A microarray patterning microfluidic culture chip which can be inserted into an insertion assembly

Abstract

The application discloses a microarray patterned microfluidic culture chip which can be inserted into an insertion assembly and belongs to the technical field of microfluidic chips, and comprises a culture substrate, wherein a culture cavity is arranged in the middle of the culture substrate, the culture cavity comprises a surrounding plate of an outer ring, and the surrounding plate and the culture substrate form an open culture solution storage cavity; a plurality of culture wells are arranged in an array on the culture substrate in the range of the surrounding plate, a micro-pit array is arranged in the culture well, the culture well can be inserted into the insertion assembly, an auxiliary hole is arranged between the transversely distributed culture wells, integrally formed flow channel plates are arranged on the two sides of the culture cavity, and sealing plates are connected to the bottom of the flow channel plates. The microarray patterned microfluidic culture chip is matched with the insertion assembly and a gas pressure control device, liquid is driven to reciprocate between the culture well and the cylinder of the insertion assembly through periodic gas pressure change, local microenvironment is forced to be uniform, and a nutrition gradient and a waste gradient are weakened; the chip as a whole keeps absolute stillness, real-time microscopic imaging can be directly carried out from the bottom of the bottom plate, and stopping and standing are not needed, and observation is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a microarray patterned microfluidic culture chip with insertable components. Background Technology

[0002] Biomedical research is gradually shifting from traditional two-dimensional cell culture models to more biologically relevant three-dimensional culture methods. Three-dimensional cell culture technology can more realistically replicate the three-dimensional structure, biomechanical properties, and intercellular interactions of cells in vivo, providing an important experimental platform for disease modeling, drug discovery, and tissue engineering. Organoids are a prime example of three-dimensional cell culture.

[0003] However, conventional culture methods typically rely on stasis, where cells exist within a liquid microenvironment where there are no changes other than diffusion, and no processes such as convection or advection that enhance the possibility of material exchange. This limits the application of three-dimensional cell culture in high-throughput analysis and industrial production.

[0004] To overcome stagnant culture, various liquid perturbation strategies have been developed in existing technologies. For example, shaking incubators or rotating drums drive liquid flow through overall mechanical motion. However, these methods cause overall movement of the cell culture device, severely interfering with real-time microscopic imaging, requiring operators to stop the machine and allow it to stand still before observation. Another example is the external direct tubing perfusion system, which drives liquid flow through multiple pairs of inlet and outlet tubing and a peristaltic pump. However, its structure is fragmented and complex, and the absolute flow velocity can easily wash away cell pellets. To prevent this, cell pellets must be suspended within the gel matrix, resulting in fresh liquid only indirectly supplying nutrients from the side of the gel, leading to low mass transfer efficiency. Furthermore, existing microfluidic chips mostly use external tubing to connect the flow channels, resulting in a complex chip structure that is difficult to integrate with standard automated equipment.

[0005] In addition, existing microfluidic chip culture devices typically require pipettes or pumps for liquid transfer during drug administration, which is cumbersome.

[0006] Therefore, there is an urgent need for a microfluidic culture chip system that can achieve localized liquid disturbance mass transfer while keeping the overall device stationary, and facilitates real-time observation and precise drug delivery. Summary of the Invention

[0007] The purpose of this invention is to provide a microarray patterned microfluidic culture chip with an insertable component. An internal flow channel system is formed by the integrated flow channel plate and sealing plate, enabling uniform liquid supply to multiple cells simultaneously. A reversible adapter allows for rapid docking with external liquid supply systems or sealing devices. A linked design between the cover and the insertable component allows for a single, integrated insertion and removal operation. The near-U-shaped microculture pits with a large opening and a small bottom promote cell spheroid formation and, when used with the matching insertable component, address the technical problems of uneven mass transfer caused by static culture and the inconvenience of real-time imaging with certain perturbation methods in existing technologies.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A microarray patterned microfluidic culture chip with an insertable component includes a culture medium plate. The culture medium plate has a culture chamber in its center, and the culture chamber includes an outer surrounding plate. The surrounding plate and the culture medium plate form a top-open culture medium storage chamber. A plurality of culture wells are arrayed on the culture medium plate within the area of ​​the surrounding plate. The culture wells are interconnected or non-interconnected hole structures for inserting the insertable component. Auxiliary holes are provided between the laterally distributed culture wells. Each culture well has a micro-culture pit group at its bottom, the micro-culture pit group comprising a plurality of arrayed micro-culture pits with openings facing inwards towards the culture well. The culture chamber has integrally formed flow channel plates on both sides. The lower surface of each flow channel plate has several open branch channels, each branch channel converging at a vertically penetrating liquid inlet. The end of each branch channel connects to the culture chamber through a vertically penetrating liquid outlet. A sealing plate is connected to the bottom of the flow channel plate, sealing the bottom openings of the liquid inlet, branch channels, and outlet, forming a single unit with the culture medium plate. An adapter or sealing device is connected above the liquid inlet.

