A vascular organoid perfusion culture chip and methods of use thereof

Abstract

The present application relates to the technical field of vascular organoid perfusion culture, and specifically discloses a vascular organoid perfusion culture chip and a use method thereof. The chip is assembled by an upper cover plate, a middle chip and a bottom chip. The organoid culture hole of the bottom chip is provided with a central planting area and a micro-groove array structure, and the channel is provided with a flow field diameter reduction joint and an anti-bubble chamfer structure. The present application cooperates the local fluid shear stress generated by the micro-groove physical structure in the hole and the diameter reduction of the bottom channel to provide double synergistic guidance of topological morphology and fluid mechanics for the germination of the vascular organoid, and overcomes the defect of disordered sprouting in the traditional well plate. The anti-bubble design and the multi-stage buffer flow path ensure the stability of long-term dynamic perfusion and promote the maturity of the in-vitro capillary network model.

Description

Technical Field

[0001] This invention relates to the field of vascular organoid perfusion culture technology, specifically to a vascular organoid perfusion culture chip and its usage method. Background Technology

[0002] In fields such as angiogenesis mechanism research, disease simulation, drug screening, and tissue engineering, vascular organoids play an important role in highly biomimetic in vitro simulation of in vivo vascular structure and function. However, traditional static plate culture lacks a dynamic blood perfusion environment and cannot simulate in vivo blood flow and mechanical stimulation, resulting in a significant gap between the growth and development of vascular organoids and the actual in vivo state. Furthermore, there are stability and habitat simulation issues such as random and chaotic vascular budding direction and low maturity of capillary networks. Therefore, it is necessary to introduce a technical approach that combines dynamic microfluidics and physical topology guidance to improve this.

[0003] Existing technologies, such as Chinese invention patent CN104630059A "A microfluidic chip and method for establishing an in vitro co-culture model of three types of cells", are characterized by using microfluidic channels to infuse liquid and combining it with matrix gel to form a three-dimensional culture space, which enables dynamic observation of cell growth. However, they have shortcomings such as lack of precise physical structural guidance for the three-dimensional growth direction of vascular organoids, difficulty in matching the local flow field shear force with physiological stress, and easy stomatal retention at the intersection of right angle interfaces.

[0004] The existing scheme has a specific drawback: the simple bottom structure of the organoid culture well leads to isotropic mechanical and topological stimulation of endothelial cells in the substrate, resulting in vascular sprouting extending radially and randomly, which makes it impossible to qualitatively and directionally induce the orderly connection of the vascular network. Summary of the Invention

[0005] This invention provides a vascular organoid perfusion culture chip and its usage method, aiming to solve the problem in related technologies where the vascular buds of vascular organoid perfusion culture chips extend radially and randomly, making it impossible to qualitatively and directionally induce the orderly connection of vascular networks.

[0006] A perfusion culture chip for vascular organoids includes an upper cover plate for sealing and preventing contamination, a middle chip for primary distribution of storage liquid and fluid, a bottom chip for vascular organoid colonization and culture, and a perfusion inlet and outlet for perfusion culture. The middle chip has multiple storage holes, and the bottom chip is divided into corresponding alignment holes and organoid culture holes. The upper cover plate covers the top of the middle chip. The organoid culture holes include a central colonization area located at the geometric center and a microgroove array structure extending from the central colonization area to the surrounding edges. A bottom channel connecting the storage holes and the organoid culture holes is provided between the middle chip and the bottom chip. The bottom channel has a flow field narrowing structure in the area below the organoid culture holes. An anti-bubble chamfer structure is provided at the connection between the vertical channel inside the middle chip and the bottom channel of the bottom chip.

[0007] The effects are as follows: By introducing a central colonization zone and microgroove array structure inside the organoid culture wells of the bottom chip, the isotropic and chaotic growth state of organoid budding in traditional flat-bottomed culture wells is broken. The microgroove array provides a clear three-dimensional physical space track for the migration and luminal extension of vascular endothelial cells, realizing directional budding of blood vessels by utilizing the contact guidance characteristics of cells. The flow field narrowing structure 17 set at the bottom channel can change the cross-sectional area of ​​the fluid entering the culture well region, forming a gradient of fluid shear stress above the organoid culture well and inside the microgrooves without increasing the overall pumping flow rate. The dual synergy of physical topology and local fluid dynamic stimulation promotes the orderly extension and maturation of the capillary network of vascular organoids. The anti-bubble chamfer structure 18 eliminates the right-angle dead volume region in the microfluidic channel, avoids the retention of microbubbles at the interface, and ensures the continuity of the fluid microenvironment and cell survival rate during long-term dynamic perfusion culture. The bottom-layer chip is designed with alignment wells and organoid culture wells, allowing for spatial alignment with the liquid storage wells of the middle-layer chip during assembly, facilitating the storage and supply of culture medium. The sealed, anti-contamination design of the top cover plate blocks the entry of microorganisms from the external environment, maintaining a sterile internal culture environment.

