Microfluidic culture chip for vascularized organoids and culture method of three-dimensional vascularized organoids

Abstract

The invention belongs to the technical field of three-dimensional vascularized organoid culture, and mainly provides a vascularized organoid microfluidic culture chip and a three-dimensional vascularized organoid culture method.The culture chip comprises a base body, and the base body is provided with a first liquid storage cavity, a second liquid storage cavity and a central annular cavity communicated with the first liquid storage cavity and the second liquid storage cavity; the base body is provided with an organoid culture cavity surrounded by a central annular cavity, and the organoid culture cavity is communicated with the central annular cavity; a sliding plate is slidably arranged in the first liquid storage cavity, and the volume of the first liquid storage cavity is changed by driving the sliding plate to move. The sliding plate is introduced into the vascularized organ-like microfluidic culture chip to adjust the liquid level difference between the first liquid storage cavity and the second liquid storage cavity to drive the cell culture fluid to flow, so that long-term stable perfusion of the cell culture fluid is realized under the condition that an external pump control system is not needed, dependence of a traditional external pump control system is avoided, and the operation is simple and convenient. The experimental operation steps are simplified, and the overall complexity and the use cost of the device are reduced.

Description

Technical Field

[0001] This invention belongs to the field of three-dimensional vascularized organoid culture technology, and in particular relates to a microfluidic culture chip for vascularized organoids and a method for culturing three-dimensional vascularized organoids. Background Technology

[0002] In recent years, with the development of tissue engineering and tumor biology, three-dimensional vascularized organoid models have received widespread attention in disease research and drug screening. Compared with traditional two-dimensional cell culture systems, three-dimensional vascularized organoids can better simulate the extracellular matrix environment and intercellular spatial structure in vivo, thus having significant advantages in terms of physiological relevance and the accuracy of experimental results. However, while existing two-dimensional culture systems are still widely used, they lack vascular structures, making it impossible to achieve effective delivery of nutrients, oxygen, and drugs. Furthermore, the planar substrate cannot reflect the complex three-dimensional spatial tissue structure, thus making it difficult to realistically reproduce the in vivo microenvironment.

[0003] The rapid development of microfluidic technology has provided new solutions for the construction of three-dimensional vascularized organoids. Three-dimensional culture systems integrating vascular networks exhibit unique advantages in drug delivery, immune cell penetration, and dynamic microenvironment monitoring, enabling real-time observation and periodic assessment of changes in the organoid's internal environment. In particular, when the vascular network within the organoid can maintain stable perfusion, it can significantly improve the structural integrity and functional maintenance of the organoid, thus more closely resembling the physiological state of in vivo tissues.

[0004] Although existing research has shown that vascular perfusion plays a crucial role in vascular network stabilization, current three-dimensional vascularized organoid models still have certain limitations. Chinese patent application CN114591836A discloses an in vitro vascular bed microfluidic chip and its application. The microfluidic chip in this patent application includes a first channel, a second channel, a third channel, and a fourth channel. A first flow-limiting section is provided between the second channel and the first channel, and between the third channel and the first channel. The first flow-limiting section is configured to allow cell culture medium injected into the second and third channels to flow through the vascular bed culture chamber, while simultaneously confining endothelial cell gel within the vascular bed culture chamber. A second flow-limiting section is provided between the fourth channel and the second channel, configured to confine the fibroblasts within the fourth channel while allowing fibroblast secretions to diffuse into the second channel.

[0005] The structure and operation of the extracorporeal vascular bed microfluidic chip disclosed in this patent application are quite complex, and it also requires an external pump control system, which increases the system's operating cost. Therefore, how to achieve simple, low-cost, and reliable perfusion remains an urgent technical problem to be solved. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a microfluidic culture chip for vascularized organoids and a method for culturing three-dimensional vascularized organoids, in order to solve the problems that the structure and operation of microfluidic chips in the prior art are relatively complex, rely on external pump control systems, and have high system usage costs.