[0009] Preferably, the adapter includes an adapter sleeve connected to the culture medium plate and a silicone connector. The adapter sleeve includes a central tube located at the center, an annular connecting part connected to the middle of the outer ring of the central tube, and a U-shaped rotating body connected to the outer ring of the annular connecting part. One end of the central tube is an adapter flat nozzle, and the other end is provided with spline teeth to form an adapter spline nozzle. Both ends of the central tube have a tapered inward structure, and an annular locking groove is formed by an inward concavity on the outer side of the near end face.

[0010] Preferably, the height of the adapter flat nozzle protruding from the rotating body is slightly less than the thickness of the flow channel plate, and the height of the adapter nozzle protruding from the rotating body is equal to the thickness of the flow channel plate. The end face of the adapter nozzle can extend into the bottom of the filling port. This size design ensures that the adapter nozzle and the bottom of the filling port are tightly connected, eliminating dead volume, while the end face of the rotating body fits against the upper surface of the flow channel plate, forming an end face seal.

[0011] Preferably, the outer periphery of the filling port is provided with an annular boss, and the inner ring of the annular boss is provided with a raised snap-fit ​​portion. The snap-fit ​​portion engages with the snap-fit ​​groove of the central tube of the adapter sleeve for a snap-fit ​​seal. The interference fit between the snap-fit ​​portion and the snap-fit ​​groove achieves axial positioning, and the mating silicone head fills the gap between the rotating body and the annular boss to form a radial seal.

[0012] Preferably, the culture medium plate and the sealing plate are bonded together by adhesive, adhesive tape, or welded by ultrasonic, laser, or hot-press welding. The sealing plate closes the open groove on the lower surface of the flow channel plate into a closed flow channel, which is simple to process and provides a reliable seal.

[0013] Preferably, the outer ring of the enclosure is provided with a gasket, and the cover sits on the gasket to cover the entire culture chamber, which not only preserves the possibility of gas exchange, but also prevents the evaporation of the culture medium and external contamination.

[0014] Preferably, the inner end face of the cover is provided with a snap-fit ​​portion for fixing the insert assembly or the dispensing device and a limiting block for limiting the position. The snap-fit ​​portion is an elastic clamping structure used to clamp the support leg or reinforcing rib of the insert assembly, so that the insert assembly and the cover are integrated into one piece; the limiting block is used to abut against the end face of the insert assembly when it is inserted in the reverse direction, thereby limiting the insertion depth.

[0015] Preferably, the diameter of the culture well is 1mm-50mm and the height of the surrounding plate is 1mm-30mm, to accommodate different specifications of insertion components and cell culture requirements.

[0016] Preferably, each culture well contains 0-500 micro-culture pits at its bottom, with each micro-culture pit having a diameter of 0.1mm-2mm and a spacing of 0.02mm-3mm between adjacent micro-culture pits. When the number is 0, the bottom of the culture well is flat and can be used for adherent cell culture or special experiments.

[0017] Preferably, the number of flow channels is 1-50, and the cross-sectional length of each flow channel is equal. The equal length design ensures that the flow resistance of each flow channel is consistent.

[0018] Preferably, the inner wall of the microculture pit is coated with a low-adhesion coating or an extracellular matrix coating. The low-adhesion coating promotes spontaneous formation of cell spheroids; the extracellular matrix coating enhances the adhesion and spreading ability of specific cells.

[0019] Preferably, the side profile of the microculture pit has a configuration with a large opening and a small bottom, including but not limited to V-shape, quadratic function shape, conic section shape, and catenary shape. These near-U-shaped configurations have a smooth transition at the bottom, without stress concentration, which is conducive to cell deposition and aggregation into spheres, and facilitates the complete removal of cell spheres.

