Vascularized organ-like chip based on seal microstructure patterning and preparation method of vascularized organ-like chip

Abstract

The invention provides a vascularized organ-like chip based on seal microstructure patterning and a preparation method thereof, the chip comprises a micro-channel layer, the upper surface of the micro-channel layer is provided with a gel perfusion channel, a central tissue chamber and culture medium flow channels at two sides which are connected, and tumor spheres or colon cells are inoculated in the central tissue chamber; the liquid storage pool layer is arranged on the upper surface of the micro-channel layer and corresponds to the inlet and outlet of the gel filling channel, the inlet and outlet of the central tissue chamber and the inlet and outlet of the culture medium runner, the liquid storage pool layer is provided with a plurality of liquid storage pools and a plurality of through holes, and the liquid storage pools contain culture solutions to provide nutrients for cultured tissues; and the seal module is arranged above the central tissue chamber, the seal module is provided with a tumor seal and a colon seal, and the tumor seal and the colon seal are respectively provided with a microstructure three-dimensional module and are respectively used for simulating the culture environment of tumor spheres or colon cells. According to the invention, in-vitro construction of the vascularized tumor model and the colon model is realized, and a guide is provided for construction of the vascularized organ-like model with specific organ three-dimensional microstructure characteristics.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a vascularized organoid chip based on stamp microstructure patterning and a preparation method thereof. Background Art

[0002] Cancer is the leading cause of death worldwide, and the scientific community is working hard to find effective solutions. As the market for anti-cancer drug development grows, the development of more advanced drug screening systems has become a current research focus, aiming to narrow the gap between preclinical research and actual clinical trials. As an emerging in vitro research tool, microfluidic organ chips have shown great potential in the fields of life sciences and engineering. This platform can build physiological models of organs in tiny chips, thus providing a new research method for cancer research.

[0003] In the tumor microenvironment, blood vessels are key players in the delivery of anticancer drugs and play an indispensable role. After blood vessel formation, they are randomly recruited to the tumor site and become twisted or leaky under the influence of the tumor microenvironment, thereby providing the required nutrients and oxygen to the tumor tissue to meet its metabolic needs. However, these leaky blood vessels trigger enhanced permeability and retention effects, resulting in increased interstitial fluid pressure, which hinders the smooth delivery of drug molecules through the endothelial barrier. Therefore, there is an urgent need for a more physiological in vitro cancer model that covers the tumor stroma to effectively reflect the complex tumor microenvironment (TME) so as to be better used for microfluidic drug screening. Joonha Park et al. wrote an article in Lab on a chip, 2022, 22, 4335 titled Enabling perfusion through multicellular tumor spheroids promotinglumenization in a vascularized cancer model. The tumor microenvironment reconstruction method developed successfully constructed a perfusable tumor vascular model and applied it to a new drug screening platform. Nevertheless, they perfused tumor spheroids into the microfluidic chip together with endothelial cells, which is inconsistent with the order of real tumor growth. In the natural environment, vascular networks usually develop before tumors form to provide necessary nutrients and oxygen. Therefore, to more accurately simulate the complex microenvironment in the body, vascular networks should be established first and then tumor cells should be introduced.

[0004] The inner wall surface of the small intestine is composed of dense "crypt-villus" units. The villi are finger-like protrusions, and the crypts are located in the depressions at the base of the villi. The normal function of the small intestine depends on the continuous renewal of epithelial cells, which is achieved by the migration and differentiation of intestinal stem cells along the crypt-villus axis. However, the traditional colon barrier model, which is usually a two-dimensional monolayer 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, it is particularly important to develop an in vitro intestinal model with a more complex colon microstructure to achieve research under more physiological conditions.

[0005] The traditional three-dimensional tissue culture models reported so far are complex and inefficient in simulating the in vivo microenvironment, and usually require redesign of chips and platforms. In addition, vascular tumor models are usually constructed using a single or co-culture approach, while colon models are mostly constructed using static culture. These traditional methods have many limitations, including insufficient flexibility in model construction and poor reproducibility of the real microenvironment, which limits their widespread application.

