Vasculature organoid chip based on stamp microstructure patterning and preparation method thereof

Abstract

This invention provides a vascularized organoid chip based on stamp microstructure patterning and its fabrication method. The chip includes: a microchannel layer with interconnected gel perfusion channels, a central tissue chamber, and two culture medium channels on its upper surface, wherein tumor spheroids or colon cells are seeded in the central tissue chamber; a reservoir layer located on the upper surface of the microchannel layer, corresponding to the inlet and outlet positions of the gel perfusion channels, the central tissue chamber, and the culture medium channels, wherein the reservoir layer has multiple reservoirs and multiple through holes, the reservoirs containing culture medium to provide nutrients for the cultured tissue; and a stamp module located above the central tissue chamber, the stamp module having a tumor stamp and a colon stamp, each with a three-dimensional microstructure module, used to simulate the culture environment of tumor spheroids or colon cells respectively. This invention enables the in vitro construction of vascularized tumor models and colon models, providing a guide for constructing vascularized organoid models with specific organ three-dimensional microstructure characteristics.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a vascularized organoid chip based on stamp microstructure patterning and its preparation method. Background Technology

[0002] Cancer is a leading cause of death worldwide, and the scientific community is working tirelessly to find effective solutions. With the growing market for anti-cancer drug development, developing more advanced drug screening systems has become a current research focus, aiming to bridge the gap between preclinical research and actual clinical trials. Microfluidic organ-on-a-chip, as an emerging in vitro research tool, has shown great potential in the life sciences and engineering fields. This platform enables the construction of physiological models of organs within tiny chips, thus providing a completely new research approach for cancer research.

[0003] In the tumor microenvironment, blood vessels play a crucial and indispensable role in the delivery of anticancer drugs. After formation, blood vessels are randomly recruited to the tumor site, where they become distorted or leaky due to the influence of the tumor microenvironment, thus providing the tumor tissue with the necessary nutrients and oxygen to meet its metabolic needs. However, these leaky vessels trigger enhanced permeability and retention effects, leading to increased interstitial fluid pressure and hindering the smooth delivery of drug molecules across the endothelial barrier. Therefore, there is an urgent need for a more physiologically representative in vitro cancer model that encompasses the tumor stroma to effectively reflect the complex tumor microenvironment (TME) and thus better facilitate microfluidic drug screening. Joonha Park et al., in their paper "Enabling perfusion through multicellular tumor spheroids promoting lumenization in a vascularized cancer model" published in Lab on a chip, 2022, 22, 4335, successfully constructed a perfusionable tumor vascular model using a tumor microenvironment reconstruction method and applied it to a novel drug screening platform. However, they co-perfused tumor spheres with endothelial cells into a microfluidic chip, which is inconsistent with the order of real tumor growth. In the natural environment, vascular networks typically develop before tumor formation to provide essential nutrients and oxygen. Therefore, to more accurately simulate the complex microenvironment within the body, a vascular network should be established first, followed by the introduction of tumor cells.

[0004] The inner wall of the small intestine is composed of densely packed crypt-villi units. Villi are finger-like projections, and crypts are located in depressions at the base of the villi. Normal small intestinal function depends on the continuous renewal of epithelial cells, which is achieved through the migration and differentiation of intestinal stem cells along the crypt-villi axis. However, traditional colonic barrier models, typically two-dimensional monolayers formed by culturing human colorectal cancer cell lines (Caco-2) on standard Transwell inserts, cannot accurately simulate the three-dimensional microenvironment of the small intestine. Therefore, developing in vitro intestinal models with more complex colonic microstructures is crucial for research that more closely approximates physiological conditions.

[0005] Existing traditional 3D tissue culture models are complex and inefficient in simulating the in vivo microenvironment, often requiring redesigned chips and platforms. Furthermore, vascular tumor models are typically constructed using single or co-culture methods, while colon models are mostly constructed using static culture. These traditional methods have numerous limitations, including insufficient flexibility in model construction and poor reproducibility of the real microenvironment, restricting their widespread application.

[0006] Therefore, there is an urgent need to develop a vascularized organoid chip that can be flexibly applied to various in vitro models, so as to overcome the shortcomings of existing three-dimensional tissue culture methods, including but not limited to tumor models and colon models. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a vascularized organoid chip based on stamp microstructure patterning and its preparation method, thereby enhancing the ability to simulate the microenvironment and meeting the needs of three-dimensional tissue culture.

[0008] According to one aspect of the present invention, a vascularized organoid chip based on stamp microstructure patterning is provided, comprising:

[0009] The microchannel layer has a gel perfusion channel, a central tissue chamber and a culture medium flow channel on its upper surface. The central tissue chamber is connected to the gel perfusion channel and the culture medium flow channel. One or more tissues or tissue cells are inoculated in the central tissue chamber.

[0010] A reservoir layer is provided on the upper surface of the microchannel layer and corresponds to the inlet and outlet of the gel perfusion channel, the central tissue chamber and the inlet and outlet of the culture medium flow channel. The reservoir layer is provided with multiple reservoirs and multiple through holes. The reservoir contains culture medium to provide nutrients for the cultured tissues or tissue cells.