[0008] Preferably, the microgroove array structure is radially distributed, with the width of a single microgroove being 10-50 μm and the depth being 20-100 μm, and the center angle between adjacent microgrooves being 15°-45°. The size range of the microgrooves matches the natural diameter of human microvessels and capillaries, physically restricting the random expansion of cells in non-target directions, so that the fluid entering the groove forms a microscale laminar flow, promoting the targeted delivery of nutrients along the extension direction of endothelial cells.

[0009] Preferably, the flow field narrowing structure is an inwardly protruding arc-shaped protrusion on the sidewall of the channel, which reduces the local channel cross-sectional area by 30%-60% to generate a local wall shear stress of 1-15 dyn / cm² in the fluid flowing above the organoid culture well. This shear stress range simulates the blood flow shear force level inside real human capillaries and venules, stimulates the expression of mechanoreceptors on the surface of vascular endothelial cells, and enhances the integrity of the vascular barrier function of organoids.

[0010] Preferably, the anti-bubble chamfer structure is located at the corner of the fluid flow direction and has a smooth transition slope with an inclination angle of 15°-60°. The smooth transition slope eliminates the stagnation zone at the fluid's forward advance, allowing the culture medium to smoothly pass over the channel corner during the initial injection and subsequent circulation, thus eliminating the risk of organoid ischemia and necrosis caused by air blockage.

[0011] Preferably, the top four corners of the middle layer chip have contact support pillars, and the upper cover plate maintains a gap of 0.1-0.5mm with the middle layer chip through the contact support pillars; this tiny gap prevents suspended particulate matter and microorganisms from the external environment from falling into the chip, while allowing carbon dioxide and oxygen in the incubator to diffuse into the space above the liquid storage hole, maintaining the pH balance of the culture medium.

[0012] Preferably, the bottom of the liquid storage hole of the middle layer chip is connected to a bottom channel on both sides, the upper end of the bottom channel is connected to a vertical channel, and the upper end of the vertical channel is connected to a horizontal channel to form a through flow path; the folded arrangement of the multi-level channels allows the fluid to be buffered and rectified before entering the final culture well, eliminating the high-frequency fluid oscillation caused by the external peristaltic pump pulse.

[0013] Preferably, the outer interface of the horizontal channel is provided with a threaded structure and connected with a Luer connector to form the irrigation inlet and irrigation outlet; the combination of the thread and the Luer connector provides mechanical connection and liquid seal, enabling the chip to interface with commercial infusion lines and drive pump equipment.

[0014] Preferably, the upper cover plate is a grooved plate with flanges, with side reinforcing ribs and vertical reinforcing ribs on its outer side and horizontal reinforcing ribs on its top surface; the reinforcing ribs make the upper cover plate less prone to warping and deformation when subjected to long-term temperature and humidity changes in the incubator or the hot-press sterilization process, thus ensuring the long-term dimensional stability of the entire chip shell structure.

[0015] Preferably, the central implantation area is a concave hemispherical or frustum-shaped groove with a maximum inner diameter slightly larger than the diameter of the pre-cultured vascular organoids. In the initial stage of sample addition, the organoids naturally settle and slide into the central implantation area under the action of gravity, avoiding the asymmetric phenomenon of subsequent microgroove budding guidance caused by inoculation position deviation.

[0016] Preferably, the upper cover plate, middle chip and bottom chip are made of polycarbonate material that has undergone surface hydrophilication plasma treatment, and the surface water contact angle is less than 45°. The high light transmittance material facilitates bright field and fluorescence real-time observation under an inverted microscope, and the hydrophilic surface reduces fluid propulsion resistance and enhances the adhesion of extracellular matrix gel to the bottom of the pore.