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

[0007] In view of the defects in the prior art, the purpose of the present invention is to provide a vascularized organoid chip based on stamp microstructure patterning and a preparation method thereof, so as to enhance the simulation ability of the microenvironment and meet 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] A microchannel layer, the upper surface of which is provided with a gel perfusion channel, a central tissue chamber and a culture medium flow channel, wherein the central tissue chamber is connected to the gel perfusion channel and the culture medium flow channel, and one or more tissues or tissue cells are inoculated in the central tissue chamber;

[0010] A liquid 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 liquid reservoir layer is provided with a plurality of liquid reservoirs and a plurality of through holes. The liquid reservoirs contain culture fluid to provide nutrients for the cultured tissues or tissue cells.

[0011] The stamp module is arranged above the central tissue chamber. The stamp module is provided with one or more tissue or tissue cell stamps. Each of the tissue or tissue cell stamps 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 flow channel is connected and communicated with the central tissue chamber via a phase guiding structure, and the culture medium flow channel is located on both sides of the gel perfusion channel.

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

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

[0015] The reservoir layer includes a plurality of first reservoirs and a second reservoir, the position of the second reservoir corresponds to the central tissue chamber, and the tissue or tissue cells are inoculated into the central tissue chamber through the second reservoir; the plurality of first reservoirs correspond to the inlet and outlet positions of the culture medium flow channel through a plurality of reservoir through holes, and the culture medium in the first reservoirs flows into the culture medium flow channel through the reservoir through holes;

[0016] The liquid reservoir layer is also provided with a seal module matching hole, and the seal module is provided with a seal matching module. The seal matching module is matched with the seal module matching hole through the second liquid reservoir and communicated with the central tissue chamber for imprinting.

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

[0018] Furthermore, in the tumor stamp, the microstructure three-dimensional module is a tumor microstructure, the cross-section of the tumor microstructure is a rectangular with chamfers, and the three-dimensional feature is a convex hemispherical shape. The three-dimensional fillet 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 a trapezoid, which can ensure that the tumor microstructure is accurately imprinted to the center of the central tissue chamber.

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

[0021] A microfluidic chip mold having a microchannel layer and a reservoir layer is made by 3D printing resin material, and a three-dimensional stamp having a microstructure is made by 3D printing resin material;

[0022] Pour PDMS material into the mold, perform mold curing, cut the PDMS mold material from the mold to obtain a microchannel PDMS layer and a reservoir PDMS layer, and punch holes 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 are bonded and cured by oxygen plasma, and then sterilized at high temperature and high pressure after curing to obtain a vascularized organoid chip with a stamp microstructure pattern.

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

[0025] 1. The present invention provides a vascularized organoid chip based on stamp microstructure patterning. The microfluidic chip is designed by a reservoir layer and a microchannel layer to enhance the simulation capability of the microenvironment for culturing various tissues or tissue cells. The chip can be used to create a dynamic culture model, thereby improving the repeatability and reliability of the model constructed in the microfluidic chip. For example, by combining a tumor stamp with a colon stamp, a tumor vascular model and a colon epithelial barrier model under dynamic culture were successfully created using the organoid chip.

[0026] 2. The method for preparing a vascularized organ-on-chip based on stamp microstructure patterning provided by the present invention prepares stamps with different microstructures through high-precision 3D printing, thereby enhancing the ability to simulate the microenvironment; providing a richer microenvironment in cell culture and tissue engineering research, promoting the research and application of cell behavior, and improving the flexibility of designing different organ models.

[0027] In summary, the present invention overcomes the problems of insufficient flexibility in existing traditional model construction and poor reproducibility of the real microenvironment, can be flexibly applied to the construction of various in vitro models, provides new ideas and methods for the design of various organ chips, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0031] Figure 3 This is a schematic diagram of the use process of a vascularized organoid chip based on stamp microstructure patterning according to an embodiment of the present invention;

[0032] Figure 4 A three-dimensional fluorescence image of a vascularized organoid chip imprinted based on a stamp microstructure pattern according to an embodiment of the present invention;

[0033] Figure 5 The characterization results of different days of the in vitro vascular tumor model constructed based on the vascularized organoid chip with stamp microstructure patterning according to one embodiment of the present invention;

[0034] Figure 6 This is a characterization result diagram of an in vitro model of a colon model constructed based on a vascularized organoid chip patterned with a stamp microstructure according to an embodiment of the present invention;

[0035] Figure 7 The local magnification results of different focal planes of the in vitro model of the colon model built based on the vascularized organoid chip with stamp microstructure patterning according to one embodiment of the present invention;

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

[0037] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope 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 "include" and "comprise" used indicate the presence of the described features, wholes, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections.