[0011] The stamp module is located above the central tissue chamber. The stamp module is provided with one or more tissue or tissue cell stamps. Each tissue or tissue cell stamp is provided with a microstructure three-dimensional module corresponding to the tissue or tissue cell, which is used to simulate the culture environment of the tissue or tissue cell.

[0012] Optionally, the culture medium channel is connected and communicates with the central tissue chamber through a phase guiding structure, and the culture medium channel is located on both sides of the gel perfusion channel.

[0013] Optionally, the liquid storage layer and the microchannel layer are made of PDMS (polydimethylsiloxane) and bonded together by oxygen plasma.

[0014] Optionally, the plurality of through holes includes a first through hole and a second through hole for gel infusion, wherein the first through hole and the second through hole correspond to and communicate with the inlet and outlet of the gel infusion channel;

[0015] The liquid storage layer includes multiple first liquid storage pools and one second liquid storage pool. The position of the second liquid storage pool corresponds to the central tissue chamber. The tissue or tissue cells are inoculated into the central tissue chamber through the second liquid storage pool. The multiple first liquid storage pools correspond to the inlet and outlet positions of the culture medium flow channel through multiple liquid storage pool through-holes. The culture medium in the first liquid storage pools flows into the culture medium flow channel through the liquid storage pool through-holes.

[0016] The liquid storage tank layer is also provided with a stamp module matching hole. The stamp module is provided with a stamp matching module. The stamp matching module matches with the stamp module matching hole through the second liquid storage tank and communicates with the central tissue chamber for imprinting.

[0017] Optionally, the three-dimensional tissue is vascular tumor tissue or colonic barrier tissue; the stamp module includes a tumor stamp and a colonic stamp.

[0018] Furthermore, in the tumor stamp, the three-dimensional microstructure module is a tumor microstructure with a cross-section of a chamfered rectangle and a three-dimensional feature of a convex hemisphere. The three-dimensional rounded corners formed by the chamfers can optimize the injection path and fluid distribution of the hydrogel.

[0019] Furthermore, in the tumor stamp, the microstructure three-dimensional module is a tumor microstructure, and the cross-section of the tumor stamp is trapezoidal, which ensures that the tumor microstructure is accurately imprinted onto the center of the central tissue chamber.

[0020] According to another aspect of the present invention, a method for fabricating the above-described microstructure-patterned vascularized organoid chip is provided, the method comprising:

[0021] A microfluidic chip mold with a microchannel layer and a liquid reservoir layer was made by 3D printing resin material; a three-dimensional stamp with a microstructure was also made by 3D printing resin material.

[0022] PDMS material is poured into the mold and cured by molding. The PDMS molding material is then cut off from the mold to obtain a microchannel PDMS layer and a reservoir PDMS layer. Holes are punched at the gel inlet and outlet and the culture medium inlet and outlet corresponding to the reservoir layer.

[0023] The microchannel PDMS layer and the reservoir PDMS layer were bonded together by oxygen plasma bonding and cured. After curing, they were sterilized by high temperature and high pressure to obtain a vascularized organoid chip with a stamp-like microstructure pattern.

[0024] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0025] 1. This invention provides a vascularized organoid chip based on stamp microstructure patterning. By designing a microfluidic chip with a reservoir layer and a microchannel layer, the simulation capability of the microenvironment of various tissues or tissue cell cultures is enhanced. The chip can be used to create dynamic culture models, improving the repeatability and reliability of model construction in microfluidic chips. For example, by combining tumor stamps and colon stamps, a stepwise culture model of tumor vessels and a colonic epithelial barrier model under dynamic culture was successfully created using this organoid chip.

[0026] 2. The method for preparing vascularized organoid chips based on stamp microstructure patterning provided by this invention enhances the ability to simulate the microenvironment by preparing stamps with different microstructures through high-precision 3D printing; it provides a richer microenvironment in cell culture and tissue engineering research, promotes the research and application of cell behavior, and improves the flexibility of designing different organ models.

[0027] In summary, this invention overcomes the problems of insufficient flexibility and poor reproducibility of real microenvironments in existing traditional model construction. It can be flexibly applied to the construction of various in vitro models, providing new ideas and methods for the design of various organ-on-a-chip, and has broad application prospects. Attached Figure Description

[0028] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0029] Figure 1 This is a schematic diagram of a vascularized organoid chip structure based on stamp microstructure patterning provided in an embodiment of the present invention;

[0030] Figure 2 This is a schematic diagram of the microstructure of a seal in one embodiment of the present invention;

[0031] Figure 3 This is a schematic diagram illustrating the usage process of a vascularized organoid chip based on the microstructure patterning of a seal, according to an embodiment of the present invention.

[0032] Figure 4 This is a three-dimensional fluorescence image of a vascularized organoid chip imprint based on a microstructure patterned seal, according to an embodiment of the present invention.