[0017] A method for using a perfusion culture chip for vascular organoids includes the following steps: S1. Chip fabrication and bonding: The middle layer chip and the bottom layer chip are bonded together using biocompatible adhesive or double-sided tape; the bottom channel section retains a hollow structure to ensure the channel's permeability; the liquid storage holes of the middle layer chip and the alignment holes of the bottom layer chip completely overlap and their external contours perfectly match; the internal channels of the assembled chip are hydrophilicated by introducing a modifying solution. S2. Use human pluripotent induced stem cells to differentiate vascular organoids, and then resuspend the vascular organoids in a mixture of collagen and Matrigel. S3. The vascular organoids resuspended in the matrix gel are successively seeded into the vascular organoid culture wells. The concave shape of the culture wells is used to allow the organoids to settle naturally and center along the central implantation area. Depending on the size of the vascular organoids, ensure that there are 1-3 vascular organoids per well. S4. Cover the top with the lid to keep it sealed, and place it upside down in a 37℃, 5% CO2 incubator for 60-90 minutes to allow the matrix adhesive to fully cross-link and cure.

[0018] S5. Connect one end of the perfusion culture tube to the perfusion inlet, and inject culture medium into the other end of the perfusion tube to fill the perfusion tube with liquid. During this process, use the anti-bubble chamfer structure in the channel to smoothly remove the remaining air; so that the liquid reservoir is filled with liquid and the height is maintained at 1 / 2-3 / 4 of the chip height. Then connect the liquid injection end of the perfusion tube to the perfusion outlet. S6. Connect the perfusion tube to the peristaltic pump and control the perfusion rate between 0.1 μL / min and 5 ml / min. At this time, the fluid is locally accelerated when it flows through the bottom channel under the action of the flow field narrowing structure, which applies physiological constant shear stress to the organoids in the culture well. Together with the microgroove array structure at the bottom, it qualitatively induces the vascular organoids to bud and mature in a directional manner.

[0019] The above method has the following beneficial effects: This method combines precise biological manipulation with the micro-topology and local flow field control of the chip's underlying structure. The inverted placement in step S4, combined with the central implantation in step S3, ensures the optimal position of the organoids in three-dimensional space. The dynamic perfusion in steps S5 and S6 not only effectively eliminates the dead zones that are prone to bubble formation in microfluidic chips, but also, within a suitable flow rate range, stimulates physiological-level shear stress through the narrowing structure, supplemented by the physical guidance of microgrooves. This results in longer branches, more complete lumens, and highly controllable orientation of the vascular organoid budding network generated in vitro, significantly improving the reliability of disease models and drug testing data.

[0020] By adopting the above technical solution, the beneficial effects of the present invention are as follows: 1. By combining the microgrooves of the bottom layer with the flow obstruction features of the local flow field, the directional guidance of the budding and extension of vascular organoids is realized in both the physical spatial morphology and the hydrodynamic stress dimensions, thereby improving the orderliness of the capillary network construction and the simulation accuracy of the model.

[0021] 2. By combining multi-stage flow channel buffering, diameter reduction flow obstruction design and anti-bubble slope, the fluid continuity and shear force stability of the microfluidic system under long-term dynamic perfusion are ensured, avoiding physical damage to fragile organoids caused by air blockage and flow field turbulence.

[0022] 3. The overall chip adopts a modular three-layer assembly mode, with the upper layer being breathable and pollution-proof, the middle layer being liquid storage and buffer, and the bottom layer being for colonization and germination. It is compatible with the hole spacing design of automated pipetting workstations, reducing human operation errors in high-throughput drug screening experiments. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the perfusion culture chip for vascular organoids of the present invention.

[0024] Figure 2 This is a schematic diagram of the mid-layer chip structure of the present invention.

[0025] Figure 3 This is a schematic diagram of the cross-sectional structure of the middle layer chip in this invention.

[0026] Figure 4 This is a schematic diagram of the underlying chip structure of the present invention.

[0027] Figure 5 This is a schematic diagram of the upper cover plate structure of the present invention.

[0028] Figure 6 This is a microscope image of organoids cultured on a chip in the example.

[0029] Figure 7 This is a schematic diagram of the micro-trench array structure on the underlying chip of the present invention.

[0030] Figure 8This is a schematic diagram of the flow field narrowing structure and the anti-bubble chamfered junction structure of the present invention.