[0039] It should also be understood that the terms used in this specification are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. Embodiments All drawings are only for the convenience of explaining the technical content of the present invention, and the technical features such as the numbers, the positions of the parts, the mutual relations between the parts, and the sizes of the parts used in the optimal implementation mode do not constitute a limitation on the technical solution itself. In the description of this specification, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc. is based on the orientation or positional relationship shown in the drawings, only for the convenience of description and simplified description, rather than indicating or suggesting that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0040] Reference Figure 1 As shown, a vascularized organoid chip based on stamp microstructure patterning provided by one embodiment of the present invention includes a reservoir layer 1, a microchannel layer 2 and a stamp module 3, wherein the reservoir layer 1 is located on the upper surface of the microchannel layer 2, and 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 flow channels 5, the gel perfusion channel 4 is connected to the central tissue chamber, and one or more tissues or tissue cells are inoculated in the central tissue chamber; the two-side culture medium flow channels 5 are connected to the central tissue chamber through a phase guide structure, and nutrients and nutrients are transported thereto. The stamp module 3 with a microstructure pattern is arranged above the central tissue chamber, and the stamp module 3 can be provided with one or more tissue or tissue cell stamps, and each 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 each tissue or tissue cell.

[0041] In this embodiment, the reservoir 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 reservoir layer 2 is provided with a plurality of liquid reservoirs and a plurality of through holes, wherein the liquid reservoirs contain culture fluid to provide nutrients for the cultured tissues or tissue cells. Specifically, the plurality of through holes include a first through hole 8 and a second through hole 11 for gel perfusion, and 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 the tissue or tissue cells are inoculated into the central tissue chamber through the second reservoir 7; the multiple first reservoirs 6 correspond to the entrance and exit 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 reservoir layer is also provided with a seal module matching hole, and the seal module is provided with a seal matching module. The seal matching module is matched with the seal matching module hole 9 through the second liquid reservoir 7 and communicated with the central tissue chamber for imprinting.

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

[0046] In addition, in other embodiments, the seal module 3 is further equipped with a seal handle module 35, which is designed for clamping and stamping operations using tweezers, further improving the operability and ease of use of the seal.

[0047] In some embodiments, the reservoir layer 1 and the microchannel layer 2 are both made of polydimethylsiloxane and bonded as one by oxygen plasma. Specifically, as an embodiment, the thickness of the microchannel layer 2 can be 1.5-2.5mm, and the central tissue chamber is designed to be circular, which is convenient for exchanging nutrients with the culture medium flow channel 5. Due to the need to transfer the three-dimensional microstructure of the stamp, the height of the central tissue chamber is designed to be 300-500μm, and the radius is 1-1.5mm, which is used for filling the hydrogel. Its patterned structural design limits cell growth to a restricted area. The height of the central tissue chamber determines the height of the 3D extracellular matrix, that is, the space where the three-dimensional capillary network can be spontaneously formed. The larger central tissue chamber height can form a three-dimensional capillary network with a more complex spatial hierarchy. The thickness of the reservoir layer 1 can be 6-10mm, and the radius of the reservoir is 4-5mm, so as to provide sufficient volume for storing culture medium. Of course, in other embodiments, it can also be other sizes, which are selected according to actual conditions.

[0048] In some embodiments, the culture medium flow channels 5 on both sides are connected to the central tissue chamber through a phase guide structure, and the width of the microchannel layer phase guide structure is 200-250 μm, and the height is 150-250 μm. Correspondingly, the shape of the central tissue chamber is circular, the height is 300-500 μm, and the radius is 1-1.5 mm. Of course, in other embodiments, other sizes can also be used, which are selected according to actual conditions.

[0049] In the above embodiments, the tissue or tissue cell can be any tissue or tissue cell that can be cultured in 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 by different stamps through the stamp module.