[0033] Figure 5 The above are the characterization results of an in vitro vascular tumor model constructed based on a microstructure patterned vascular organoid chip according to an embodiment of the present invention at different times.

[0034] Figure 6 This is a characterization result of an in vitro model of a colonic model constructed based on a microstructure patterned vascular organoid chip according to an embodiment of the present invention.

[0035] Figure 7 This is a magnified view of different focal planes of an in vitro colon model constructed based on a microstructure patterned vascularized organoid chip according to an embodiment of the present invention.

[0036] The corresponding labels in the figure are as follows: 1 is the reservoir layer, 2 is the microchannel layer, 3 is the stamp module, 4 is the gel perfusion channel, 5 is the culture medium flow channel, 6 is the first reservoir, 7 is the second reservoir, 8 is the first through hole for gel perfusion, 9 is the hole for the stamp matching module, 10 is the through hole for the reservoir, 11 is the second through hole for gel perfusion, 31 is the three-dimensional module of the tumor stamp microstructure, 32 is the tumor stamp matching module, 33 is the three-dimensional module of the colon stamp microstructure, 34 is the stamp reservoir matching module, and 35 is the stamp handle module. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0038] It should be understood that the terms "first," "second," etc., in the following embodiments are used to distinguish different objects, rather than to describe a specific order. The terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or collections thereof.

[0039] It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the invention. All accompanying drawings are merely for illustrative purposes, and the numbers, positions of components, relationships between components, and dimensions of components used in the preferred embodiments do not constitute a limitation on the technical solution itself. In the description of this specification, it should be understood that the terms "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for ease of description and simplification, not to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention.

[0040] Reference Figure 1 As shown, an embodiment of the present invention provides a vascularized organoid chip based on stamp microstructure patterning, comprising a reservoir layer 1, a microchannel layer 2, and a stamp module 3. The reservoir layer 1 is located on the upper surface of the microchannel layer 2. The upper surface of the microchannel layer 2 is provided with a gel perfusion channel 4, a central tissue chamber, and two side culture medium channels 5. The gel perfusion channel 4 is connected to the central tissue chamber, in which one or more tissues or tissue cells are seeded. The two side culture medium channels 5 are connected to the central tissue chamber through a phase guiding structure and deliver nutrients to it. The stamp module 3 with microstructure patterning is located above the central tissue chamber. The stamp module 3 may have one or more tissue or tissue cell stamps, each stamp having a corresponding three-dimensional microstructure module of the tissue or tissue cell, used to simulate the culture environment of each tissue or tissue cell.

[0041] In this embodiment, the liquid storage layer 1 and the microchannel layer 2 are made of PDMS and bonded together by oxygen plasma. The structure of the central tissue chamber and the stamp module 3 can be precisely matched.

[0042] In one embodiment, the liquid storage layer 2 is provided with multiple liquid storage tanks and multiple through holes. The liquid storage tanks contain culture medium to provide nutrients for cultured tissues or tissue cells. Specifically, the multiple through holes include a first through hole 8 and a second through hole 11 for gel perfusion. The first through hole 8 and the second through hole 11 correspond to and communicate with the inlet and outlet of the gel perfusion channel 4.

[0043] Specifically, the reservoir layer 1 includes multiple first reservoirs 6 and one second reservoir 7. The position of the second reservoir 7 corresponds to the central tissue chamber, and tissues or tissue cells are inoculated into the central tissue chamber through the second reservoir 7. The multiple first reservoirs 6 correspond to the inlet and outlet positions of the culture medium flow channels 5 on both sides through multiple reservoir through holes, and the culture medium in the first reservoirs 6 flows into the culture medium flow channels 5 through the reservoir through holes.

[0044] In addition, the liquid storage tank layer is also provided with a stamp module matching hole. The stamp module is provided with a stamp matching module. The stamp matching module matches with the stamp matching module hole 9 through the second liquid storage tank 7 and communicates with the central tissue chamber for imprinting.

[0045] In one embodiment, the two culture medium channels can be culture medium perfusion channels, the gel perfusion channel 4 can be a cell gel perfusion channel, and the culture medium channel 5 can be a tissue culture medium perfusion channel. The culture medium channel 5 is located on both sides of the gel perfusion channel 4, and all three channels are connected to the central tissue chamber. Figure 1 As shown, the culture medium flow channels 5 and gel perfusion channels 4 on both sides can be designed symmetrically with the central tissue chamber as the center.

[0046] In addition, in other embodiments, the stamp module 3 is also equipped with a stamp handle module 35, which is designed for picking up and pressing the stamp with tweezers, further improving the operability and ease of use of the stamp.

