company. Then, based on the existing lithography machine in the laboratory, a silicon wafer with the microfluidic structure is made using soft lithography (the flow channel height selected for flow channel layer 1 is 100μm, and the flow channel height selected for cell culture layer 2 is 200μm). Finally, the unstructured side of the silicon wafer is pasted into a culture dish to form a mold.

[0228] Polydimethylsiloxane (PDMS) prepolymer and curing agent were uniformly mixed in a 10:1 ratio and poured into a petri dish. Through vacuuming, bubble blowing, drying, and peeling, PDMS sheets with microchannel structures were obtained. Figure 2 The flow channel layer 1 and cell culture layer 2 are shown in the diagram.

[0229] Perforation is performed on the flow channel layer 1 to obtain all through holes: A specific perforator is used to punch through the cell culture chamber, and perforations are performed at preset positions on the cell culture layer 2 to obtain the first culture chamber liquid extraction hole 214, the first culture chamber through hole 216, the second culture chamber liquid extraction hole 224, the second culture chamber through hole 226, the third culture chamber liquid extraction hole 234, the third culture chamber through hole 236, the fourth culture chamber liquid extraction hole 244, and the fourth culture chamber through hole 246.

[0230] Subsequently, oxygen plasma bonding is performed on the flow channel layer 1 and the cell culture layer 2 to ensure that the corresponding pores on the flow channel layer 1 and the cell culture layer 2 are in the positions described above.

[0231] The porous membrane layer 3 is a polycarbonate (PC) porous membrane.

[0232] Metabolite collection layer 4 is a silicon wafer with a microchannel structure (channel height selected is 500μm) fabricated using a photolithography mask method. Specifically, a SAF100 photosensitive polymer film (each layer is 100μm thick) is cut into a square shape, the protective film is removed, and the entire silicon wafer is covered. After two hot-pressing reinforcements, the surface protective film is removed, and the film is re-attached to 500μm. After baking, the microchannels are formed by photolithography. A 2% sodium carbonate solution is used for development, and the film is hardened on a hot plate to form a mold. Then, the unstructured side of the silicon wafer is pasted into a petri dish to form the mold. Polydimethylsiloxane (PDMS) prepolymer and curing agent are uniformly mixed in a 10:1 ratio and poured into a petri dish. Through vacuuming, bubble blowing, drying, and peeling operations, a PDMS sheet with a microchannel structure is obtained. Figure 2 The metabolic product collection layer 4 is shown in the figure.

[0233] Subsequently, a PC porous membrane is attached to the bottom surface of the cell culture chamber, and the metabolite collection layer 4 and the cell culture layer 2 are subjected to oxygen plasma bonding treatment to ensure that the corresponding pores on the metabolite collection layer 4 and the cell culture layer 2 are in the positions described above.

[0234] By sequentially bonding the chip layers through the above steps, a vascularized chip capable of repeatedly collecting organoid metabolites in situ can be obtained. To accelerate the adhesion effect after bonding, the bonded chip is placed in a hot plate and heated for 5 minutes for reinforcement. Before the experiment, a 10μL pipette tip is used to pre-fill the first cell ball injection well 113, the second cell ball injection well 123, the third cell ball injection well 133, and the fourth cell ball injection well 143 of the chip. Then, high-temperature sterilization is performed, followed by ultraviolet sterilization in a clean bench for 30 minutes, and then proper storage is carried out.

[0235] Example 3: Use of Vascularized Organ-on-a-Chip

[0236] First, the required experimental cells are cultured until the confluence reaches over 80%, then the cells are collected and counted. The corresponding number of cells is taken based on the number of microarrays required for the experiment (preferably, the concentration of human umbilical vein endothelial cells (HUVECs) is 9 × 10⁻⁶). 6 The concentration of NHLF in human lung fibroblasts is 7 × 10⁶ cells / mL. 6 After removing the supernatant, the mixture is mixed with fibrinogen to form the cell stock solution (the cell stock solution must be used within 15 minutes, otherwise it will coagulate on its own). A certain amount of the cell stock solution is then mixed with thrombin to form a bio-hydrogel (the ratio of thrombin to cell stock solution is 1:10).