[0050] In the above-mentioned embodiment of the present invention, a microfluidic chip is designed by a liquid reservoir layer and a microchannel layer to enhance the simulation capability of the microenvironment for culturing various tissues or tissue cells. By designing and planning the microstructure of the stamp module 3 and combining it with a matching organoid chip, efficient construction of different biological models can be achieved using a vascularized organoid chip with a patterned stamp microstructure.

[0051] The following uses the stamp module 3 including a tumor stamp and a colon stamp as an example to further illustrate the vascularized organoid chip of the present application. Figure 1 and Figure 2 , a vascularized organoid chip based on stamp microstructure patterning provided by one embodiment of the present invention comprises a reservoir layer 1, a microchannel layer 2 and a stamp module 3, wherein the reservoir layer 1 is located on the upper surface of the microchannel layer 2, and 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 flow channels 5, the gel perfusion channel 4 is connected to the central tissue chamber, and tumor spheroids or colon cells are inoculated in the central tissue chamber; the two-side culture medium flow channels 5 are connected to the central tissue chamber through a phase guiding structure, and nutrients and nutrients are transported thereto. The stamp module 3 is arranged above the central tissue chamber, and the stamp module 3 is provided with a tumor stamp and a colon stamp, and the tumor stamp and the colon stamp are provided with different microstructure three-dimensional modules, which are 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 through hole 8 for gel perfusion and a second through hole 11 for gel perfusion, which correspond to the inlet and outlet of the gel perfusion channel 4, respectively, so as to facilitate the perfusion of the 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 a plurality of first reservoirs 6 and a second reservoir 7, wherein the position of the second reservoir 7 corresponds to the central tissue chamber, and corresponds to the central tissue chamber through the tumor stamp matching module hole 9, so as to facilitate the culture medium in the second reservoir 7 to flow into the central tissue chamber under the action of gravity and transport nutrients and nutrients to it, and the tumor spheroid or colon cell is inoculated into the central tissue chamber of the microchannel layer 2 through the second reservoir 7; the plurality of first reservoirs 6 correspond to the inlet and outlet positions of the culture medium flow channel 5 on both sides through the plurality of reservoir through holes 10, so as to facilitate the culture medium in the first reservoir 6 to flow into the culture medium flow channel 5 in the microchannel layer 2 through the reservoir through holes 10.

[0053] In this embodiment, the seal module 3 is located above the central tissue chamber and corresponds precisely to the central tissue chamber, that is, the seal module 3 and the central tissue chamber are of the same size and have corresponding positions.

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

[0055] The reservoir layer 1 also includes a plurality of matching holes, including a tumor stamp module matching hole 9 and a colon stamp module matching hole. The tumor stamp includes a tumor stamp matching module 32, which is used for accurate stamping of the stamp, and has a trapezoidal cross-section design, and a trapezoidal rotating body, which matches the tumor stamp module matching hole 9, thereby ensuring that the tumor microstructure can be accurately stamped to the center of the central tissue chamber.

[0056] At the same time, in this embodiment, the colon stamp also includes a microstructure three-dimensional module, namely the colon microstructure three-dimensional module 33, the cross section of which is a trapezoidal array with an interval of 100-200 μm, and the longitudinal section is composed of 9 to 36 circular arrays with an interval of 200-600 μm. This three-dimensional structure (colon microstructure three-dimensional module) is transferred to the hydrogel to simulate the microstructure of the crypts in the colon, thereby constructing a bionic microenvironment.

[0057] The seal module 3 is also provided with a seal reservoir matching module 34, the size of which is slightly smaller than the second reservoir 7, so as to facilitate accurate calibration during stamping and ensure smooth discharge of excess gas during stamping. Specifically, the positions of the second reservoir 7 and the seal reservoir matching module 34 (when in use) correspond vertically, and the small size of the seal reservoir matching module 34 allows for smooth stamping and is also convenient for discharging excess gas.

[0058] The vascularized organoid chip based on stamp microstructure patterning in the above embodiment of the present invention, combined with the tumor stamp and the colon stamp, can be used to create a tumor vascular model and a colon epithelial barrier model that are cultured step by step under dynamic culture.