[0047] In some embodiments, both the reservoir layer 1 and the microchannel layer 2 are made of polydimethylsiloxane and bonded together by oxygen plasma. Specifically, as an example, the thickness of the microchannel layer 2 can be 1.5-2.5 mm, and the central tissue chamber is designed to be circular to facilitate nutrient exchange with the culture medium channels 5. Due to the need for transferring the three-dimensional microstructure of the stamp, the height of the central tissue chamber is designed to be 300-500 μm, and the radius is 1-1.5 mm, for filling the hydrogel. Its patterned structure design confines cell growth within a restricted area. The height of the central tissue chamber determines the height of the 3D extracellular matrix, i.e., the space in which the three-dimensional capillary network can spontaneously form. A larger central tissue chamber height can form a more complex three-dimensional capillary network. The thickness of the reservoir layer 1 can be 6-10 mm, and the radius of the reservoir is 4-5 mm, thus providing sufficient volume for storing the culture medium. Of course, in other embodiments, other dimensions are also possible, depending on the specific circumstances.

[0048] In some embodiments, the two side culture medium channels 5 are connected to the central tissue chamber via a phase-guided structure. The width of the microchannel layer phase-guided structure is 200-250 μm, and the height is 150-250 μm. Correspondingly, the central tissue chamber is circular in shape, with a height of 300-500 μm and a radius of 1-1.5 mm. Of course, in other embodiments, other dimensions may also be used, depending on the specific circumstances.

[0049] In the above embodiments, the tissue or tissue cell can be any tissue or tissue cell that can be cultured using a culture medium, and the specific culture medium can be selected and determined according to the cultured tissue or tissue cell. Different tissue or tissue cell models are cultured separately and can be constructed using different stamps through the stamp module.

[0050] The above embodiments of the present invention design a microfluidic chip through a reservoir layer and a microchannel layer to enhance the simulation capability of the microenvironment of various tissues or tissue cell cultures. By designing and planning the microstructure of the stamp module 3 and combining it with a matching organoid chip, it is possible to achieve efficient construction of different biological models using a vascularized organoid chip with a patterned stamp microstructure.

[0051] The following example, using the stamp module 3 containing a tumor stamp and a colon stamp, further illustrates the vascularized organoid chip of this application. (Refer to...) Figure 1 and Figure 2 An embodiment of the present invention provides a vascularized organoid chip based on stamp microstructure patterning, comprising a reservoir layer 1, a microchannel layer 2, and a stamp module 3. The reservoir layer 1 is located on the upper surface of the microchannel layer 2. The upper surface of the microchannel layer 2 is provided with a gel perfusion channel 4, a central tissue chamber, and two side culture medium channels 5. The gel perfusion channel 4 connects to the central tissue chamber, in which tumor spheroids or colon cells are seeded. The two side culture medium channels 5 are connected to the central tissue chamber through a phase guiding structure and deliver nutrients to it. The stamp module 3 is located above the central tissue chamber and includes a tumor stamp and a colon stamp. The tumor stamp and colon stamp have different three-dimensional microstructure modules, respectively used to simulate the culture environment of tumor spheroids or colon cells.

[0052] In one embodiment, the reservoir layer 1 is provided with a first gel perforation hole 8 and a second gel perforation hole 11, which correspond to the inlet and outlet of the gel perforation channel 4, respectively, to facilitate the perfusion of cell gel into the central tissue chamber of the microchannel layer 2 through the reservoir layer 1. Furthermore, the reservoir layer 1 is also provided with multiple first reservoirs 6 and one second reservoir 7, wherein the position of the second reservoir 7 corresponds to the central tissue chamber and is also connected to the central tissue chamber through the tumor stamp matching module hole 9, facilitating the flow of culture medium in the second reservoir 7 into the central tissue chamber under gravity, thus delivering nutrients and nourishing substances. Tumor spheroids or colon cells are inoculated into the central tissue chamber of the microchannel layer 2 through the second reservoir 7. The multiple first reservoirs 6 correspond to the inlet and outlet positions of the culture medium flow channels 5 on both sides through multiple reservoir holes 10, facilitating the flow of culture medium in the first reservoirs 6 into the culture medium flow channels 5 in the microchannel layer 2 through the reservoir holes 10.

[0053] In this embodiment, the stamp module 3 is located above the central tissue chamber and corresponds precisely to the central tissue chamber, that is, the stamp module 3 and the central tissue chamber are the same size and correspond in position.

[0054] Specifically, the tumor stamp includes a microstructure three-dimensional module, namely the tumor stamp microstructure three-dimensional module 31. This module provides a convex hemispherical three-dimensional structure, which can be transferred into the hydrogel, thereby forming a concave hemispherical microstructure in the hydrogel for tumor spheres to be inoculated. The cross-section of this microstructure is a chamfered rectangle, and the three-dimensional rounded corner design formed by the chamfer effectively optimizes the injection path and fluid distribution of the hydrogel, ensuring that cells penetrate the hydrogel more uniformly.

[0055] The reservoir layer 1 also includes multiple matching holes, including matching holes 9 for the tumor stamp module and matching holes for the colon stamp module. The tumor stamp includes a tumor stamp matching module 32, which is used for precise imprinting. The cross-section of the tumor stamp is designed as a trapezoid, and its three-dimensional structure is a trapezoidal rotating body. It matches with the matching hole 9 of the tumor stamp module, thereby ensuring that the tumor microstructure can be accurately imprinted to the center of the central tissue chamber.