[0007] To achieve the above and other related objectives, the present invention provides a vascularized organoid microfluidic culture chip, comprising a substrate, wherein the substrate is provided with a first liquid storage chamber and a second liquid storage chamber, and a central annular chamber connecting the first liquid storage chamber and the second liquid storage chamber; The substrate is provided with an organoid culture chamber, and the central annular chamber is arranged around the periphery of the organoid culture chamber, and the organoid culture chamber is connected to the central annular chamber. A sliding plate is slidably arranged inside the first liquid storage chamber. By driving the sliding plate to move, the volume of the first liquid storage chamber is changed, so that a liquid level difference is formed between the first liquid storage chamber and the second liquid storage chamber, so as to generate an irrigation driven by the liquid level difference in the central annular chamber.

[0008] In some embodiments of the present invention, slides are provided on opposite sides of the first liquid storage cavity, and sliders that slide in cooperation with the slides are provided on opposite sides of the sliding plate.

[0009] In some embodiments of the present invention, a sealing strip is provided on the side of the sliding plate that contacts the cavity wall of the first liquid storage chamber, and the sealing strip is used to seal the gap between the sliding plate and the cavity wall of the first liquid storage chamber.

[0010] In some embodiments of the present invention, the substrate includes a chip body, patterned double-sided adhesive, and cover glass stacked sequentially from top to bottom; The chip body has a first channel corresponding to the first liquid storage chamber, a second channel corresponding to the second liquid storage chamber, and the organoid culture chamber. The patterned double-sided adhesive has a notch. When the chip body, the patterned double-sided adhesive, and the cover glass are stacked together, the notch, the chip body, and the cover glass together form the central annular cavity.

[0011] In some embodiments of the present invention, the thickness of the patterned double-sided adhesive is between 100 μm and 300 μm, preferably 200 μm.

[0012] In some embodiments of the invention, the central annular cavity is used to form a vascular network.

[0013] A method for three-dimensional vascularized organoid culture using the aforementioned vascularized organoid microfluidic culture chip includes the following steps: Step 1: Inject the cell-fibrinogen-thrombin gel into the central annular chamber; Step 2: Inject the cell-free fibrinogen gel into the organoid culture chamber; Step 3: Add cell culture medium to the first and second reservoirs, so that the liquid level of the cell culture medium in the first reservoir is different from the liquid level of the cell culture medium in the second reservoir. Step 4: A three-dimensional vascular network is cultured and formed in the central annular chamber, and then the organoid is seeded into the organoid culture chamber; Step 5: Move the sliding plate to create a liquid level difference between the first and second liquid storage chambers, thereby generating perfusion in the three-dimensional vascular network of the central annular chamber and realizing long-term dynamic co-culture of the three-dimensional vascular network and organoids.

[0014] In some embodiments of the present invention, in step five, when the liquid level heights of the first liquid storage chamber and the second liquid storage chamber tend to be consistent, the sliding plate is pushed again to re-establish the liquid level difference, and this operation is repeated to maintain long-term stable perfusion.

[0015] In some embodiments of the present invention, the cell-fibrinogen-thrombin gel is a fibrin gel containing human umbilical vein endothelial cells and fibroblasts.

[0016] In some embodiments of the present invention, the method for preparing the organoids is as follows: tumor cells from patients or other sources are mechanically sheared, enzymatically digested and screened to obtain a single-cell suspension, which is then embedded in a matrix gel and cultured in a culture medium containing specific growth factors to form organoids with cavity and vacuolar structures.