[0020] Compared with the prior art, the beneficial effects of this application are as follows: The chip of this invention can be used with an insertion component and a gas pressure control device to drive the liquid to flow back and forth between the culture well and the cylinder of the insertion component through periodic gas pressure changes, thereby achieving forced homogenization of the local microenvironment and weakening the nutrient gradient and waste gradient; at the same time, the chip as a whole remains absolutely still, and real-time microscopic imaging can be performed directly from below the base plate without stopping the machine, which is convenient for observation. The culture wells and auxiliary holes are staggered between rows to maximize the number of culture sites within a limited area. The auxiliary holes also serve multiple functions, such as helping to form the culture well wall, balancing gas pressure, accommodating the local space of the plug-in, and providing spatial separation. The size and structure of the culture well are precisely matched with the matching insertion components, which can be inserted in the forward direction to achieve liquid oscillation mass transfer. The operation is simple and easy to observe. The open distribution channel on the lower surface of the flow channel plate is sealed by the sealing plate to form an internal flow path. Fresh culture medium enters from the liquid inlet, is evenly distributed through equal-length distribution channels, and then flows into the culture chamber from the liquid outlet, realizing the gentle renewal of the culture medium. The entire liquid supply system is integrated into the chip body, with a compact structure. The adapter sleeve, with its snap-fit ​​groove and annular boss, provides a snap-fit ​​seal, allowing for quick connection to external injection pumps, storage tanks, or automated liquid workstations. When not supplying liquid, the filling port can be sealed with a plugging device, offering flexible switching. The adapter sleeve adopts a central tube double-nozzle structure, namely the adapter flat nozzle and the adapter spline nozzle. When the adapter flat nozzle is facing up, the end face of the flat nozzle is flat, and when it is connected with silicone connectors or automated liquid workstation pipelines, the contact area is large, and after compression, an end face seal is formed, which is not easy to leak. When the adapter spline nozzle is facing up, it can be directly adapted to conventional medical silicone tubing without the need for an additional adapter. The inner end face of the cover is provided with a snap-fit ​​part for the insertion part. The snap-fit ​​part on the cover clamps and fixes multiple plug-in units or dosing devices, so that all plug-ins and the cover are integrated into one unit, simplifying the operation process and shortening the operation time. The limiting part forms a physical limit on the cover, ensuring that the plug-in can be fully inserted into the bottom of the cultivation well when inserted in the forward direction, providing perfect oscillation mass transfer conditions. Attached Figure Description

[0021] Figure 1 This is a top view of the microarray patterned microfluidic culture chip of the present invention; Figure 2 for Figure 1 Schematic diagram of cross-section structure; Figure 3 This is a bottom view schematic diagram of the flow channel layout on the lower surface of the flow channel plate of the present invention; Figure 4 This is a schematic diagram of the cover structure of the present invention; Figure 5 This is a schematic diagram of the cover structure of the present invention from a bottom view; Figure 6 This is a schematic diagram of the adapter sleeve structure of the present invention; Figure 7 This is a bottom view of the adapter sleeve structure of the present invention; Figure 8 This is a schematic cross-sectional view of the adapter sleeve of the present invention; Figure 9 This is a schematic diagram of the silicone head structure of the present invention; Figure 10 This is a cross-sectional view of the combined cover and insertion assembly of the present invention; List of identifiers in attached diagrams: 1-Culture plate; 2-Surrounding plate; 3-Cultivation well; 4-Auxiliary hole; 5-Micro-cultivation pit assembly; 501-Micro-cultivation pit; 6-Flow channel plate; 601-Diverter channel; 7-Liquid inlet; 701-Annular boss; 7011-Snap-fit ​​part; 8-Liquid outlet; 9-Sealing plate; 10-Sealing device; 11-Adapter sleeve; 1101-Central tube; 1102-Annular connection part; 1103-Rotating body; 1104-Snap-fit ​​groove; 12-Gasket; 13-Cover; 1301-Snap-fit ​​block; 1302-Limiting block; 14-Insert assembly; 15-Drug dispensing device; 16-Silicone head.

[0022] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] It should be noted that the terms "upper," "lower," "left," "right," "front," and "rear" used in the following description refer to the directions shown in the accompanying drawings, while the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections or electrical connections; they can refer to direct connections or indirect connections through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. Example 1

[0024] like Figures 1 to 10 As shown, this embodiment provides a microarray patterned microfluidic culture chip with insertable components, including a culture medium plate 1, a surrounding plate 2, a culture well 3, an auxiliary hole 4, a microculture pit group 5, a flow channel plate 6, a flow channel 601, a liquid inlet 7, a liquid outlet 8, a sealing plate 9, and a sealing device 10.

[0025] The culture medium plate 1 can be made of a transparent material such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), or glass. In this embodiment, the culture medium plate 1 has a rectangular plate structure.

[0026] The culture medium plate 1 has a culture chamber in the middle. The culture chamber includes an outer ring plate 2, which together with the culture medium plate 1 forms a top-open culture medium storage chamber. The height of the ring plate 2 is 1mm-30mm, preferably 5mm in this embodiment, and the wall thickness is 1mm-3mm. A gasket 12 is provided on the outer ring of the ring plate 2. The gasket 12 is a partial protrusion on the outer wall of the ring plate 2 with a height of 0.2mm-3mm, used to support the cover 13.

[0027] A plurality of culture wells 3 are arrayed on the culture medium plate 1 within the area of ​​the surrounding plate 2. In this embodiment, the number of culture wells 3 is 30, such as... Figure 1 or Figure 3 As shown, they are arranged in an array of 5 rows × 6 columns. Each culture well 3 has a circular hole structure with a diameter of 1mm-50mm and a depth of 1mm-30mm. The deep hole structure of the culture well 3 allows it to accommodate the inserted insertion component 14.