[0237] The bio-hydrogel requires the chip injection process to be completed within 20 seconds: it is injected through the first hydrogel injection hole 112 on the upper surface of the channel layer 1, and flows through the first culture chamber hydrogel injection hole 212, the first cell culture chamber 21, and the first culture chamber hydrogel outlet hole 215 on the upper surface of the channel layer 1 to the first hydrogel outlet hole 115 on the upper surface of the channel layer 1. Due to the surface tension of the liquid in the rhomboid channel 1711 of the channel layer 1, cells will not leak into the channel layer 1. The other three chip units are used in the same way as the first chamber unit; after injecting the bio-hydrogel, they are placed in an incubator for 15 minutes to allow the hydrogel to solidify.

[0238] Remove the chip from the incubator and inject 15 μL of laminin into injection well 15. The laminin will flow from injection well 15 through culture medium channel 17 to outlet well 16 at the other end. Remove the 10 μL pipette tip used for placement, and then place the chip in the cell culture incubator for 15 min to allow the laminin to modify the cell culture chamber.

[0239] After the laminin fully infiltrates the cell culture chamber, remove the chip from the cell culture incubator and inject a small amount of EGM-2 culture medium into outlet well 16. Observe whether the culture medium flows rapidly through the cell culture chamber and emerges from the opposite inlet well 15. After the culture medium emerges, use a peristaltic pump to pump the culture medium from inlet well 15 (flow rate of 1 μL / min) and return it to the cell culture incubator.

[0240] After the vascular network is pre-formed in the first cell culture chamber 21, the second cell culture chamber 22, the third cell culture chamber 23, and the fourth cell culture chamber 24, the cell spheres or organoids that need to be pre-vascularized are placed into the cell culture chambers through the first cell sphere injection hole 113, the second cell sphere injection hole 123, the third cell sphere injection hole 133, and the fourth cell sphere injection hole 143, respectively, for co-culture of vascularized tumor spheres.

[0241] During this period, the metabolites will pass through the porous membrane layer 3 to reach the collection chamber. To obtain the metabolites produced during cell growth, the first collection chamber extraction hole 411, the second collection chamber extraction hole 421, the third collection chamber extraction hole 431, and the fourth collection chamber extraction hole 441 of the metabolite collection layer 4 can be extracted using an injection syringe.

[0242] Example 4: Collection and Detection of Metabolites

[0243] After the laminin culture medium is perfused into channel 17, the reserved 10 μL pipette tip is removed. When angiogenesis occurs within the chip and shows a tendency to connect (approximately around day four), cell spheres / organoids are added for co-culture. Tumor spheres with vascular networks will form approximately three to four days after co-culture. To detect the components of metabolites in the tumor spheres / organoids, 10 μL (200 μL) pipette tips can be inserted into the first collection chamber wells 411, 421, 431, and 441 for analysis. To analyze the effect of drugs on the tumor spheres, the drug can be delivered from the blood vessels to the tumor spheres via perfusion, and 10 μL (200 μL) pipette tips can be inserted into the first collection chamber wells 411, 421, 431, and 441 for analysis. Specific liquid analysis methods include enzyme-linked immunosorbent assay (ELISA) testing.

[0244] Example 5: Preparation of another vascularized organ-on-a-chip

[0245] The rest of this embodiment is the same as that in embodiment 2, except that the metabolite collection layer 4 in embodiment 5 is prepared by soft lithography and the flow channel height is 200 μm.

[0246] Example 6: Another application of vascularized organ-on-a-chip

[0247] The rest of this embodiment is the same as that in Embodiment 3, except that the concentration of injected HUVEC endothelial cells is 8 × 10⁻⁶. 6 / mL, the NHLF concentration in lung fibroblasts was 6×10 6 / mL. After the microvascular network is formed, the cell spheres or organoids that need to be pre-vascularized are placed into the central cell sphere culture chamber through the cell sphere injection well for pre-vascularization culture.