[0017] As described above, the present invention has the following beneficial effects: by introducing a sliding plate into the vascularized organoid microfluidic culture chip to adjust the liquid level difference between the first and second liquid reservoirs to drive the flow of cell culture medium, long-term stable perfusion of cell culture medium can be achieved without the need for an external pump control system, avoiding dependence on traditional external pump control systems, which not only simplifies experimental operation steps, but also reduces the overall complexity and cost of the device. Attached Figure Description

[0018] Figure 1 The diagram shown is a structural schematic from a first perspective of the present invention. Figure 2 The diagram shown is a structural schematic from a second perspective of the present invention. Figure 3 Shown as an exploded view of the present invention; Figure 4 The image shown is a top view of the present invention. Figure 5 Displayed as Figure 4 Sectional view along the middle AA direction; Figure 6 Displayed as Figure 5 Enlarged view of point B in the middle; Figure 7 The images are shown as a view of the organoid manufacturing process, (a) showing the organoid manufacturing process, and (b) showing the view of the organoids after culture. Figure 8 The diagram shows the liquid level difference between the first and second liquid storage chambers. (a) shows the change in liquid level height before and after pushing the sliding plate, and (b) shows the liquid level difference curve. Figure 9 The images show the vascularization process: (a) the injection process of fibrin gel from human umbilical vein endothelial cells and fibroblasts, and (b) the growth process of fibrin gel from human umbilical vein endothelial cells and fibroblasts. Figure 10 Displayed as a three-dimensional vascularized organoid performance map with and without perfusion.

[0019] Explanation of icon numbers: 1. Substrate; 11. Cover glass; 12. Patterned double-sided adhesive; 121. Notch; 13. Chip body; 131. Track; 2. First liquid storage chamber; 21. First channel; 3. Second liquid storage chamber; 31. Second channel; 4. Organoid culture chamber; 5. Sliding plate; 51. Slider; 6. Central annular chamber. Detailed Implementation

[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0021] Please see Figures 1-10 It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0022] like Figure 1 , Figure 2As shown, the first aspect of the present invention provides a vascularized organoid microfluidic culture chip, comprising a substrate 1, wherein the substrate 1 has a first reservoir 2 and a second reservoir 3 for placing cell culture medium to maintain a continuous supply. The substrate 1 also has a central annular chamber 6 connecting the first reservoir 2 and the second reservoir 3, the central annular chamber 6 for placing a cell-fibrinogen-thrombin gel. The substrate 1 further has an organoid culture chamber 4 for placing a cell-free fibrinogen gel, the central annular chamber 6 being arranged around the periphery of the organoid culture chamber 4, and the organoid culture chamber 4 being connected to the central annular chamber 6, thereby allowing the cell-fibrinogen-thrombin gel to contact the cell-free fibrinogen gel. After the cell-fibrinogen-thrombin gel in the central annular chamber 6 forms a vascularized structure, organoids are added to the cell-free fibrinogen gel in the organoid culture chamber 4, thereby establishing an interaction between the organoids and the vascularized structure.

[0023] In some embodiments of the present invention, the central annular chamber 6 surrounds the lower periphery of the organoid culture chamber 4.

[0024] In some embodiments of the present invention, the central annular chamber 6 surrounds the central periphery of the organoid culture chamber 4.

[0025] A sliding plate 5 is slidably arranged within the first reservoir 2. By driving the sliding plate 5 to move, the liquid level of the cell culture medium in the first reservoir 2 is changed, thereby creating a liquid level difference between the cell culture medium in the second reservoir 3. That is, when the liquid levels on both sides are the same, by adjusting the position of the sliding plate 5 in the first reservoir 2, the liquid level in the first reservoir 2 changes, resulting in a liquid level difference between the liquid levels in the first reservoir 2 and the second reservoir 3, thus generating perfusion between the first reservoir 2 and the second reservoir 3 within the central annular chamber 6. This allows the cell-fibrinogen-thrombin gel within the central annular chamber 6 to be continuously subjected to shear stress driven by the cell culture medium, thereby achieving long-term stable perfusion of the cell culture medium without the need for an external pump control system, i.e., forming a continuous interstitial fluid flow environment within the central annular chamber 6.

[0026] In some embodiments of the present invention, by pushing the sliding plate 5 to move, the liquid level in the first liquid storage chamber 2 can be raised, thereby forming a flow from the first liquid storage chamber 2 to the second liquid storage chamber 3.

[0027] In some embodiments of the present invention, by pushing the sliding plate 5 to move, the liquid level in the first liquid storage chamber 2 can be lowered, thereby forming a flow from the second liquid storage chamber 3 to the first liquid storage chamber 2.