[0028] Auxiliary holes 4 are provided between the laterally distributed culture wells 3. The auxiliary holes 4 can be deep holes, shallow holes, or even non-formed holes in special cases. Preferably, the diameter of the auxiliary holes 4 is no larger than the diameter of the culture well 3. The culture wells 3 and auxiliary holes 4 are staggered between rows, with adjacent rows of culture wells 3 offset laterally, and the auxiliary holes 4 located below these offset positions, forming a close arrangement. The auxiliary holes 4 serve to collect the culture medium when the insertion component 14 is inserted into the culture well 3, preventing overflow and contamination. They also serve multiple functions, including pressure balancing, providing functional space for the insertion component, and creating spatial separation.

[0029] Each culture well 3 has a micro-culture pit group 5 at its bottom. The micro-culture pit group 5 contains several micro-culture pits 501 arranged in an array. Each culture well 3 has 0-500 micro-culture pits 501 at its bottom, with the diameter of the micro-culture pits 501 ranging from 0.1 mm to 2 mm, and the spacing between adjacent micro-culture pits 501 is 0.02-3 mm. The openings of the micro-culture pits 501 face the interior of the culture well 3. After the cell suspension is added, the cells are deposited in the micro-culture pits 501 under the action of gravity. Due to the combined effect of the aggregate morphology of the micro-culture pits and the coating, they spontaneously aggregate to form cell spheres of uniform size.

[0030] Depending on the type of cells being cultured, the inner wall of the micro-culture pit 501 can be selectively coated with different functional coatings.

[0031] When 3D suspension culture of tumor cell spheres, organoids, etc., is required, a low-adhesion coating is applied to the inner wall of the microculture pit 501. The low-adhesion coating uses a polyhydroxyethyl methacrylate (PHEMA) coating or a coating based on a certain polyethylene glycol (PEG) substrate. The specific coating method is as follows: an ethanol solution containing 2-methoxy-polyethylene glycol methacrylate monomer or a PEG substrate is dropped onto the inner wall of the microculture pit 501. Air is expelled from the pit using a centrifuge to ensure the coating solution permeates every point on the surface. After UV curing or solvent evaporation, a thin film is formed. This coating significantly reduces the adhesion between cells and the pit wall, promoting spontaneous aggregation of cells into spheres within the pit.

[0032] When three-dimensional culture of adherent cells such as hepatocytes and endothelial cells is required, an extracellular matrix (ECM) coating is applied to the inner wall of the microculture pit 501. The ECM coating uses Matrigel, type I collagen, or fibronectin. The coating method is as follows: Matrigel or collagen solution is applied to the inner wall of the microculture pit 501 and incubated at 37°C for 30 minutes to form a gel layer, or fibronectin solution is incubated overnight at 4°C for adsorption. This coating enhances cell adhesion and spreading, guiding cells to form specific adherent three-dimensional structures within the micropit. Example 2

[0033] The culture chamber has integrally molded flow channel plates 6 on both sides. The flow channel plates 6 and the culture medium plate 1 are integrally injection molded and made of the same material. The lower surface of the flow channel plates 6 has several open branch channels 601, such as... Figure 3 As shown. The branch channel 601 is a groove formed on the lower surface of the flow channel plate 6. The cross-section of the groove can be rectangular, semi-circular, or trapezoidal, with a width of 0.1mm-2mm and a depth of 0.1mm-1mm. The branch channels 601 on each side converge at the vertically penetrating liquid inlet 7. The liquid inlet 7 is a circular through hole with a diameter of 1mm-5mm, which extends vertically from the upper surface of the flow channel plate 6 to the lower surface.

[0034] The end of each branch channel 601 is connected to the outlet 8 located in the culture chamber. The outlet 8 is a circular through hole that vertically penetrates the channel plate 6 and the culture medium plate 1, directly connecting the end of the branch channel 601 to the culture medium storage chamber.

[0035] The number of branch channels 601 ranges from 1 to 50. In this embodiment, there are 5 branch channels 601 on each side, for a total of 10. All branch channels 601 are of equal length, that is, the centerline length of the channel from the inlet 7 to the outlet 8 is the same. This equal-length design ensures that the flow resistance of each branch channel 601 is consistent, making the liquid flow rate from each outlet 8 uniform and avoiding the problem of large flow rate at the near end and small flow rate at the far end in traditional branched channels.

[0036] A sealing plate 9 is connected to the bottom of the flow channel plate 6. The sealing plate 9 is a flat plate made of PS, PMMA, or COC, with a thickness of 0.2mm-3mm. The sealing plate 9 is bonded to the lower surface of the flow channel plate 6 by adhesive, adhesive tape, or by ultrasonic, laser, or hot-press welding. The sealing plate 9 completely seals the bottom opening of the distribution channel 601, forming a closed flow channel; it also seals the bottom periphery of the liquid inlet 7 and the liquid outlet 8 to prevent liquid leakage. After sealing, the flow channel plate 6, the sealing plate 9, and the culture medium plate 1 form a non-removable whole.