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

[0060] S100, using 3D printing resin material to make a microfluidic chip mold having a microchannel layer and a liquid reservoir layer; using 3D printing resin material to make a three-dimensional stamp having a microstructure;

[0061] S200, pouring PDMS material into the microfluidic chip mold, performing mold curing to obtain PDMS mold material, cutting the PDMS mold material from the microfluidic chip mold to obtain a microchannel layer and a liquid reservoir PDMS layer; punching holes at the gel inlet and outlet and the culture medium inlet and outlet corresponding to the liquid reservoir PDMS layer;

[0062] S300, bonding the two layers of PDMS by oxygen plasma and curing them; sterilizing them by high temperature and high pressure after curing to obtain a vascularized organoid chip with a stamp microstructure pattern.

[0063] In a specific embodiment, a stamp microstructure patterned vascularized organoid chip uses a resin material with a printing accuracy of 0.025 mm, and a stamp module with different microstructures, a microchannel layer microfluidic chip mold, and a microfluidic chip mold with a reservoir layer are prepared by 3D printing. The mold is then re-molded using a PDMS material with high biocompatibility to obtain a microchannel PDMS layer and a reservoir PDMS layer with a microstructure.

[0064] Specifically, you can follow the steps below to achieve this:

[0065] S1. Use AutoCAD and Solidworks tools to design the microchannel layer, reservoir layer and stamp module of the vascularized organoid chip with stamp microstructure patterning;

[0066] S2. Use high-precision 3D printing resin materials to make a microfluidic chip mold with a microchannel layer and a reservoir layer, as well as a tumor stamp module and a colon stamp module. Soak the printed resin module in deionized water and perform ultrasonic cleaning; after cleaning and drying, dry it in an oven at 60-70°C overnight and place it under ultraviolet light for 10 minutes, so that the PDMS can be easily demolded from the resin mold;

[0067] S3. Evenly mix the PDMS prepolymer and curing agent in a mass ratio of 10:1, evacuate the vacuum pot to remove the bubbles in the PDMS, pour the bubble-free PDMS material into the microfluidic chip mold of the microchannel layer and the reservoir layer. In this process, it is important to avoid the generation of bubbles, and then transfer to an oven at 70-80℃ for curing for 4-8h.

[0068] S4. Cut the PDMS mold material from the resin mold to obtain a microchannel PDMS layer and a liquid reservoir PDMS layer, and punch holes at the gel inlet and outlet and the culture medium inlet and outlet corresponding to the PDMS liquid reservoir PDMS layer; specifically, the diameter of the hole punched at the gel inlet and outlet is 1 mm, and the diameter of the hole punched at the culture medium inlet and outlet is 2 mm. After the punching is completed, seal and store with a disposable isolation blue film.

[0069] S5. Use a glass slide as a carrier, attach the microchannel PDMS layer with the microstructure facing upwards to the surface of the glass slide, and attach the PDMS reservoir PDMS layer with the corresponding perforated side facing upwards to the surface of the glass slide, then place it in a plasma device for treatment, start the ignition at 0.7 mbar pressure for 45 seconds, bond the treated microchannel PDMS layer and the reservoir PDMS layer surface accordingly, and use tweezers to gently press out the bubbles between the PDMS. Then place the bonded organ chip on a 120°C hot plate and heat for 2 hours to enhance the bonding effect.

[0070] In this step, after the microchannel layer and the reservoir PDMS layer are bonded to each other, the first reservoir is connected to the microchannel layer culture medium channel 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 microchannel layer gel perfusion channel. Other areas are not connected, and the stamp modules (tumor stamp module and colon stamp module) exist independently, are capped before tissue culture, and then removed.

[0071] S6. After solidification, transfer to an autoclave for high temperature and high pressure sterilization to obtain a vascularized organoid chip with a stamp microstructure pattern.

[0072] The above-mentioned preparation method of the present invention prepares stamps with different microstructures through high-precision 3D printing, which can enhance the simulation ability of the microenvironment, provide a richer microenvironment in cell culture and tissue engineering research, and improve the flexibility of designing different organ models.

[0073] The vascularized organoid chip with stamp microstructure patterning in the above embodiment of the present invention can be used for constructing a variety of in vitro models. For better explanation, specific instructions for using the vascularized organoid chip for constructing in vitro models are provided.