[0028] In some embodiments of the present invention, slide rails 131 are provided on opposite sides of the first liquid storage chamber 2, and sliders 51 that slide in cooperation with the slide rails 131 are provided on opposite sides of the sliding plate 5. When the sliding plate 5 is driven to move, the sliders 51 slide within the slide rails 131, ensuring the stability of the sliding plate 5.

[0029] In some embodiments of the present invention, the sliding plate 5 can be moved by pushing it with the hand of a user.

[0030] In some embodiments of the present invention, the sliding plate 5 can be moved by a drive unit. The drive unit includes a stepper motor and a lead screw that are connected together. The lead screw is rotatably connected to the base 1. The lead screw and the sliding plate 5 are threaded together. When the stepper motor runs, it drives the lead screw to rotate, and the lead screw drives the sliding plate 5 to move along the slide rail 131.

[0031] In some embodiments of the present invention, the driving unit is a cylinder, and the telescopic shaft of the cylinder is connected to the sliding plate 5. When the telescopic shaft of the cylinder moves, it drives the sliding plate 5 to move.

[0032] In some embodiments of the present invention, a sealing strip is provided on the side of the sliding plate 5 that contacts the cavity wall of the first liquid storage chamber 2. The sealing strip is used to seal the gap between the sliding plate 5 and the cavity wall of the first liquid storage chamber 2. The sealing strip ensures the airtightness between the sliding plate 5 and the cavity wall of the first liquid storage chamber 2, preventing cell culture medium from leaking from the cavity wall of the sliding plate 5 and the first liquid storage chamber 2, reducing the waste of cell culture medium, and ensuring that all cell culture medium can be utilized.

[0033] like Figures 3-6 As shown, in some embodiments of the present invention, the substrate 1 includes a chip body 13, a patterned double-sided adhesive 12 and a cover glass 11 stacked sequentially from top to bottom. The chip body 13 is fixed to the cover glass 11 by the patterned double-sided adhesive 12. That is, the substrate 1 is a split structure. After the culture is completed, the split structure facilitates the cleaning of each part.

[0034] In some embodiments of the present invention, the chip body 13 is a 3D-printed polymethyl methacrylate structure.

[0035] like Figures 3-6 As shown, the chip body 13 has a first channel 21 and a second channel 31. Both ends of the first channel 21 and the second channel 31 are open. When the chip body 13 is fixed on the cover glass 11, the openings on the same side of the first channel 21 and the second channel 31 are blocked by the cover glass 11, leaving only the opening on the other side. Thus, the first channel 21 and the second channel 31 become the first liquid storage chamber 2 and the second liquid storage chamber 3, respectively. The organoid culture chamber 4 is arranged on the chip body 13.

[0036] like Figures 3-6As shown, the chip body 13 has a spacer wall with a first channel 21 and a second channel 31 on both sides. The organoid culture chamber 4 is arranged on the spacer wall. The patterned double-sided adhesive 12 has the same shape as the bottom surface of the chip body 13, but it has a notch 121 and a certain thickness, thus leaving a gap between the annular cavity wall of the organoid culture chamber 4 and the cover glass 11 below it, forming the central annular chamber 6 that is open on the side. When the cell-fibrinogen-thrombin gel is injected into the central annular chamber 6, the cell-fibrinogen-thrombin gel is uniformly filled by capillary action.

[0037] In some embodiments of the present invention, the notch 121 is preferably recessed relative to the edge of the organoid culture cavity 4, so that the central annular cavity 6 forms an annular shape.

[0038] In some embodiments of the present invention, the substrate 1 is a single integral structure, with a central annular chamber 6 disposed at the bottom of the first liquid storage chamber 2 and the second liquid storage chamber 3, and communicating with the first liquid storage chamber 2 and the second liquid storage chamber 3. An organoid culture chamber 4 is disposed on the substrate 1 between the first liquid storage chamber 2 and the second liquid storage chamber 3, and communicates with the central annular chamber 6.