[0248] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A vascularized organ-on-a-chip, characterized in that, The vascularized organ chip comprises, in sequence, a flow channel layer (1), a cell culture layer (2), a porous membrane layer (3), and a metabolite collection layer (4). The flow channel layer (1) has a culture medium flow channel (17) on the side near the cell culture layer, and the culture medium flow channel (17) is connected to the outside through the culture medium hole; The cell culture layer (2) is provided with several cell culture chambers that penetrate the cell culture layer (2); the vascularized organ chip is provided with several hydrogel channels and cell ball injection holes, and the hydrogel channels and cell ball injection holes respectively connect each cell culture chamber to the outside. The porous membrane layer (3) covers at least the bottom of all cell culture chambers; The metabolite collection layer (4) has several collection chambers on the side near the porous membrane layer, and each cell culture chamber has at least one collection chamber below it; the vascularized organ-on-a-chip also has several liquid extraction channels, and each collection chamber is connected to the outside through the liquid extraction channels.

2. The vascularized organ-on-a-chip according to claim 1, characterized in that, The hydrogel channel includes a hydrogel injection channel and a hydrogel discharge channel, and the hydrogel injection channel, cell culture chamber and hydrogel discharge channel form a hydrogel pathway that communicates with the outside world. And / or, the culture medium pore is located in the flow channel layer (1), the culture medium pore includes an injection hole (15) and an outlet hole (16) penetrating the flow channel layer (1), the injection hole (15) and the outlet hole (16) are respectively located at both ends of the culture medium flow channel (17), and the vertical projection of the injection hole (15) and the outlet hole (16) is located outside the cell culture chamber; And / or, the culture medium channel (17) includes a serpentine channel (171), the serpentine channel (171) includes a plurality of alternating curved channels (1712) and rhomboid channels (1711), the vertical projection of the rhomboid channels (1711) is located inside the cell culture chamber, and the vertical projection of the curved channels (1712) is located outside the cell culture chamber; the rhomboid channel (1711) includes a rhomboid outer edge (17111) and a plurality of non-connected rhomboid micropillars (17112), and a pathway for culture medium flow is formed between the rhomboid outer edge (17111) and the rhomboid micropillars (17112); And / or, the vascularized organ-on-a-chip also has a capillary burst valve channel and a chip channel, and the cell culture chamber is also connected to the outside through the capillary burst valve channel.

3. The vascularized organ-on-a-chip according to claim 2, characterized in that, The shortest distance between the outer edge of the rhombus (17111) and the rhombus micropillar (17112) is 20~25μm; the shortest distance between two adjacent rhombus micropillars (17112) is 35~40μm; the width of the curved flow channel (1712) is 0.1~0.15mm; And / or, the culture medium channel (17) further includes straight channels (172) extending outward from both ends of the serpentine channel (171). And / or, the hydrogel injection channel on the vascularized organ chip is composed of the flow channel layer hydrogel injection hole on the flow channel layer (1) and the culture chamber hydrogel injection hole on the cell culture layer (2); And / or, the hydrogel injection channel on the vascularized organ chip is composed of the hydrogel injection hole on the flow channel layer (1) and the hydrogel injection hole on the culture chamber on the cell culture layer (2); And / or, the liquid collection channel on the vascularized organ chip is composed of a liquid collection hole on the flow channel layer (1), a liquid collection hole on the culture chamber on the cell culture layer (2), and a liquid collection hole on the collection chamber on the metabolite collection layer (4); And / or, the capillary burst valve channel on the vascularized organ chip is composed of the flow channel capillary burst valve hole on the flow channel layer (1) and the culture chamber capillary burst valve hole on the cell culture layer (2); And / or, the chip channel on the vascularized organ chip is composed of flow channel through holes on the flow channel layer (1), culture chamber through holes on the cell culture layer (2), and collection chamber through holes on the metabolite collection layer (4).