[0031] Reference numerals: 1. Upper cover plate; 2. Middle chip; 3. Bottom chip; 4. Liquid storage hole; 5. Irrigation inlet; 6. Irrigation outlet; 7. Contact support column; 8. Bottom channel; 9. Vertical channel; 10. Horizontal channel; 11. Luer connector; 12. Alignment hole; 13. Organoid culture well; 14. Side reinforcing rib; 15. Vertical reinforcing rib; 16. Horizontal reinforcing rib; 17. Flow field diameter reduction structure; 18. Anti-bubble chamfer; 19. Microgroove array structure. Detailed Implementation

[0032] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0033] like Figures 1-6 As shown, a perfusion culture chip for vascular organoids consists of an upper cover plate 3, a middle chip 2, a bottom chip 1, and external connecting components. The general process is as follows: fluid enters the buffer channel inside the middle chip 2 through the external connecting components, and then flows downward through the anti-bubble chamfer structure 18 into the bottom chip 1. After local throttling and acceleration in the bottom chip 1, physiological shear force is applied to the organoids in the central colonization area, guiding the cells to develop along the microgroove array structure 19. After the fluid collects, it flows out of the chip to form a cycle.

[0034] The upper cover plate 3 includes a cover plate body, side reinforcing ribs 14, vertical reinforcing ribs 15, horizontal reinforcing ribs 16, and edge flanges.

[0035] The cover plate body has an integrally recessed, shallow groove-shaped rectangular thin-walled structure, made of high-transmittance medical-grade polystyrene. The amorphous structure of polystyrene gives the plate excellent optical transmittance, stable refractive index, low autofluorescence background, and transmittance of over 90%. Side reinforcing ribs 14 and vertical reinforcing ribs 15 are perpendicularly arranged along the outer edges of the long and short sides of the cover plate body, and their cross-sections are mostly chamfered rectangles.

[0036] Horizontal reinforcing ribs 16 are distributed in a grid pattern on the top surface of the cover plate body, and the grid nodes are rounded to reduce stress concentration. Edge flanges are set around the lower edge of the cover plate body, and their inner contour dimensions are slightly larger than the outer edge dimensions of the middle layer chip 2 described later. The length of the flanges extending inward is sufficient to cover the chip seams.

[0037] The effects or functions of the components under the upper cover plate 3 are as follows: the overall shallow groove and edge flange form a covering barrier, physically blocking the intrusion path of airborne sediment colonies and dust particles, maintaining the sterility of the system; the side reinforcing ribs 14, vertical reinforcing ribs 15 and horizontal reinforcing ribs 16 improve the bending section modulus of the thin-walled cover plate without obstructing the top observation view, avoiding warping deformation caused by material internal stress release and frequent handling when placed in a 37-degree Celsius constant temperature incubator for a long time, ensuring the flatness of the focal plane during optical detection; the high light transmittance material meets the direct transmission imaging requirements of non-invasive optical microscopes, allowing researchers to conduct in-situ bright-field or fluorescence tracking observation of the angiogenesis process in the culture well for several consecutive days.

[0038] The middle layer chip 2 includes a chip main frame, a liquid storage hole 4, a contact support post 7, a horizontal channel 10, and a vertical channel 9.

[0039] The chip main frame is a rectangular plate with a certain thickness, and its length and width ratio conforms to the standard microporous plate specifications. Contact support posts 7 are located at the four corners of the top surface of the chip main frame. They are tiny cylindrical protrusions with a height maintained between 0.1 and 0.5 millimeters.

[0040] The liquid storage hole 4 consists of multiple large-diameter circular holes that vertically penetrate the main frame of the chip. These holes are arranged in a straight line or rectangular array, and the hole walls are polished. The contact support post 7 has a positional fit with the edge flange and the cover plate body of the upper cover plate 3 mentioned above. When the upper cover plate 3 is fastened, the contact support post 7 presses against the inner surface of the cover plate body, so that a gap for gas exchange is maintained between the upper cover plate 3 and the top surface of the main frame of the chip. At the same time, the edge flange forms a labyrinth-like barrier on the outside.

[0041] The horizontal channel 10 is located inside the main chip frame near the end, with its two ends leading to the outside and inside of the chip respectively, and is in the shape of a cylindrical hollow tube. The vertical channel 9 is vertically connected to the inner end of the horizontal channel 10 and extends downward to the bottom surface of the main chip frame. The effects or functions of the components under the middle layer chip 2 are as follows: the main chip frame constitutes the core supporting skeleton of the chip, providing solid support for all microfluidic channels and housing chambers; the liquid storage hole 4 contains a volume of culture medium during the culture process, increasing the fluid capacity inside the chip, and acting as a kind of intravenous reservoir, buffering the fluctuations in osmotic pressure of the culture medium caused by cell metabolism and natural water evaporation, and extending the time cycle for changing the culture medium; The contact support column 7 not only bears the weight of the upper cover plate 3, but the gaps it forms, combined with the labyrinthine barrier design, not only prevent external bacteria from falling in, but also allow the carbon dioxide gas required for maintaining the physiological concentration in the incubator to smoothly diffuse into the liquid surface of the storage hole 4, maintaining the pH balance of the internal buffer solution. The horizontal channel 10 and the vertical channel 9 constitute the internal water supply network for fluid to enter the culture unit from the outside. The 90-degree bend of the pipeline folds the path of the incoming culture medium and dissipates kinetic energy, which plays a role in fluid rectification and reducing pressure pulses.