[0039] In some embodiments of the present invention, the base 1 is an integral structure, and the central annular chamber 6 is arranged at the middle height of the first liquid storage chamber 2 and the second liquid storage chamber 3, and is connected to the first liquid storage chamber 2 and the second liquid storage chamber 3.

[0040] In some embodiments of the present invention, the thickness of the patterned double-sided adhesive 12 is between 100 μm and 300 μm. Preferably, the thickness of the patterned double-sided adhesive 12 is 200 μm.

[0041] A second aspect of the present invention provides a method for three-dimensional vascularized organoid culture using the above-mentioned vascularized organoid microfluidic culture chip, comprising the following steps: Step 1: Inject the cell-fibrinogen-thrombin gel into the central annular chamber 6, and achieve uniform filling of the cell-fibrinogen-thrombin gel in the central annular chamber 6 through capillary action.

[0042] Step 2: Inject cell-free fibrinogen gel into organoid culture cavity 4 to facilitate the inoculation and culture of organoids from tumors or other sources.

[0043] Step 3: Inject different amounts of cell culture medium into the first reservoir 2 and the second reservoir 3 respectively, so that the liquid level of the cell culture medium in the first reservoir 2 is different from that in the second reservoir 3, thus creating a liquid level difference.

[0044] Step 4: After forming a three-dimensional vascular network in the central annular chamber 6, the organoids are seeded into the organoid culture chamber 4.

[0045] Step 5: When the liquid levels of the cell culture medium in the first reservoir 2 and the second reservoir 3 are consistent, push the sliding plate 5 to move, so that a liquid level difference is formed between the first reservoir 2 and the second reservoir 3, so as to generate perfusion in the three-dimensional vascular network of the central annular chamber 6, and realize the long-term dynamic co-culture of the three-dimensional vascular network and organoids.

[0046] This invention drives the flow of cell culture medium by setting a sliding plate 5 to adjust the liquid level difference between the first liquid storage chamber 2 and the second liquid storage chamber 3, avoiding reliance on traditional external pump control systems. This not only simplifies experimental operation steps but also reduces the overall complexity and cost of the device.

[0047] In some embodiments of the present invention, in step three, by pushing the sliding plate 5, the liquid level of the cell culture medium in the first liquid storage chamber 2 can be higher or lower than the liquid level of the cell culture medium in the second liquid storage chamber 3, thereby generating perfusion of the cell culture medium in different flow directions.

[0048] In some embodiments of the present invention, in step five, when the liquid levels of the first liquid storage chamber 2 and the second liquid storage chamber 3 tend to be consistent, the sliding plate 5 is pushed again to re-establish the liquid level difference, and this operation is repeated to maintain long-term stable perfusion.

[0049] In some embodiments of the present invention, the cell culture medium includes the following components: Basic culture medium: HEPES and bicarbonate buffer; Serum: 10% fetal bovine serum (FBS); Added factor: 1% penicillin-streptomycin (double antibiotic); Buffer conditions: pH 7.4 after equilibration in an incubator environment with 5% CO2 and 95% air.

[0050] In some embodiments of the present invention, the cell-fibrinogen-thrombin gel is a fibrin gel containing human umbilical vein endothelial cells and fibroblasts. The fibrin gel containing human umbilical vein endothelial cells and fibroblasts is injected into the central annular chamber 6, achieving uniform filling through capillary action. Fibroblasts provide support to endothelial cells by secreting pro-angiogenic factors and remodeling the extracellular matrix, thereby promoting the self-assembly of endothelial cells in the three-dimensional matrix to form a three-dimensional vascular network with luminal structures.

[0051] In some embodiments of the present invention, the fibrin gel containing human umbilical vein endothelial cells and fibroblasts comprises the following components: Human umbilical vein endothelial cells (HUVECs), 2×105 cells / mL, uniformly dispersed in the gel; Human skin fibroblasts (HDFs), 5×10 5 cells / mL, forming a synergistic effect with HUVECs; Fibrinogen solution, 5 mg / mL; Thrombin solution, 1 U / mL; Calcium chloride (CaCl2) solution, 2.5 mmol / L; Vascular endothelial growth factor (VEGF), 20 ng / mL, promotes the formation of tubular structures in HUVECs.