[0056] In this embodiment, the colonic imprint also includes a microstructure three-dimensional module, namely the colonic microstructure three-dimensional module 33. The cross-section of this module is a trapezoidal array spaced 100-200 μm apart, while the longitudinal section consists of 9 to 36 circular arrays spaced 200-600 μm apart. This three-dimensional structure (colonic microstructure three-dimensional module) is transferred into the hydrogel to simulate the microstructure of the colonic crypts, thereby constructing a biomimetic microenvironment.

[0057] The stamp module 3 also includes a stamp liquid reservoir matching module 34. The size of the stamp liquid reservoir matching module 34 is slightly smaller than that of the second liquid reservoir 7, which facilitates precise calibration during stamping and ensures the smooth discharge of excess gas during the stamping process. Specifically, the second liquid reservoir 7 and the stamp liquid reservoir matching module 34 are vertically aligned (in use). The smaller size of the stamp liquid reservoir matching module 34 allows for smooth stamping and also facilitates the discharge of excess gas.

[0058] The vascularized organoid chip based on microstructure patterning of stamps in the above embodiments of the present invention, combined with tumor stamps and colon stamps, can be used to create a tumor vascular model and a colonic epithelial barrier model under dynamic culture and stepwise culture.

[0059] In another embodiment of the present invention, a method for preparing the above-mentioned microstructure patterned vascularized organoid chip is provided, the method comprising:

[0060] S100: A microfluidic chip mold with a microchannel layer and a reservoir layer is made using 3D printing resin material; a three-dimensional stamp with a microstructure is made using 3D printing resin material.

[0061] S200. Pour PDMS material into the microfluidic chip mold, perform mold curing to obtain PDMS mold material, cut the PDMS mold material from the microfluidic chip mold to obtain a microchannel layer and a reservoir PDMS layer; punch holes in the gel inlet and outlet and culture medium inlet corresponding to the reservoir PDMS layer;

[0062] S300: Two layers of PDMS are bonded together by oxygen plasma bonding and cured; after curing, they are sterilized by high temperature and high pressure to obtain a vascularized organoid chip with a stamp-like microstructure pattern.

[0063] In one specific embodiment, the vascularized organoid chip with microstructure patterned stamp is prepared by using resin material with a printing accuracy of 0.025 mm through 3D printing to prepare stamp modules, microchannel layer microfluidic chip molds, and reservoir layers with different microstructures. The microstructure is then molded using PDMS material with high biocompatibility, thereby obtaining a microchannel PDMS layer and a reservoir PDMS layer with microstructures.

[0064] Specifically, you can achieve this by following these steps:

[0065] S1. Using AutoCAD and Solidworks tools, a microchannel layer, a reservoir layer, and a stamp module of a vascularized organoid chip with a microstructure patterned stamp were designed.

[0066] S2. A microfluidic chip mold with a microchannel layer and a reservoir layer, as well as a tumor stamp module and a colon stamp module, were fabricated using high-precision 3D printing resin material. The printed resin modules were immersed in deionized water and ultrasonically cleaned; after cleaning and drying, they were dried overnight in an oven at 60-70℃ and then placed under a UV lamp for 10 minutes, which facilitated the demolding of PDMS from the resin mold.

[0067] S3. Mix the PDMS prepolymer and curing agent evenly at a mass ratio of 10:1. Vacuum the PDMS in a vacuum pot to remove air bubbles. Pour the bubble-free PDMS material into the microfluidic chip mold of the microchannel layer and the reservoir layer. During this process, avoid generating air bubbles. Then transfer it to an oven at 70-80℃ for curing for 4-8 hours.

[0068] S4. Cut the PDMS molding material from the resin mold to obtain the microchannel PDMS layer and the reservoir PDMS layer. Make holes in the gel inlet and outlet and the culture medium inlet and outlet corresponding to the PDMS reservoir PDMS layer. Specifically, the diameter of the hole in the gel inlet and outlet is 1 mm, and the diameter of the hole in the culture medium inlet and outlet is 2 mm. After the hole is made, seal it with a disposable isolation blue film.

[0069] S5. Using a glass slide as a carrier, the microchannel PDMS layer with the microstructure side facing up is attached to the glass slide surface, and the PDMS reservoir layer with the corresponding perforated side facing up is attached to the glass slide surface. The slide is then placed in a plasma device for processing, and ignition is performed at 0.7 mbar for 45 seconds. The processed microchannel PDMS layer and reservoir PDMS layer are then bonded together, and air bubbles between the PDMS layers are gently pressed out with tweezers. The bonded organ-on-a-chip is then placed on a 120°C hot plate and heated for 2 hours to enhance the bonding effect.