[0042] The bottom layer chip 1 includes a substrate, alignment holes 12, organoid culture wells 13, a microgroove array structure 19, a bottom channel 8, a flow field diameter reduction structure 17, and an anti-bubble chamfer structure 18. The substrate is a transparent thin plate with a flat bottom surface, which is injection molded from the same polycarbonate material as the middle layer chip 2 mentioned above, and is treated with an oxygen plasma surface modification process.

[0043] Alignment wells 12 are distributed on the substrate, and their positions and diameters correspond one-to-one with the liquid storage wells 4 of the middle layer chip 2 mentioned above. Organoid culture wells 13 are located at the center of the bottom of each alignment well 12, and each well is a depression with a central implantation area, with a smooth transition at the bottom edge of the depression.

[0044] The microgroove array structure 19 consists of multiple tiny radial grooves etched into the bottom and surrounding areas of the organoid culture wells 13. The width of a single microgroove is set to 10 to 50 micrometers, and the depth is set to 20 to 100 micrometers. The bottom channel 8 is a horizontally oriented microchannel located inside the substrate, with both ends opening upwards. One end connects to the vertical channel 9 of the middle layer chip 2 mentioned above, and the other end connects to the bottom sidewall of the liquid storage hole 4.

[0045] The flow field narrowing structure 17 is located on the side wall of the middle section of the bottom channel 8, near the organoid culture well 13, and appears as an inwardly narrowing protrusion that narrows the channel width. The anti-bubble chamfer structure 18 is located at the junction of the vertical channel 9 and the bottom chip 1, and is a transition slope with an inclination angle. The bottom contours of the bottom chip 1 and the middle chip 2 are matched and bonded together with biocompatible adhesive.

[0046] The effects or functions of each component under the bottom chip 1 assembly are as follows: The substrate seals the bottom contour of the middle chip 2; plasma modification imparts high hydrophilicity to the surface, allowing the culture medium and extracellular matrix gel to achieve a smaller contact angle on this surface, enhancing the adhesion of the gel; the alignment well 12 and the reservoir well 4 are spliced ​​together to form a complete three-dimensional containment cavity, providing space for the operation of large-volume culture media; the concave morphology of the organoid culture well 13 uses gravity to guide the initially inoculated spherical organoids suspended in the uncrosslinked matrix gel to automatically slide down the slope and be centrally implanted, eliminating the uneven placement caused by the position deviation of the pipette during manual spotting; the microgroove array structure 19, through the micron-level undulations on the surface, provides three-dimensional spatial anchor points for the extension of pseudopodia of vascular endothelial cells, based on cells... The contact guidance effect forces endothelial cells to grow in an orderly, mesh-like pattern along the groove direction, preventing cells from spreading disorderly to the surrounding flat areas. The bottom channel 8 connects the liquid inlet point to the culture pool, constructing a closed-loop liquid path at the bottom layer. The flow field narrowing structure 17 increases the local flow velocity when the fluid passes through the narrowing section according to the continuity equation of fluid mechanics, forming microscale wall shear stress in the organoid culture well 13 that conforms to the physiological environment of human capillaries. This mechanical stimulation can upregulate the expression of endothelial cell layer-related adhesion junction proteins, stimulating vascular maturation and lumen formation. The anti-bubble chamfer structure 18 guides the smooth movement of the liquid surface front during the perfusion bed construction stage, preventing air from being trapped in the right-angle blind zone due to surface tension and forming long-term stagnant bubbles, thus eliminating the hidden danger of air blockage blocking the flow path.

[0047] The external connection components include a threaded interface, a Luer connector 11, and a flexible irrigation tube. The threaded interface is integrally formed on the outer opening surface of the horizontal channel 10 of the middle layer chip 2 mentioned above.

[0048] The Luer connector 11 features internal and external threads that match the threaded interface and a conical sealing structure with a one-sixth taper. A flexible irrigation tube, made of medical-grade silicone, is fitted onto the other end of the Luer connector 11 and connects to an external constant-flow peristaltic pump via a soft polymer material.