[0052] The cell culture medium flows through the tiny gaps in the endothelial cell gel, creating interstitial flow. The mechanical forces generated during this process further stimulate the vascularization process of the endothelial cells. After the vascular network is formed, the flow of the cell culture medium and the shear forces generated inside the blood vessel lumen continuously stimulate the division and proliferation of endothelial cells, forming a richer and more biomimetic vascular network.

[0053] In some embodiments of the present invention, the organoid preparation method involves: obtaining a single-cell suspension from patient-derived or other sources through mechanical shearing, enzymatic digestion, and screening; embedding the suspension in a matrix gel; and culturing it in a culture medium containing specific growth factors to form organoids with cavitation and vacuolar structures. The organoids are then seeded into the organoid culture chamber 4, establishing an interaction with the three-dimensional vascular network in the central annular chamber 6. Long-term stable co-culture of the three-dimensional vascular network and the organoids is achieved under dynamic fluid flow conditions, thereby constructing three-dimensional vascularized organoids with higher physiological relevance.

[0054] The following specific embodiments illustrate the method for three-dimensional vascularized organoid culture using the aforementioned vascularized organoid microfluidic culture chip.

[0055] Example A method for three-dimensional vascularized organoid culture using the above-mentioned vascularized organoid microfluidic culture chip includes the following steps: Step 1: Inject fibrin gel containing human umbilical vein endothelial cells and fibroblasts into the central annular chamber 6, and achieve uniform filling of the cell-fibrinogen-thrombin gel in the central annular chamber 6 through capillary action.

[0056] Step 2: Inject cell-free fibrinogen gel into organoid culture cavity 4 to facilitate the inoculation and culture of organoids from tumors or other sources.

[0057] Step 3: Inject cell culture medium into the first reservoir 2 and the second reservoir 3, then incubate at 37°C and 5% CO2. 2The cells are incubated statically in an incubator. During this process, fibroblasts support the self-assembly of endothelial cells by secreting pro-angiogenic factors and remodeling the extracellular matrix, ultimately forming a vascular network with interconnected cavities within the gel matrix. This drives the sliding plate 5 to move, raising the liquid level in the first reservoir 2 to a higher level than the liquid level in the second reservoir 3. This level difference generates initial directional perfusion in the central annular chamber 6, providing preliminary fluid shear force stimulation for the formation of the three-dimensional vascular network.

[0058] Step Four: As Figure 7 As shown, organoids are seeded into the gel in organoid culture chamber 4. The organoid preparation method specifically involves: obtaining a single-cell suspension from patient-derived or other sources through mechanical shearing, enzymatic digestion, and screening; embedding the suspension in a matrix gel; and culturing it in a culture medium containing specific growth factors to form organoids with cavitation and vacuolar structures. Once a vascular network forms in the central annular chamber 6, the pre-prepared organoids are seeded into the gel in organoid culture chamber 4. During culture, the organoids and the vascular network automatically establish an interaction.

[0059] Step 5: As Figure 8 As shown, after 24 hours, when the levels of the cell culture medium in the first reservoir 2 and the second reservoir 3 are equal, the sliding plate 5 is moved to raise the level of the cell culture medium in the first reservoir 2 above that in the second reservoir 3. The level is recorded. By adjusting the level difference between the first reservoir 2 and the second reservoir 3 by moving the sliding plate 5, long-term stable perfusion of the cell culture medium is achieved without an external pump control system, forming a continuous interstitial fluid flow environment in the central annular chamber 6. Repeating this operation provides a long-term, stable interstitial fluid flow environment for the co-culture system throughout the entire culture cycle without an external pump control system, and three-dimensional vascularized organoids are constructed after 7 days. These three-dimensional vascularized organoids are cultured in a dynamic fluid flow environment, thus exhibiting higher physiological relevance.