[0070] In this step, after the microchannel layer and the PDMS reservoir layer are bonded together, the first reservoir is connected to the culture medium channel of the microchannel layer through the reservoir through-hole; the second reservoir is connected to the central chamber of the microchannel layer through the stamp matching hole; the first and second gel channels are connected to the inlet and outlet of the gel perfusion channel of the microchannel layer. Other areas are not connected. The stamp modules (tumor stamp module and colon stamp module) exist independently, are stamped before tissue culture, and are removed afterwards.

[0071] S6. After curing, the sample is transferred to an autoclave for high-temperature and high-pressure sterilization to obtain a vascularized organoid chip with a microstructure patterned from a stamp.

[0072] The above-described preparation method of the present invention can enhance the ability to simulate the microenvironment by preparing stamps with different microstructures through high-precision 3D printing, providing a richer microenvironment in cell culture and tissue engineering research, and improving the flexibility of designing different organ models.

[0073] The vascularized organoid chip with microstructure patterning of the stamp in the above embodiments of the present invention can be used for the construction of various in vitro models. For better illustration, specific instructions for using the vascularized organoid chip for in vitro model construction are provided.

[0074] refer to Figure 3 , Figure 3 The left column shows a top view of the organ-on-a-chip, and the right column shows a cross-sectional view of the organ-on-a-chip. They are presented in pairs from top to bottom. Taking the construction of a vascularized tumor model as an example, the main process is as follows:

[0075] S1. The prepared microstructure patterned vascularized organoid chip is sterilized, and the tumor stamp module is soaked in cell anti-adhesion solution and then air-dried. This helps the stamp not stick to the cell gel during the demolding step.

[0076] In this step, sterilization is performed by irradiation with ultraviolet light for 2 hours, followed by soaking the tumor stamp module in a cell anti-adhesion solution for 2 hours and then air drying. Of course, in other embodiments, other times may be used, and it is not limited to this parameter.

[0077] S2. Place the semi-circular embossed stamp above the central tissue chamber. Then, place the gel containing human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (NHLF), as well as the cell-free gel, on crushed ice. Aspirate the fibrin gel containing cells and quickly mix it with thrombin several times to ensure there are no air bubbles. Then, insert the pipette tip into the gel inlet of the stamp chip and gently pour the gel. Observe that the gel will spontaneously fill the entire central tissue chamber under capillary action. Release the pipette, being careful not to apply too much pressure during pouring.

[0078] In this step, the inoculation concentrations of HUVEC and NHLF can be 7 × 10⁻⁶. 6 Cells / ml. Aspirate 6 μl of fibrin gel containing cells and quickly mix it with 2 μl of thrombin 2-3 times, then check for air bubbles.

[0079] S3. After the gel in the central circular tissue chamber of the microchannel layer has completely polymerized and solidified, use a pipette to add EGM-2 culture medium to one side of the reservoir. Then add a small amount of EGM-2 to the opposite side of the reservoir to cover the bottom of the reservoir and create a liquid level difference. This will generate a fluid pressure difference in the culture medium channels on both sides of the hydrogel, allowing nutrients to be transported into the central tissue chamber. Place the cell in a CO2 cell culture incubator for incubation. In the central tissue chamber, HUVECs form vascular fragments and initially connect under the induction of growth factors produced by NHLF.

[0080] In this step, use a pipette to add 300-800 μl of EGM-2 culture medium to one side of the reservoir. Then, add a small amount of EGM-2 to the opposite reservoir to cover the bottom, creating a liquid level difference of about 4-6 mm. Incubate in a CO2 cell culture incubator for 1 day, or any other time depending on the specific conditions.

[0081] S4. On the second day, the tumor stamp was removed, and the cell-free fibrin gel was aspirated into a sterile culture dish. The cultured mature cell spheres were slowly aspirated into the gel in the culture dish using a dispensing needle. The cell spheres were obtained by mixing human non-small cell lung cancer cells (A549) with NHLF in a 1:1 ratio and culturing them in an ultra-low adsorption plate. Then, a new dispensing needle was used to pick up the cell spheres and gel together and gently add them into the central well. The cell spheres slowly sank into the surface of the microstructured hydrogel under their own gravity.

[0082] In this step, 100 μl of cell-free fibrin gel can be aspirated into a sterile culture dish.

[0083] S5. After the hydrogel containing tumor cell spheres added above the central circular tissue chamber has polymerized and solidified, use a pipette to add EGM-2 culture medium to the central reservoir. This allows the culture medium to apply pressure to the hydrogel from above, which helps promote the growth of tumors and vascular networks. After adding the culture medium, place the chip device in a glass dish, pour a small amount of sterile water into a sterile centrifuge tube cap, and place it in the pipette tip box to maintain humidity for vascular culture. Observe the changes in the culture medium level in the reservoir periodically, and replace the culture medium as needed, adjusting the direction of the pressure difference.

[0084] In this step, use a pipette to add 200-500 μl of EGM-2 culture medium to the central reservoir. A 10-15 mm glass dish can be used. Observe the change in the culture medium level in the reservoir every 24 hours.

[0085] In the above embodiments of the present invention, referring to steps S1-S5, vascularized tumor models can be constructed using vascularized organoid chips. Other tumor models can also be implemented similarly.