4. The vascularized organ-on-a-chip according to claims 1-3, characterized in that, The vascularized organ-on-a-chip has one or more of the following features: 1) The flow channel layer (1) includes a culture medium flow channel (17), a liquid injection hole (15), a liquid outlet hole (16), and a plurality of flow channel layer pore groups. Each flow channel layer pore group includes a flow channel layer hydrogel injection hole, a cell ball injection hole, a flow channel layer hydrogel outlet hole, a flow channel layer liquid extraction hole, a flow channel layer capillary burst valve hole, and a flow channel layer through hole. 2) The cell culture layer (2) includes several cell culture chambers and cell culture layer channel groups. Each cell culture layer channel group includes a culture chamber hydrogel injection hole, a culture chamber hydrogel outlet hole, a culture chamber liquid extraction hole, a culture chamber capillary burst valve, a culture chamber capillary burst valve hole, and a culture chamber through hole. 3) The porous membrane of the porous membrane layer (3) is selected from polycarbonate membrane, polydimethylsiloxane membrane, and polyethylene terephthalate membrane; 4) The metabolite collection layer (4) includes several collection chambers and metabolite collection channel groups. Each metabolite collection channel group includes a collection chamber liquid extraction hole, a collection chamber through hole and a collection chamber flow channel. The collection chamber liquid extraction hole and the collection chamber through hole are respectively connected to the collection chamber through the collection chamber flow channel.

5. The vascularized organ-on-a-chip according to claim 4, characterized in that, The vascularized organ-on-a-chip also has one or more of the following features: 11) The hydrogel injection hole, cell ball injection hole, hydrogel discharge hole, liquid extraction hole, capillary burst valve hole, and through hole of the flow channel layer all penetrate the flow channel layer (1). 12) The position of the capillary burst valve hole in the flow channel layer corresponds to the position of the capillary burst valve hole in the culture chamber; 13) The position of the hydrogel injection hole in the flow channel layer corresponds to the position of the hydrogel injection hole in the culture chamber; 14) The vertical projection of the cell ball injection hole is located within the cell culture chamber; 15) The position of the hydrogel injection hole in the flow channel layer corresponds to the position of the hydrogel injection hole in the culture chamber; 16) The position of the liquid intake hole in the flow channel layer corresponds to the position of the liquid intake hole in the culture chamber and the liquid intake hole in the collection chamber; 17) The positions of the through holes in the flow channel layer correspond to the positions of the through holes in the culture chamber and the through holes in the collection chamber; 18) The thickness of the flow channel layer (1) is 0.5~0.6mm, the length is 55~60mm, and the width is 24~26mm; the height of the culture medium flow channel (17) is 100~200μm; 19) The diameter of the hydrogel injection hole, cell ball injection hole, hydrogel injection hole, liquid extraction hole, capillary burst valve hole, through hole, injection hole (15), and liquid outlet hole (16) of the flow channel layer is 1~1.5mm; 21) The liquid extraction hole and the through hole of the culture chamber penetrate the cell culture layer (2); 22) The hydrogel injection hole and hydrogel discharge hole of the culture chamber are respectively connected to both ends of the cell culture chamber, and the lateral width of the hydrogel injection hole and / or hydrogel discharge hole of the culture chamber decreases from away from the cell culture chamber to near the cell culture chamber. 23) The capillary burst valve of the culture chamber is connected to the connection between the hydrogel injection hole of the culture chamber and the cell culture chamber; 24) The valve port width of the capillary burst valve in the culture chamber is greater than the shortest distance between two adjacent rhomboid micropillars (17112); 25) The cell culture layer (2) has a thickness of 0.4~0.5 mm, a length of 55~60 mm, and a width of 24~26 mm; 26) The height of the hydrogel injection hole, hydrogel discharge hole, capillary burst valve, and capillary burst valve hole in the culture chamber is 1~1.5mm. 27) The maximum lateral width of the hydrogel injection hole and hydrogel discharge hole in the culture chamber is 1~1.5mm, and the diameter of the liquid extraction hole, capillary burst valve hole, and through hole in the culture chamber is 1~1.5mm. 28) The cell culture chamber is rectangular or truncated rectangular, and the length of the cell culture chamber is 7~8mm and the width is 1.8~2.0mm; 29) The pipe width of the capillary rupture valve in the culture chamber is 0.25~0.35mm; 31) The length of the porous membrane layer (3) is 25~35mm, the width is 8~10mm, the pore size of the porous membrane is 5.0~20.0μm, the thickness is 30~40μm, and the porosity is 10%~15%; 41) The thickness of the metabolite collection layer (4) is 0.6~0.8 mm, the length is 55~60 mm, and the width is 24~26 mm; 42) The height of the liquid collection hole, the through hole of the collection chamber, and the flow channel of the collection chamber is 0.2~0.5mm; 43) The length of the collection chamber is 6.4~7.4mm, and the width is 3~4mm; 44) The width of the flow channel in the collection chamber is 0.1~0.2mm; 45) The diameter of the liquid collection hole and the through hole of the collection chamber is 1~1.5mm.