[0060] Validation of three-dimensional vascular network perfusion function To verify the patency and function of the three-dimensional vascular network constructed in this invention, a fluorescent microbead perfusion experiment was conducted after the three-dimensional vascular network was formed.

[0061] Fluorescent microbead perfusion experiment procedure: Dimethyloxaloylglycine (DMOG) was added to the cell culture medium. After 7 days of culture, 1 μm diameter red fluorescent microbeads were added to the cell culture medium and perfused for 30 minutes. Imaging was performed as follows. Figure 9 As shown.

[0062] like Figure 9As shown in (a), fibroblasts provide support for endothelial cells by secreting pro-angiogenic factors and remodeling the extracellular matrix, thereby promoting the self-assembly of endothelial cells in the three-dimensional matrix to form a three-dimensional vascular network with cavitation structures.

[0063] like Figure 9 As shown in (b), the results indicate that fluorescent microspheres are uniformly distributed in the blood vessels within the formed three-dimensional vascular network, suggesting that the formed blood vessels possess good perfusion channels and stability.

[0064] The promoting effect of dynamic perfusion on the formation of three-dimensional vascular network To demonstrate the effectiveness of dynamic perfusion in this invention, a comparative experiment was conducted on the formation of three-dimensional vascular networks under dynamic perfusion and static conditions. In the experiment, fibrin gel containing human umbilical vein endothelial cells and fibroblasts was seeded into the aforementioned vascularized organoid microfluidic culture chip (dynamic perfusion conditions) and conventional well plates (static conditions) for parallel culture.

[0065] Static conditions: Select 6-well plates. The 6-well plates should be rinsed 1-2 times with sterile phosphate-buffered saline (PBRS) to remove residual coating solution and air-dried before use. Remove the required cell culture medium, PBRS buffer, and trypsin from the 4°C freezer and allow them to equilibrate to room temperature for 15-30 minutes. Neutralize the trypsin with serum-containing culture medium; prepare the neutralization solution in advance. Then, add the cell culture medium, PBRS buffer, and trypsin to the 6-well plates. Next, add fibrin gel containing human umbilical vein endothelial cells and fibroblasts to the 6-well plates to form a cell suspension. Gently agitate the 6-well plates horizontally 3-5 times in a laminar flow hood to ensure the cell suspension evenly covers the bottom of the wells, avoiding cell aggregation in the center or at the edges. Then, add organoids and place the 6-well plates in a 37°C, 5% CO2 incubator. 2 The three-dimensional vascular network under static conditions was obtained by incubating the cells in an incubator for 7 days.

[0066] The results showed that endothelial cells cultured under static conditions only formed incomplete and unstable two-dimensional structures, while in the vascularized organoid microfluidic culture chip of the present invention, endothelial cells were able to form a more stable and interconnected three-dimensional vascular network under the action of dynamic fluid flow.

[0067] like Figure 10 As shown, comparisons using fluorescence imaging and image analysis software revealed that the number of nodes, branches, and connection complexity of the three-dimensional vascular network in the culture system of this invention were significantly higher than those in the static culture system. Statistical analysis further demonstrated that the three-dimensional vascular network formed under dynamic flow conditions was more dense and complex, exhibiting characteristics closer to the real vascular system in vivo.

[0068] Therefore, the dynamic flow environment controlled by the sliding plate 5 in this invention can maintain long-term stable perfusion without the need for external pump control, and significantly promote the three-dimensionality and complexity of the vascular network, overcoming the shortcomings of traditional static culture methods in terms of vascular stability and physiological relevance.