[0086] The following describes the actual culture effect of the vascularized organoid chip with microstructure patterned stamps in the above embodiments to demonstrate the feasibility of using it for in vitro culture of vascular tumor models and colon models and its preparation process.

[0087] refer to Figure 4 As shown, 65 kDa FITC-Dextran, labeled with green fluorescence, was mixed with a hydrogel. Then, 6 μl of fibrin gel solution containing dextran was thoroughly mixed with thrombin and injected into an organoid microarray with a tumor stamp. Within the microarray, the hydrogel constructed a three-dimensional microstructure under the imprinting of the tumor stamp, and the microarray was then placed in a CO2 incubator for 15 minutes for gel solidification. After solidification, observation of the three-dimensional XZ and YZ interfaces clearly showed that the tumor stamp microstructure had been successfully transferred into the hydrogel, forming a precise three-dimensional structure. Furthermore, the guiding structures on both sides of the tumor stamp were also clearly visible at the YZ interface. These guiding structures ensure that the culture medium exchanges substances with the hydrogel through specific phase-guided interfaces, thus providing an effective channel for nutrient supply and waste metabolism in cells. This experimental procedure not only successfully constructed a microscopic 3D structure but also verified the precise transfer effect of the stamping technology in hydrogels, laying the foundation for subsequent cell culture and model studies.

[0088] Reference Figure 5As shown, a vascularized organoid microarray with microstructure patterning successfully constructed a vascular tumor model after seeding human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (NHLF), and tumor spheres, and acquired bright-field and fluorescence images at different growth days. During culture, HUVECs and NHLFs self-assembled to form a capillary network. On the second day, after injecting a cell-mixed fibrin gel into the central tissue chamber, HUVECs initially formed vascular fragments under the induction of growth factors and began to connect with each other. By the third day, the cells further organized, initially forming a three-dimensional capillary network; and by the seventh day of culture, the fluorescence image of the blood vessels in the central tissue chamber showed that relatively thicker and longer vascular lumens had formed. Figure 5 The dashed lines in the fluorescence image below correspond to the locations of tumor spheres introduced on the second day. Agua Sobrino et al., in their paper "3D microtumors invitro supported by perfused vascular networks" published in Scientific Reports, 2016, 6, 31589, suggest that the perfusionable vascular network surrounding the tumor facilitates nutrient and oxygen supply. Furthermore, the fibroblasts within the tumor spheres help maintain their shape and sphericity. Analysis of phase-contrast images and manual detection of diameter changes revealed that the tumor sphere diameter increased from approximately 540 μm on day three to approximately 780 μm on day seven, showing a significant growth trend while maintaining its initial sphericity throughout the culture process.

[0089] Reference Figure 6 , 7 As shown, a cell-free fibrin hydrogel was first used to fill the bottom microchannel layer as the base of the colon model. After transferring the crypt microstructure pattern from the colonic stamp to the hydrogel, high-concentration human colon adenocarcinoma cells (Caco-2) were seeded onto the solidified hydrogel through a central reservoir. During this process, the culture medium in the reservoir was connected to the culture medium in the microchannel layer through the Caco-2 cells via the hydrogel, achieving efficient material transfer. A dynamic culture environment was formed due to the difference in liquid levels between the outer and central culture media. Using fibrinogen hydrogel as the base facilitated cell adhesion, proliferation, and differentiation. During culture, Caco-2 cells differentiated into features similar to small intestinal epithelial cells, such as microvilli and tight junctions. Once the cells merged, they spontaneously differentiated into mature intestinal brush border cells, forming an endothelial barrier. Figure 6 As shown in the figure. Since the intestinal crypt model is a three-dimensional structure, this embodiment of the invention also characterized the bottom, middle, and barrier surface of the intestinal crypts accordingly. Figure 7The images show magnified bright-field microscopy images of the colonic barrier at different focal planes. The images clearly show that Caco-2 cells are evenly distributed and completely covered throughout the microstructure, indicating that the complete endothelial barrier has been initially constructed.

[0090] The above embodiments of the present invention describe a vascularized organoid chip based on stamp microstructure patterning and its fabrication method. By designing a microfluidic chip with a reservoir layer and a microchannel layer, and combining tumor and colon stamps, this organoid chip successfully created a dynamically cultured, stepwise cultured tumor vascular model and a colonic epithelial barrier model. The organoid chip of the present invention improves the reproducibility and reliability of biological model construction; the high-precision 3D printing of stamps with different microstructures provides a richer microenvironment in cell culture and tissue engineering research, promotes the study and application of cell behavior, increases the flexibility of designing different organ models, provides guidance for constructing vascularized organoid models with specific organ three-dimensional microstructural characteristics, and provides a reliable testing platform in the early stages of drug development.