[0037] The sealing method for culture medium plate 1 and sealing plate 9 is determined based on the material selection: When the material is PS or PMMA, ultrasonic welding is used; the ultrasonic welding frequency is 20kHz-40kHz, the welding time is 0.5s-3s, and the welding pressure is 0.2MPa-0.6MPa. Before welding, a small amount of ethanol or special welding accelerator is applied to the lower surface of the flow channel plate 6 and the upper surface of the sealing plate 9 to improve the welding strength.

[0038] When the material is PDMS, oxygen plasma bonding is used; the lower surface of the flow channel plate 6 and the upper surface of the sealing plate 9 are simultaneously placed in an oxygen plasma cleaning machine for 30s-120s (power 50W-100W, oxygen flow rate 50sccm-200sccm), and then immediately bonded together and baked at 80℃ for 30min to cure the bonding.

[0039] When the material is COC, hot pressing welding is used; the flow channel plate 6 and the sealing plate 9 are aligned and stacked, and placed in a hot press. The heating temperature is 120℃-150℃, the pressure is 0.5MPa-2MPa, and the holding time is 10s-60s.

[0040] When a removable seal is required, use double-sided adhesive tape (such as 3M medical tape) or UV-cured optical adhesive.

[0041] The operation is as follows: Fresh culture medium is injected through the inlet 7 on one side and distributed to each distribution channel 601. From the end of the distribution channel 601, it flows through the outlet 8 to the culture medium storage chamber; within the culture medium storage chamber, it is distributed into each culture well 3 of the chip. If there is excess culture medium, it can be drawn away through the inlet on the other side, allowing the liquid to slowly flow into the culture wells and exchange substances with the cell spheres in the micro-culture pits 501 through diffusion. The inlet and outlet ports serve both the function of liquid inflow and outflow. Example 3

[0042] like Figure 2 , Figures 7 to 9 As shown, an adapter or sealing device 10 is connected above the liquid inlet 7. The adapter includes an adapter sleeve 11 and a mating silicone head 16.

[0043] The adapter sleeve 11 is injection molded from materials such as polypropylene (PP) or thermoplastic elastomer (TPE) and is a one-piece structure. The adapter sleeve 11 includes: Center tube 1101: Located at the center of adapter sleeve 11, with an outer diameter preferably 1mm-8mm. One end of the center tube 1101 is an adapter flat nozzle with a flat end face, used to connect to an external silicone tube or connector; the other end is provided with spline teeth to form an adapter nozzle, with 3-8 spline teeth, which is used to directly connect to a regular silicone tube when the adapter nozzle is facing upwards.

[0044] Annular connecting part 1102: connected to the middle of the outer ring of the central tube 1101, and has a disc-shaped or ring-shaped structure.

[0045] Rotating body 1103: Connected to the outer ring of the annular connecting part 1102, with a U-shaped cross-section on one side. In this embodiment, the cross-section opens towards the adapter nozzle side of the central tube 1101. The U-shaped cross-section gives the rotating body 1103 elastic deformation capability. When subjected to axial pressure, the U-shaped groove can produce micro-elastic deformation, enhancing the tightness of the fit with the mating silicone head 16.

[0046] The two ends of the central tube 1101 have a tapered inward structure, that is, the outer diameter of the end is slightly smaller than the outer diameter of the middle, and the cone angle is 1°-15°, which facilitates insertion into the liquid filling port 7 or the external connector. The central tube 1101 has an annular retaining groove 1104 formed by the inward concavity on both sides near the annular connection part.

[0047] The dimensional relationships of the components of the adapter are as follows: the height H1 of the adapter flat nozzle protruding from the rotating body 1103 is less than the thickness T of the flow channel plate 6 (H1 < T), which facilitates the outflow of culture medium during liquid addition; the height H2 of the adapter spline nozzle protruding from the rotating body 1103 is equal to the thickness T of the flow channel plate 6 (H2 ≤ T), ensuring that when the adapter sleeve 11 is inserted into the liquid inlet 7, the end face of the adapter spline nozzle can extend into the bottom of the liquid inlet 7 and be flush with the bottom end of the liquid inlet 7, so that the culture medium flows out from the middle of the spline teeth, thereby eliminating the dead volume at the bottom of the liquid inlet 7 and avoiding liquid residue and bubble accumulation.