[0069] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A vascularized organoid microfluidic culture chip, characterized in that, The application relates to a chip for long-term dynamic co-culture of three-dimensional blood vessel network and organoids, which comprises a base (1) provided with a first liquid storage cavity (2) and a second liquid storage cavity (3) and a central annular chamber (6) communicating the first liquid storage cavity (2) and the second liquid storage cavity (3); the base (1) is provided with an organoid culture cavity (4), the central annular chamber (6) is arranged around the periphery of the organoid culture cavity (4), and the organoid culture cavity (4) communicates with the central annular chamber (6); a sliding plate (5) is slidably arranged in the first liquid storage cavity (2), the volume of the first liquid storage cavity (2) is changed by driving the sliding plate (5) to move, a liquid level difference is formed between the first liquid storage cavity (2) and the second liquid storage cavity (3), and perfusion driven by the liquid level difference is generated in the central annular chamber (6).

2. The vascularized organoid microfluidic culture chip of claim 1, wherein, Sliding grooves (131) are arranged on opposite sides of the first liquid storage cavity (2), and sliding blocks (51) which are in sliding cooperation with the sliding grooves (131) are arranged on opposite sides of the sliding plate (5).

3. The vascularized organoid microfluidic culture chip of claim 2, wherein, A sealing strip is arranged on the side of the sliding plate (5) in contact with the cavity wall of the first liquid storage cavity (2), and the sealing strip is used for sealing the gap between the sliding plate (5) and the cavity wall of the first liquid storage cavity (2).

4. The vascularized organoid microfluidic culture chip of claim 1, wherein, The base (1) comprises a chip body (13), a patterned double-sided adhesive (12) and a cover glass (11) which are sequentially stacked from top to bottom; The chip body (13) is provided with a first hole (21) corresponding to the first liquid storage cavity (2), a second hole (31) corresponding to the second liquid storage cavity (3) and the organoid culture cavity (4); The patterned double-sided adhesive (12) has a notch (121), and when the chip body (13), the patterned double-sided adhesive (12) and the cover glass (11) are combined in layers, the notch (121) cooperates with the chip body (13) and the cover glass (11) to form the central annular chamber (6).

5. The vascularized organoid microfluidic culture chip of claim 4, wherein, The thickness of the patterned double-sided adhesive (12) is between 100 mu m and 300 mu m, and is preferably 200 mu m.

6. The vascularized microfluidic culture chip of claim 5, wherein, The central annular chamber (6) is used for forming a blood vessel network.

7. A method for culturing three-dimensional vascularized organoids using the vascularized organoid microfluidic culture chip according to any one of claims 1-6, wherein, The application comprises the following steps: Step one: injecting a cell-fibrinogen-thrombin gel into the central annular chamber (6); Step two: injecting a cell-free fibrinogen gel into the organoid culture cavity (4); Step three: adding cell culture solution into the first liquid storage cavity (2) and the second liquid storage cavity (3), so that the liquid level height of the cell culture solution in the first liquid storage cavity (2) is different from the liquid level height of the cell culture solution in the second liquid storage cavity (3); Step four: culturing a three-dimensional blood vessel network in the central annular chamber (6), and then inoculating organoids into the organoid culture cavity (4); Step five: moving the sliding plate (5) to form a liquid level difference between the first liquid storage cavity (2) and the second liquid storage cavity (3), so as to generate perfusion in the three-dimensional blood vessel network of the central annular chamber (6), and realize long-term dynamic co-culture of the three-dimensional blood vessel network and the organoids.

8. The method of culturing three-dimensional vascularized organoids according to claim 7, wherein, In step five, when the liquid level of the first liquid storage cavity (2) and the second liquid storage cavity (3) tend to be consistent, the sliding plate (5) is pushed again to reestablish the liquid level difference, and the operation is repeated to maintain long-term stable perfusion.

9. The method of culturing three-dimensional vascularized organoids according to claim 7, wherein, The cell-fibrinogen-thrombin gel is a fibrin gel containing human umbilical vein endothelial cells and fibroblasts.

10. The method of culturing three-dimensional vascularized organoids of claim 7, wherein, The preparation method of the organoid: tumor cells of patient origin or other origin are obtained in single cell suspension through mechanical shearing, enzymolysis and screening, and then embedded in Matrigel and cultured in a culture solution containing specific growth factors to form the organoid with a cavity and a vacuole structure.

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