[0049] The effects or functions of each component in the external connection assembly are as follows: The threaded interface and Luer connector 11 realize the mechanical transition and tight locking between the microfluidic chip's microchannel and the external large-size macro fluid pipeline. Its conical interference fit provides sufficient sealing performance to resist working hydraulic pressure, eliminating the risk of leakage under long-term operation and ensuring the biosafety of long-term culture experiments; The flexible perfusion tube's good elastic modulus isolates the mechanical vibration of the external peristaltic pump rotor from the chip body, maintains the stability of the focal plane of the microscopic imaging, and prevents image shaking and blurring when shooting under high magnification. At the same time, its high air permeability also helps to replenish dissolved oxygen in the liquid inside the pipeline.

[0050] Working principle: In the experimental preparation stage, the bottom chip 1 and the middle chip 2 were first aligned and bonded by applying biocompatible adhesive or using medical double-sided tape, and then cured for a certain period of time to ensure complete sealing of the junctions of each internal channel and prevent adhesive overflow from clogging the microchannels. After the chip assembly was completed, the chip channels were rinsed, dried, and sterilized using deionized water and ethanol solution. The surface of the substrate, especially the organoid culture wells 13 and the microgroove array structure 19, was immersed and modified with a surface modification solution. After standing and washing, the inner walls of the channels acquired hydrophilic properties, improving the adhesion of subsequent matrix adhesive materials. In the organoid inoculation stage, spherical vascular organoids, pre-differentiated to specific germ layers and maturity levels using human pluripotent induced stem cells, are collected. The organoids are resuspended in a liquid matrix gel solution prepared from a mixture of collagen and relevant extracellular matrix extracts. Using a precision pipette, droplets of gel carrying the organoids are aspirated and individually dropped into the organoid culture wells 13 of the bottom layer chip 1. Due to the concave shape of the central implantation area of ​​the culture well, before the gel undergoes thermal cross-linking and solidification in its liquid state, the spherical vascular organoids settle under gravity and slide down the slope of the concave pit, eventually settling firmly at the geometric center of the well bottom. At this point, each well contains a small number of individual vascular organoids. Subsequently, the upper cover plate 3 is placed on top of the middle layer chip 2, and the entire chip is inverted and placed in a constant-temperature incubator at 37 degrees Celsius containing 5% carbon dioxide for static incubation. The purpose of inverting the placement is to prevent the suspended organoids from deforming due to excessive adhesion to the bottom before gel solidification. After standing for 60 to 90 minutes, the polypeptide chains inside the matrix gel undergo complete three-dimensional network cross-linking and solidification, realizing the phase transition of the matrix gel, thereby firmly fixing the vascular organoids in the three-dimensional space of the organoid culture well 13. During the infusion initiation phase, the flexible perfusion tube and Luer connector 11 of the external connection component are screwed into the threaded interface of the middle layer chip 2, which serves as the perfusion inlet 5. The external peristaltic pump starts operating, driving liquid culture medium containing specific growth-inducing factors and nutrients into the flexible perfusion tube, and then into the horizontal channel 10 of the middle layer chip 2. After flowing laterally along the horizontal channel 10, the culture medium turns and flows downward into the vertical channel 9. At the junction of the vertical channel 9 and the bottom channel 8 of the bottom layer chip 1, thanks to the smooth slope transition guidance of the anti-bubble chamfer structure 18 and the hydrophilic properties of the surface, the liquid gas-liquid interface maintains a continuous forward pushing state, without airflow entrainment, cavitation, or microbubble stripping at the corner. After the air is expelled, the culture medium flows into the reservoir 4 along the bottom channel 8 until the liquid level rises to most of the height of the middle layer chip 2, and then the perfusion outlet 6 on the other side is connected to the return pipeline. During the dynamic culture and induction phase, the rotational speed of the external drive pump was adjusted to set the overall perfusion velocity within a specific operating range. The bubble-free culture medium encountered a flow field narrowing structure 17 as it flowed through the middle section of the bottom channel 8. Due to the reduced cross-sectional area of ​​the physical channel, the local fluid velocity was increased. This accelerated microjet smoothly swept over the organoids encased in the solid matrix gel and the surrounding microgroove array structure 19. On one hand, the fluid wall shear stress generated by friction at the gel interface was transmitted to the internal vascular endothelial cells through the gel's three-dimensional porous network. This dynamic mechanical stimulation activated the mechanoreceptor channel mechanism on the cell membrane surface, promoting the rearrangement of endothelial cell cytoskeleton proteins and the autocrine expression of pro-angiogenic factors, increasing the distribution density of tight junction proteins between endothelial cells. On the other hand, after receiving fluid stimulation, the organoid cells began to extend filopodia outwards to explore for budding, and the bottom microgroove array structure 19 provided physical three-dimensional boundaries and spatial guidance tracks. Under the influence of the contact-guided effect, budding endothelial cells tend to adhere, proliferate, and migrate along the longitudinal extension of the microgrooves, avoiding ineffective and disorderly spread to the surrounding flat areas lacking microstructure. With continuous circulation of the culture medium, the fluid containing cellular metabolic waste, after completing its exchange of substances, tumbles upwards above the reservoir pore 4, and is eventually pushed out of the reservoir cavity, exiting the chip system through the perfusion outlet 6 and returning to the external reservoir bottle. The entire process is carried out continuously for several days in a sealed environment with the upper cover plate 3, allowing for real-time monitoring of cell morphological changes through an optical microscope via the upper cover plate 3 and the substrate.