[0091] The above embodiments use tumor vascular models and colonic epithelial barrier models as examples. The chip described in this invention can also be applied to the construction of other tissue or tissue cell models, and is not limited to tumor spheroids or colonic cells. The preferred features in the above embodiments can be used individually in any embodiment, or in any combination thereof, provided they do not conflict with each other. Furthermore, parts not described in detail in the embodiments can be implemented using existing technologies.

[0092] The specific embodiments of the present invention have been described in detail above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations without departing from the scope of the claims, which does not affect the essence of the present invention. Furthermore, the above-described preferred features can be used in any combination without conflict.

Claims

1. A vascularized organoid chip based on stamp microstructure patterning, characterized in that, include: The microchannel layer has a gel perfusion channel, a central tissue chamber and a culture medium flow channel on its upper surface. The central tissue chamber is connected to the gel perfusion channel and the culture medium flow channel. One or more tissues or tissue cells are inoculated in the central tissue chamber. A reservoir layer is provided on the upper surface of the microchannel layer and corresponds to the inlet and outlet of the gel perfusion channel, the central tissue chamber and the inlet and outlet of the culture medium flow channel. The reservoir layer is provided with multiple reservoirs and multiple through holes. The reservoir contains culture medium to provide nutrients for the cultured tissues or tissue cells. A stamp module is located above the central tissue chamber. The stamp module is provided with one or more tissue or tissue cell stamps. Each tissue or tissue cell stamp is provided with a microstructure three-dimensional module corresponding to the tissue or tissue cell, which is used to simulate the culture environment of the tissue or tissue cell. The three-dimensional tissue consists of vascular tumor tissue and colonic barrier tissue; the stamp module includes a tumor stamp and a colonic stamp. The tumor stamp includes a three-dimensional microstructure module, wherein the three-dimensional microstructure module is a tumor microstructure. The longitudinal section of the tumor microstructure is a rectangle with chamfers, and the three-dimensional feature is a convex hemisphere. The three-dimensional rounded corners formed by the chamfers can optimize the injection path and fluid distribution of the hydrogel. The tumor stamp also includes a tumor stamp matching module, wherein the longitudinal section of the tumor stamp matching module is trapezoidal and its three-dimensional structure is a trapezoidal body of revolution. The trapezoidal shape can ensure that the tumor microstructure is accurately imprinted onto the center of the central tissue chamber.

2. The vascularized organoid chip with microstructured stamp patterning according to claim 1, characterized in that, The culture medium channel is connected and communicates with the central tissue chamber through a phase guiding structure, and the culture medium channel is located on both sides of the gel perfusion channel.

3. The vascularized organoid chip with microstructured stamp patterning according to claim 2, characterized in that, The liquid storage layer and the microchannel layer are made of PDMS and bonded together by oxygen plasma.

4. The vascularized organoid chip with microstructured stamp patterning according to claim 1, characterized in that, The plurality of through holes includes a first through hole and a second through hole for gel infusion, wherein the first through hole and the second through hole correspond to and communicate with the inlet and outlet of the gel infusion channel; The liquid storage layer includes multiple first liquid storage pools and one second liquid storage pool. The position of the second liquid storage pool corresponds to the central tissue chamber. The tissue or tissue cells are seeded into the central tissue chamber through the second liquid storage pool. Multiple first liquid storage tanks correspond to the inlet and outlet positions of the culture medium flow channel through multiple liquid storage tank through holes, and the culture medium in the first liquid storage tanks flows into the culture medium flow channel through the liquid storage tank through holes; The liquid storage tank layer is also provided with a stamp module matching hole. The stamp module is provided with a stamp matching module. The stamp matching module matches with the stamp module matching hole through the second liquid storage tank and communicates with the central tissue chamber for imprinting.

5. The vascularized organoid chip with microstructured stamp patterning according to claim 1, characterized in that, The colonic stamp, wherein the microstructure three-dimensional module is a colonic microstructure, the longitudinal section of which is a trapezoidal array spaced 100-200μm apart; the cross section of which is a circular array of 9-36 units spaced 200-600μm apart.

6. The microstructure patterned vascularized organoid chip according to any one of claims 1-5, characterized in that, The stamp module also includes a stamp matching module and a stamp handle module. The height of the stamp handle module is 3-5mm. The stamp matching module is used for precise matching of the stamp, and the stamp handle module is used for gripping and pressing operations.

7. A method for preparing a vascularized organoid chip with a microstructure patterned from a stamp as described in any one of claims 1-6, characterized in that, include: A microfluidic chip mold with a microchannel layer and a liquid reservoir layer was made by 3D printing resin material; a three-dimensional stamp with a microstructure was also made by 3D printing resin material. PDMS material is poured into the mold and cured by molding. The PDMS molding material is then cut off from the mold to obtain a microchannel PDMS layer and a reservoir PDMS layer. Holes are punched at the gel inlet and outlet and the culture medium inlet and outlet corresponding to the reservoir layer. The microchannel PDMS layer and the reservoir PDMS layer were bonded together by oxygen plasma bonding and cured. After curing, they were sterilized by high temperature and high pressure to obtain a vascularized organoid chip with a stamp-like microstructure pattern.

Images