[0048] Furthermore, an annular boss 701 is provided on the outer periphery of the filling port 7. The annular boss 701 is integrally formed with the flow channel plate 6, and its height is not greater than the vertical distance from the end face of the annular boss to the annular connecting part 1102. The inner ring of the annular boss 701 is provided with a raised snap-fit ​​part 7011, which is an annular flange or a number of protrusions distributed circumferentially. The snap-fit ​​part 7011 cooperates with the snap-fit ​​groove 1104 on the central tube 1101 to form a snap-fit ​​seal. When the adapter sleeve 11 is inserted downward into the filling port 7, the snap-fit ​​part 7011 slides into the snap-fit ​​groove 1104, simultaneously achieving axial limiting and preliminary sealing, and the outer ring of the annular boss 701 and the adapter sleeve achieve an interference fit.

[0049] The silicone head 16 is a ring-shaped gasket or cap-shaped structure made of silicone material, which is connected to the top of the adapter sleeve for connecting to the liquid supply. When liquid supply is required, the external syringe or pump tube is connected to the adapter nozzle; when liquid supply is not required, the adapter can be removed and replaced with a sealing device 10 (such as a rubber stopper or screw cap) to seal the liquid supply port 7. Example 4

[0050] like Figure 2 , Figure 4 and Figure 5 As shown, the gasket 12 on the outer ring of the enclosure 2 is engaged with the cover 13. The cover 13 is a transparent sheet material, which can be PMMA, PS, PC, or glass. After the cover 13 is engaged with the gasket 12, it will seal the top opening of the culture medium storage chamber, effectively preventing the culture medium from evaporating and preventing external contaminants from entering, while allowing observation from above. The cover 13 and the enclosure 2 only need to partially overlap. However, the cover 13 and the culture medium plate 1 are not completely sealed, with sufficient gaps to allow gas flow. Therefore, when liquid oscillation mass transfer operations are required, the air pressure control device adjusts the air pressure in the working chamber, and through the gap between the cover 13 and the culture medium plate 1, it can directly act on the space above the liquid surface in the culture medium storage chamber. After inserting the plug, it is sufficient to ensure that the cover 13 and the enclosure 2 partially overlap to block external dust.

[0051] The cultivation well 3 of the chip in this invention is specifically designed for the insertion component, and the two are precisely matched in size and structure. As shown in the figure, the insertion component includes a plug.

[0052] The insert is a cylindrical structure open at one end and closed at the other, injection molded from medical-grade plastics such as polypropylene (PP), polystyrene (PS), or polycarbonate (PC). The outer diameter of the insert is no larger than the inner diameter of culture well 3, allowing the cylinder to be smoothly inserted into culture well 3. The length of the cylinder is 1.0-10.0 times the depth of culture well 3.

[0053] The outer edge of the plug opening is provided with multiple support feet, the height of which is 0.1mm-5mm. When the cylinder is inserted into the culture well 3 with the open end facing down, the support feet contact the bottom of the culture well 3, and the gap between the cylinder and the bottom of the culture well forms an oscillation space for the liquid culture medium.

[0054] Multiple plug-ins can be connected into a row structure via connecting beams. The hole spacing of the row structure is precisely matched with the array spacing of the culture wells 3 on the culture chip, which facilitates the one-time operation of the entire row or the entire plate of culture wells.

[0055] Furthermore, the inner end face of the cover 13 is provided with a snap-fit ​​block 1301 for fixing the plug-in and a limiting block 1302 for limiting the position. The snap-fit ​​block 1301 can be a multi-clawed, slightly elastic clamping and fixing plug-in or a ring snap-fit ​​structure. The snap-fit ​​block 1301 on the cover clamps and fixes multiple plug-in units or dosing devices simultaneously, so that all plug-ins are integrated with the cover, which facilitates unified operation and shortens operation time. The limiting part forms a physical limit on the cover to ensure that the plug-in can be fully inserted into the bottom of the culture well when inserted in the forward direction, providing oscillatory mass transfer conditions. The limiting block 1302 is used to ensure that the plug-in can contact the bottom of the culture well when the plug-in is inserted into the culture well with the closed end facing upward. Example 5

[0056] The diameter of the cultivation well 3 is 1mm-50mm, and in this embodiment it is 10mm. The height of the surrounding plate 2 is 1mm-30mm, and in this embodiment it is 10mm.

[0057] The culture wells 3 and auxiliary holes 4 are staggered between rows. Specifically, the culture wells 3 in adjacent rows are offset laterally by half a column spacing, and the auxiliary holes 4 are located below this offset position. This layout increases the density of culture sites within a limited area, while the auxiliary holes 4 serve the dual functions of structural spacing and pressure balance between adjacent culture wells 3.

[0058] The auxiliary holes 4 have a diameter of 2mm-5mm and their number is 0.5-1 times the number of culture wells 3. In this embodiment, 30 auxiliary holes 4 are provided for each of the 30 culture wells 3.

[0059] In this embodiment, each culture well 3 has 60 micro-culture pits 501 at its bottom. The diameter of the micro-culture pits 501 is 0.1mm-2mm, and in this embodiment, it is 0.5mm. The spacing between adjacent micro-culture pits 501 is 0.02mm-3mm, and in this embodiment, it is 0.5mm.