[0074] refer to Figure 3 , Figure 3 The left column is a top view of the organoid chip, and the right column is a cross-sectional view of the organoid chip. The two are grouped from top to bottom. Taking the construction of a vascularized tumor model as an example, the main process is as follows:

[0075] S1. Sterilize the prepared stamp microstructure patterned vascularized organoid chip, soak the tumor stamp module with cell anti-adhesion solution and air dry it, which will help prevent the stamp from adhering to the cell gel during the demolding step;

[0076] In this step, ultraviolet light is used for 2 hours for sterilization, and the tumor stamp module is soaked in cell anti-adhesion liquid for 2 hours and then air-dried. Of course, in other embodiments, other time periods may be used, and are not limited to this parameter.

[0077] S2. Place a semicircular convex stamp on the top of the central tissue chamber. Then place the gel mixed with human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (NHLF) and the gel without cells on crushed ice, aspirate the fibrin gel mixed with cells, and quickly mix it with thrombin several times. After confirming that there are no bubbles, insert the tip of the pipette into the injection port of the stamp chip and gently infuse the gel. After observing that the gel will spontaneously fill the entire central tissue chamber under the action of capillary force, release the pipette. Be careful not to over-pressure during infusion.

[0078] In this step, the inoculation concentration of HUVEC and NHLF can be 7×10 6 Aspirate 6 μl of the fibrin gel mixed with cells and quickly mix it with 2 μl of thrombin 2-3 times, and then check whether there are bubbles.

[0079] S3. After the gel in the central circular tissue chamber of the microchannel layer is completely polymerized and solidified, use a pipette to draw EGM-2 culture medium and add it to one side of the reservoir, then add a small amount of EGM-2 to the opposite reservoir to cover the bottom of the reservoir, forming a liquid level difference, thereby generating a fluid pressure difference in the culture medium channels on both sides of the hydrogel, so that nutrients are transported into the central tissue chamber, and the cells are placed 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 draw 300-800 μl of EGM-2 medium into one reservoir, then add a small amount of EGM-2 to the other reservoir to cover the bottom of the reservoir, forming a liquid level difference of about 4-6 mm. Incubate in a CO2 cell culture incubator for 1 day, or other time, depending on actual conditions.

[0081] S4. The next day, the tumor stamp was removed and the cell-free fibrin gel was sucked out into a sterile culture dish. The mature cell spheres were slowly sucked out 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) and NHLF in a 1:1 ratio and culturing them in an ultra-low adsorption well plate. A new dispensing needle was then used to suck up the cell spheres and the gel together, and gently add them into the central through hole. The cell spheres slowly sank into the microstructured hydrogel surface under their own gravity.

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

[0083] S5. After the newly added hydrogel containing tumor cell spheres above the circular central tissue chamber is polymerized and solidified, use a pipette to draw EGM-2 culture medium and add it to the central reservoir, so that the culture medium exerts pressure on 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, take a small amount of sterile water and pour it into the sterile centrifuge tube cap, and put it in the pipette tip box to maintain the humidity of the vascular culture. Observe the changes in the culture medium level in the reservoir after a period of time, replace the culture medium, and change the direction of the hydraulic pressure difference.

[0084] In this step, 200-500 μl of EGM-2 medium can be pipetted into the central reservoir. The glass dish can be a 10-15 mm glass dish. The changes in the medium level in the reservoir are observed every 24 hours.

[0085] In the above embodiment of the present invention, referring to steps S1-S5, a vascularized tumor model can be constructed by using a vascularized organoid chip. Other tumor models can also be implemented by reference.

[0086] The actual culture effect of the vascularized organoid chip with stamp microstructure patterning in the above embodiment is described below to demonstrate the feasibility of in vitro culturing of vascular tumor models and colon models and their preparation process.

[0087] refer to Figure 4 As shown, 65kDa fluorescein isothiocyanate-dextran (FITC-Dextran) labeled with green fluorescence was mixed with the hydrogel, and then 6μl of fibrin gel solution containing dextran was drawn, mixed with thrombin, and then injected into the organoid chip with the tumor stamp. In the chip, the hydrogel constructed a three-dimensional microstructure under the imprint of the tumor stamp and was placed in a CO2 incubator for a 15-minute gel curing process. After the curing was completed, through the observation of the three-dimensional XZ and YZ interfaces, it can be clearly seen that the tumor stamp microstructure has been successfully transferred to the hydrogel, forming a precise three-dimensional structure. In addition, the guiding structures on both sides of the tumor stamp are also clearly visible on the YZ interface. These guiding structures ensure that the culture medium exchanges substances with the hydrogel through a specific phase-guided interface, thereby providing an effective channel for the cell's nutrient supply and waste metabolism. This experimental procedure not only successfully constructed a microscopic 3D structure, but also verified the precise transfer effect of the stamp technology in the hydrogel, laying the foundation for subsequent cell culture and model research.