6. The vascularized organ-on-a-chip according to any one of claims 1 to 5, characterized in that, The materials of the flow channel layer (1), cell culture layer (2), and metabolite collection layer (4) are high molecular polymers. The flow channel layer (1) and the cell culture layer (2) are connected by bonding, and the cell culture layer (2) and the metabolite collection layer (4) are connected by bonding. Preferably, the high molecular polymer includes polydimethylsiloxane. The bonding is selected from hot-press bonding, solvent-assisted bonding, adhesive bonding, plasma-activated bonding, surface-modified grafting bonding, copolymer interface layer bonding, nanoimprint bonding, ultrasonic bonding, and enzyme-catalyzed bonding.

7. The method for preparing a vascularized organ-on-a-chip according to any one of claims 1 to 6, characterized in that, Includes the following steps: 1) Prepare a mask for the microchannel structure pattern of each layer; 2) Prepare a positive mold for the microchannel structure of each layer using soft photolithography; 3) Pour the polymer into the positive mold of the microchannel structure of each layer, and solidify and demold to obtain each layer with microchannel structure; 4) Drill holes in the channel layer (1) and the cell culture layer (2); 5) Bond each layer sequentially to obtain the vascularized organ chip.

8. A method for vascularized co-culture of cells or organoids, comprising culturing cells or organoids using a vascularized organ microarray as described in any one of claims 1 to 6.

9. The method for vascularized co-culture according to claim 8, characterized in that, The cultivation method comprises the following steps: S1) The cell stock solution is mixed with thrombin to obtain a bio-hydrogel, wherein the cell stock solution includes endothelial cells and fibroblasts; S2) Insert the spacer into the cell ball injection well, continuously inject the bio-hydrogel from the channel layer hydrogel injection well until the bio-hydrogel flows out from the channel layer hydrogel discharge well, and then culture. S3) Continuously inject laminin from the injection hole (15) until the laminin flows out from the outlet hole (16), remove the occupant, and culture; S4) Inject culture medium through injection hole (15) and incubate; S5) Inject cell spheres or organoids through the cell sphere injection well and culture them.

10. The method for vascularized co-culture according to claim 9, characterized in that, The co-culture method also has one or more of the following characteristics: In step S1), the volume ratio of cell stock solution to thrombin is 8-12:

1. In step S1), the endothelial cell concentration in the bio-hydrogel is 7 × 10⁻⁶. 6 ~9×10 6 / mL; S13) In step S1), the concentration of fibroblasts in the bio-hydrogel is 6 × 10⁻⁶. 6 ~8×10 6 / mL; S14) In step S1), the volume of the cell stock solution is 10~15 μL / each cell culture chamber; In step S2), the volume of the bio-hydrogel is 10-15 μL; In step S3), the amount of laminin used is 10-15 μL; In step S4), the flow rate of the culture medium is 1~1.5 μL / min.

11. A method for collecting metabolites from vascularized co-culture of cells or organoids, comprising using the vascularized co-culture method as described in any one of claims 8 to 10, and collecting the metabolites from the sampling well.

12. The application of the vascularized organ-on-a-chip as described in any one of claims 1 to 6, or the preparation method as described in claim 7, or the vascularized co-culture method as described in claims 8 to 10, or the collection method as described in claim 11, in drug development, and / or drug testing, and / or drug screening, and / or drug metabolism research.

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