[0060] The inner wall of the microculture pit 501 is coated with a low-adhesion coating or an extracellular matrix coating. The low-adhesion coating uses polyhydroxyethyl methacrylate (PHEMA) or Pluronic F127 to promote spontaneous cell spheroid formation. The extracellular matrix coating uses Matrigel, type I collagen, or fibronectin to enhance the adhesion ability of specific cells.

[0061] The side section of the micro-culture pit 501 has a configuration with a large opening and a small bottom, meaning the sidewalls are smooth curves rather than straight walls, and the bottom has a smooth transition. Specifically, it includes: Quadratic function type: The sidewall profile conforms to the parabolic equation y = ax² (a>0), and the radius of curvature at the bottom is 0.05mm-0.5mm. The sidewall slope of this configuration gradually increases from the bottom upwards, which is conducive to cell deposition at the bottom center and facilitates the pipette tip to slide along the sidewall when picking up the pellets.

[0062] Conic profile: The sidewall profile is part of an ellipse or hyperbola, and the bottom is an elliptical arc surface. This configuration provides uniform stress distribution, has no sharp corners, and reduces mechanical damage to cells.

[0063] Catenary type: The sidewall profile conforms to the catenary equation y = a·cosh(x / a), has a natural mechanical equilibrium shape, and has a high degree of matching with the spherical profile of the cell sphere, which is conducive to the formation of spheres with uniform size.

[0064] Overall, the microculture pits 501 with a large opening and a small bottom have a depth of 0.1mm-1mm, an opening diameter of 0.1mm-2mm, and a bottom curvature radius of 0.05mm-1mm. Compared to traditional straight-walled cylindrical or inverted conical micropits, the cell spheroid formation rate is greatly improved.

[0065] The working principle and workflow of the microfluidic culture chip of this invention in practical use are as follows: Step 1: Cell Seeding and Static Culture The cell suspension is added vertically into the culture medium storage chamber, with the liquid level higher than the top openings of culture well 3 and auxiliary well 4. Under the influence of gravity, the cells are deposited in the micro-culture pits 501 at the bottom of each culture well 3. After static culture under certain conditions for 10 minutes to 4 hours, the cells spontaneously aggregate to form cell spheroids.

[0066] like Figure 10 As shown, the cultivation well 3 of the chip of the present invention is designed specifically for the insertion component, and the two are precisely matched in size and structure.

[0067] Step 2: Liquid Oscillation Mass Transfer In this step, the microfluidic culture chip of the present invention works in conjunction with the insertion component and the pressure control device to form a complete microfluidic culture system. The present invention is placed inside the pressure control device, the bottom of which is made of a transparent and visible material, such as glass, to facilitate observation while ensuring the overall airtightness of the pressure control device.

[0068] Specifically, the insertion component is inserted into culture well 3 with its open end facing downwards, and the supporting feet contact the bottom of culture well 3. At this time, the inside of the cylinder is covered with liquid of a certain area and volume. The gas pressure control device is activated to apply periodic gas pressure changes to the gas space above the liquid surface in the culture medium storage chamber. When the gas pressure increases, the liquid in the culture medium storage chamber is forced into the cylinder of the insertion component, the liquid level inside the cylinder rises, and the liquid level outside drops slightly; when the gas pressure decreases to near atmospheric pressure, the liquid inside the cylinder falls back to its original position under the action of gravity. This process repeats, essentially creating a reciprocating flow of liquid inside and outside the cylinder. This liquid oscillation disturbance produces a forced homogenization effect on the microenvironment around the cell spheres, effectively weakening the nutrient gradient and waste gradient, and breaking stagnant culture.

[0069] During this process, the chip as a whole remains absolutely still, with only a slight movement of liquid within the culture well 3. Therefore, real-time, continuous microscopic imaging observation of the cell spheres can be performed directly from below the culture medium plate 1 using an inverted microscope, without stopping the machine or removing the chip, which is impossible with shakers, rollers, or other devices that involve overall movement.

[0070] After the oscillating device has been running continuously for a considerable period, the liquid inside the enclosure gradually decreases due to evaporation or loss, causing the liquid level to drop. At this point, there is no need to stop the oscillating device or open the cover 13. The external liquid replenishment pipeline is directly connected to the liquid replenishment port via an adapter and sealed. While the oscillating device continues to run and the oscillation process is ongoing, liquid is injected into the enclosure through the liquid replenishment port, raising the liquid level inside the enclosure to the preset height. After stopping the liquid replenishment, the liquid replenishment pipeline is disconnected. The oscillating device operates continuously throughout the entire liquid replenishment process.