[0088] Reference Figure 5As shown in the figure, the vascularized organoid chip patterned with stamp microstructures successfully constructed a vascular tumor model after inoculating human umbilical vein endothelial cells (HUVEC), normal human fibroblasts (NHLF) and tumor spheroids, and bright field and fluorescence images were acquired at different growth days. During the culture process, HUVEC and NHLF self-assembled to form a capillary network. On the second day, after the fibrin gel mixed with cells was injected into the central tissue chamber, HUVEC 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 and initially formed a three-dimensional capillary network; and on the seventh day of culture, the fluorescence image of the blood vessels in the central tissue chamber showed that relatively thick and longer vascular lumens had been formed. Figure 5 The dotted line in the fluorescence image below corresponds to the location of the tumor spheres accessed on the second day. Agua Sobrino et al. wrote an article titled "3D microtumors invitro supported by perfused vascular networks" in Scientific Reports, 2016, 6, 31589, and believed that the perfused vascular network around the tumor contributes to the supply of nutrients and oxygen. In addition, the fibroblasts contained in the tumor spheres help maintain the shape and roundness of the spheres. By analyzing the phase contrast images and manually detecting the diameter changes, it can be observed that the diameter of the tumor spheres increased from about 540 μm on the third day to about 780 μm on the seventh day, showing a significant growth trend and maintaining the initial roundness throughout the culture process.

[0089] Reference Figure 6 , 7 As shown, a cell-free fibrin hydrogel is first used to fill the bottom microchannel layer as the base of the colon model. After the crypt microstructure pattern on the colon stamp is transferred to the hydrogel, the prepared high-concentration human colon adenocarcinoma cells (Caco-2) are inoculated onto the solidified hydrogel through the central reservoir. During this process, the culture medium in the reservoir is connected to the culture medium in the microchannel layer through the Caco-2 cells through the hydrogel, achieving effective transfer of substances, and forming a dynamic culture environment under the action of the liquid level difference between the outer culture medium and the central culture medium. Since fibrinogen hydrogel is used as the base, it is more conducive to cell attachment, proliferation and differentiation. During the culture process, Caco-2 can differentiate into characteristics similar to small intestinal epithelial cells, such as microvilli structure and tight junctions. Once the cells converge, they spontaneously differentiate into mature intestinal epithelial brush border cells to form an endothelial barrier, such as Figure 6 As shown. Since the intestinal crypt model is a three-dimensional structure, the embodiment of the present invention also performs corresponding characterization on the bottom, middle and barrier surface of the intestinal crypt. Figure 7The local magnified bright-field microscopy images of the colon barrier at different focal planes are shown. From the images, it can be clearly seen that Caco-2 cells are evenly distributed and completely cover the entire microstructure, preliminarily achieving the construction of a complete endothelial barrier.

[0090] The above-mentioned embodiment of the present invention is based on the vascularized organoid chip with stamp microstructure patterning and its preparation method. The microfluidic chip is designed by the reservoir layer and the microchannel layer, and the tumor stamp and the colon stamp are combined to successfully create a tumor vascular model and a colon epithelial barrier model under dynamic culture. The organoid chip of the present invention improves the repeatability and reliability of the biological construction model; the stamps with different microstructures are prepared by high-precision 3D printing, providing a richer microenvironment in cell culture and tissue engineering research, promoting the research and application of cell behavior, and improving the flexibility of designing different organ models. It can also provide guidance for the construction of vascularized organoid models with the three-dimensional microstructure characteristics of specific organs, and provide a reliable testing platform in the early stages of drug development.