[0051] Ultimately, under the combined constraints of physical topological orbits and hydrodynamic shear stimuli, a highly mature capillary network model with a highly ordered morphology, long-range extension, and through-luminal structure was generated within the chip. This model can be used for subsequent fluorescence staining analysis of extracts or pathological drug droplet addition tests.

[0052] A method for using a perfusion culture chip for vascular organoids includes the following steps: S1. Chip fabrication and bonding: The middle layer chip 2 and the bottom layer chip 1 are bonded together using biocompatible adhesive or double-sided tape; the bottom channel 8 part retains a hollow structure to avoid affecting the channel's permeability; the liquid storage hole 4 of the middle layer chip 2 and the alignment hole 12 of the bottom layer chip 1 completely overlap and their external contours perfectly match; the internal channels of the assembled chip are hydrophilicated by introducing a modification solution. S2. Use human pluripotent induced stem cells to differentiate vascular organoids, and then resuspend the vascular organoids in a mixture of collagen and Matrigel. S3. The vascular organoids resuspended in the matrix gel are seeded one by one into the vascular organoid culture well 13. The concave shape of the culture well is used to allow the organoids to settle naturally and center along the central implantation area. Ensure that there are 1-3 vascular organoids per well according to the size of the vascular organoids. S4. Cover with the upper cover plate 3 to keep it sealed, and place it upside down in a 37℃, 5% carbon dioxide incubator for 60-90 minutes to allow the matrix adhesive to fully cross-link and cure.

[0053] S5. Connect one end of the perfusion culture tube to the perfusion inlet 5, and inject culture medium into the other end of the perfusion tube to fill the perfusion tube with liquid. During this process, use the anti-bubble chamfer structure 18 in the channel to smoothly remove the residual air; so that the liquid storage hole 4 is filled with liquid and the height is maintained at 1 / 2-3 / 4 of the chip height. Then connect the liquid injection end of the perfusion tube to the perfusion outlet 6. S6. Connect the perfusion tube to the peristaltic pump and control the perfusion rate to between 0.1 μL / min and 5 ml / min. At this time, when the fluid flows through the bottom channel 8, it is locally accelerated under the action of the flow field narrowing structure 17, which applies physiological constant shear stress to the organoids in the culture well. Together with the microgroove array structure 19 at the bottom, it qualitatively induces the vascular organoids to bud and mature in a directional manner.

[0054] The above method has the following beneficial effects: This method combines precise biological manipulation with the micro-topology and local flow field control of the chip's underlying structure. The inverted placement in step S4, combined with the central implantation in step S3, ensures the optimal position of the organoids in three-dimensional space. The dynamic perfusion in steps S5 and S6 not only effectively removes the dead zones that are easily generated by microfluidic chips, but also stimulates physiological-level shear stress within a suitable flow rate range by means of the diameter reduction structure. With the physical guidance of the microgrooves, the budding network of the in vitro vascular organoids has longer branches, more complete lumens, and highly controllable orientation, which greatly improves the reliability of disease models and drug testing data.