[0071] In summary, this invention provides a microfluidic culture chip that is generally static, locally perturbed, facilitates real-time imaging, and supports precise drug delivery through the staggered layout of culture wells and auxiliary holes, and the precise adaptation of the micro-culture pit array and insertion components. This invention has a compact structure, requires no external tubing, and is significantly superior to existing shakers, rollers, and external tubing perfusion systems, showing broad application prospects in three-dimensional cell culture, organoid culture, and high-throughput drug screening.

[0072] It should be noted that the accompanying drawings merely illustrate the technical concept of the present invention and should not be used to limit the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A microarray patterned microfluidic culture chip with insertable components, characterized in that: The system includes a culture medium plate (1), which has a culture chamber in the middle. The culture chamber includes an outer ring plate (2), which forms an open culture medium storage chamber with the culture medium plate (1). Several culture wells (3) are arranged in an array on the culture medium plate (1) within the range of the ring plate (2). Insertion components can be inserted into the culture wells (3). Auxiliary holes (4) are provided between the laterally distributed culture wells (3). Each culture well (3) has a micro-culture pit group (5) at the bottom. The micro-culture pit group (5) includes several micro-culture pits (501) arranged in an array with openings facing the inside of the culture well. The culture chamber is provided with integrally formed flow channel plates (6) on both sides. The lower surface of the flow channel plates (6) is provided with several open branch channels (601). The branch channels (601) on each side are respectively connected to the vertically penetrating liquid inlet (7). The end of each branch channel (601) is connected to the liquid outlet (8) located in the culture chamber. The bottom of the flow channel plate (6) is connected to a sealing plate (9), which seals the bottom of the liquid inlet (7), the flow channel (601) and the liquid outlet (8), forming a whole with the culture medium plate (1); The liquid inlet (7) is connected to an adapter or a sealing device (10).

2. The microarray patterned microfluidic culture chip with insertable components according to claim 1, characterized in that: The adapter includes an adapter sleeve (11) connected to the culture medium plate (1) and a docking silicone head (16). The adapter sleeve (11) includes a central tube (1101) located at the center, an annular connecting part (1102) connected to the middle of the outer ring of the central tube, and a U-shaped rotating body (1103) connected to the outer ring of the annular connecting part (1102). One end of the central tube (1101) is a flat adapter nozzle, and the other end is provided with spline teeth to form an adapter nozzle; The two ends of the central tube (1101) are tapered and tapered, and an annular snap-fit ​​groove (1104) is formed on the outer side of the near end face.

3. The microarray patterned microfluidic culture chip with insertable components according to claim 2, characterized in that: The height of the adapter flat nozzle protruding from the rotating body (1103) is less than the thickness of the flow channel plate (6), the height of the adapter flower nozzle protruding from the rotating body (1103) is equal to the thickness of the flow channel plate (6), and the end face of the adapter flower nozzle can extend into the bottom of the liquid inlet (7).

4. The microarray patterned microfluidic culture chip with insertable components according to claim 2, characterized in that: The liquid inlet (7) is provided with an annular boss (701) on its outer periphery, and the annular boss (701) is provided with a protruding snap-fit ​​part (7011) on its inner ring. The snap-fit ​​part (7011) is engaged with the snap-fit ​​groove (1104) of the central tube for snap-fit ​​sealing.

5. A microarray patterned microfluidic culture chip with an insertable component according to claim 1, characterized in that: The culture medium plate (1) and the sealing plate (9) are bonded by adhesive, adhesive tape or welded by ultrasonic, laser or hot pressing welding; the number of the diversion channels (601) is 1-50, and the length of each diversion channel is equal.

6. The microarray patterned microfluidic culture chip with insertable components according to claim 1, characterized in that: The outer ring of the enclosure (2) is provided with a gasket (12), and the cover (13) sits on the gasket (12) to cover the entire culture chamber.

7. A microarray patterned microfluidic culture chip with an insertable component according to claim 6, characterized in that: The inner end face of the cover (13) is provided with a snap-fit ​​block (1301) for fixing the plug-in assembly or the drug dispensing device and a limiting block (1302) for limiting the position.

8. A microarray patterned microfluidic culture chip with an insertable component according to claim 1, characterized in that: The diameter of the cultivation well (3) is 1mm-50mm, and the height of the surrounding plate is 1mm-30mm.

9. A microarray patterned microfluidic culture chip with an insertable component according to claim 1, characterized in that: Each culture well (3) has a micro-culture pit group (5) at the bottom containing 0-500 micro-culture pits (501). The diameter of the micro-culture pit (501) is 0.1mm-2mm, and the spacing between adjacent micro-culture pits (501) is 0.02mm-3mm. The side section of the micro-culture pit (501) has a configuration with a large opening and a small bottom.

10. A microarray patterned microfluidic culture chip with an insertable component according to claim 1, characterized in that: The inner wall of the microculture pit (501) is provided with a low-adhesion coating or an extracellular matrix coating.

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