[0091] The above embodiments are based on tumor vascular model and colon epithelial barrier model as examples. The above chip in the present invention can also be applied to the construction of other tissue or tissue cell models, not limited to tumor spheroids and colon cells. The preferred features in the above embodiments can be used alone in any embodiment, and can also be used in any combination without conflict. In addition, the 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 above specific embodiments, and those skilled in the art can make various modifications or deformations to the embodiments without departing from the scope of the claims of the present invention, which does not affect the essence of the present invention. In addition, the above preferred features can be used in any combination without conflicting with each other.

Claims

1. A vascularized organoid chip based on stamp microstructure patterning, characterized in that: include: A microchannel layer, the upper surface of which is provided with a gel perfusion channel, a central tissue chamber and a culture medium flow channel, wherein the central tissue chamber is connected to the gel perfusion channel and the culture medium flow channel, and one or more tissues or tissue cells are inoculated in the central tissue chamber; A liquid 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 liquid reservoir layer is provided with a plurality of liquid reservoirs and a plurality of through holes. The liquid reservoirs contain culture fluid to provide nutrients for the cultured tissues or tissue cells. The stamp module is arranged above the central tissue chamber. The stamp module is provided with one or more tissue or tissue cell stamps. Each of the tissue or tissue cell stamps 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.

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

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

4. The vascularized organoid chip with stamp microstructure patterning according to claim 1, characterized in that: The plurality of through holes include a first through hole and a second through hole for gel perfusion, wherein the first through hole and the second through hole correspond to and communicate with the inlet and outlet of the gel perfusion channel; The reservoir layer includes a plurality of first reservoirs and a second reservoir, the position of the second reservoir corresponds to the central tissue chamber, and the tissue or tissue cells are inoculated into the central tissue chamber through the second reservoir; A plurality of first liquid reservoirs correspond to the inlet and outlet positions of the culture medium flow channel through a plurality of liquid reservoir through holes, and the culture medium in the first liquid reservoirs flows into the culture medium flow channel through the liquid reservoir through holes; The liquid reservoir layer is also provided with a seal module matching hole, and the seal module is provided with a seal matching module. The seal matching module is matched with the seal module matching hole through the second liquid reservoir and communicated with the central tissue chamber for imprinting.

5. The vascularized organoid chip with stamp microstructure patterning according to claim 1, characterized in that: The three-dimensional tissue is vascular tumor tissue and colon barrier tissue; the stamp module includes a tumor stamp and a colon stamp.

6. The vascularized organoid chip with stamp microstructure patterning according to claim 5, characterized in that: The tumor stamp, wherein the microstructure three-dimensional module is a tumor microstructure, has a cross-section of a rectangular shape with chamfers, and a three-dimensional feature of a convex hemispherical shape. The three-dimensional fillet formed by the chamfers can optimize the injection path of the hydrogel and the fluid distribution.

7. The vascularized organoid chip with stamp microstructure patterning according to claim 5, characterized in that: The tumor stamp, wherein the microstructure three-dimensional module is a tumor microstructure, has a cross-section that is a trapezoid, and the trapezoid can ensure that the tumor microstructure is accurately stamped to the center of the central tissue chamber.

8. The vascularized organoid chip with stamp microstructure patterning according to claim 5, characterized in that: The colon stamp, wherein the microstructure three-dimensional module is a colon microstructure, the cross-section of the colon microstructure is a trapezoidal array with an interval of 100-200 μm; the longitudinal section of the colon microstructure is 9-36 circular arrays with an interval of 200-600 μm.

9. The vascularized organoid chip with stamp microstructure patterning according to any one of claims 1 to 8, characterized in that: The seal module also includes a seal matching module and a seal handle module. The height of the seal handle module is 3-5 mm. The seal matching module is used for accurate matching of the seal, and the seal handle module is used for clamping and stamping operations.

10. A method for preparing a vascularized organoid chip with a stamp microstructure pattern according to any one of claims 1 to 9, characterized in that: include: A microfluidic chip mold having a microchannel layer and a reservoir layer is made by 3D printing resin material, and a three-dimensional stamp having a microstructure is made by 3D printing resin material; Pour PDMS material into the mold, perform mold curing, cut the PDMS mold material from the mold to obtain a microchannel PDMS layer and a reservoir PDMS layer, and punch holes 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 are bonded and cured by oxygen plasma, and then sterilized at high temperature and high pressure after curing to obtain a vascularized organoid chip with a stamp microstructure pattern.

Images