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A vascular organoid perfusion culture chip, comprising: The structure comprises an upper cover plate (3) for sealing and preventing contamination, a middle chip (2) for primary distribution of the stored liquid and fluid, a bottom chip (1) for the colonization and culture of vascular organoids, and a perfusion inlet (5) and a perfusion outlet (6) for perfusion culture; characterized in that: The middle layer chip (2) is provided with multiple liquid storage holes (4), the bottom layer chip (1) is divided into corresponding alignment holes (12) and organoid culture holes (13), and the upper cover plate (3) covers the top of the middle layer chip (2); the organoid culture hole (13) includes a central implantation area located at the geometric center and a microgroove array structure (19) extending from the central implantation area to the surrounding edges. The middle chip (2) and the bottom chip (1) are provided with a bottom channel (8) connecting the liquid storage hole (4) and the organoid culture hole (13). The bottom channel (8) is provided with a flow field narrowing structure (17) in the area corresponding to the organoid culture hole (13). The vertical channel (9) inside the middle chip (2) and the connection between the bottom chip (1) are provided with an anti-bubble chamfer structure (18).

2. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The microgroove array structure (19) is radially distributed, with a width of 10-50 μm and a depth of 20-100 μm for each microgroove, and a center angle of 15°-45° between adjacent microgrooves.

3. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The flow field narrowing structure (17) is an arc-shaped protrusion that bulges inward on the sidewall of the channel, reducing the local channel cross-sectional area by 30%-60% to generate a local wall shear stress of 1-15 dyn / cm² in the fluid flowing above the organoid culture well (13).

4. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The anti-bubble chamfer structure (18) is located at the corner of the fluid flow direction and has a smooth transition slope with an inclination angle of 15°-60°.

5. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The top four corners of the middle layer chip (2) have contact support pillars (7), and the upper cover plate (3) maintains a gap of 0.1-0.5mm with the middle layer chip (2) through the contact support pillars (7).

6. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The liquid storage hole (4) of the middle layer chip (2) is connected to the bottom channels (8) on both sides. The upper end of the bottom channel (8) is connected to the vertical channel (9), and the upper end of the vertical channel (9) is connected to the horizontal channel (10), forming a through flow path.

7. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The outer interface of the horizontal channel (10) is provided with a threaded structure and connected with a Luer connector (11), forming the irrigation inlet (5) and irrigation outlet (6).

8. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The upper cover plate (3) is a grooved plate with a flange, and its outer side is provided with side reinforcing ribs (14) and vertical reinforcing ribs (15), and its top surface is provided with horizontal reinforcing ribs (16).

9. The vascular organoid perfusion culture chip according to claim 1, characterized in that, The central implantation area is a concave hemispherical or frustum-shaped groove, with its maximum inner diameter slightly larger than the diameter of the pre-cultured vascular organoid.

10. A method for using a vascular organoid perfusion culture chip, employing the vascular organoid perfusion culture chip of claim 9, comprising the following steps: S1. Chip fabrication and bonding: The middle layer chip (2) and the bottom layer chip (1) are bonded together using biocompatible adhesive or double-sided tape; the bottom channel (8) is kept hollow to avoid affecting the channel's permeability; the liquid storage hole (4) of the middle layer chip (2) and the alignment hole (12) of the bottom layer chip (1) completely overlap and their external contours match perfectly; the internal channels of the assembled chip are hydrophilicized by introducing a modification solution. S2. Use human pluripotent induced stem cells to differentiate vascular organoids, and then resuspend the vascular organoids in a mixture of collagen and Matrigel. S3. The vascular organoids resuspended in the matrix gel are successively seeded into the vascular organoid culture wells (13). The concave shape of the culture wells is used to allow the organoids to settle naturally and center along the central implantation area. Ensure that there are 1-3 vascular organoids per well according to the size of the vascular organoids. S4. Cover the upper cover plate (3) to keep it sealed, and place it upside down in a 37°C, 5% carbon dioxide incubator for 60-90 minutes to allow the matrix adhesive to fully cross-link and cure. S5. Connect one end of the perfusion culture tube to the perfusion inlet (5), inject culture medium into the other end of the perfusion tube, fill the perfusion tube with liquid, and use the anti-bubble chamfer structure (18) in the channel to smoothly remove the remaining air; so that the liquid storage hole (4) is filled with liquid and the height is maintained at 1 / 2-3 / 4 of the chip height. Then connect the liquid injection end of the perfusion tube to the perfusion outlet (6). S6. Connect the perfusion tube to the peristaltic pump and control the perfusion rate to be between 0.1ul / min and 5ml / min. At this time, when the fluid flows through the bottom channel (8), it is locally accelerated under the action of the flow field narrowing structure (17), which applies physiological constant shear stress to the organoids in the culture well. Together with the microgroove array structure (19) at the bottom, it qualitatively induces the vascular organoids to bud and mature in a directional manner.

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