An organoid automated perfusion culture device

Abstract

The utility model discloses an organoid automatic perfusion culture device, and the device comprises: upper cover structure, upper cover structure is equipped with liquid inlet and a plurality of shunt passageways for guiding perfusion liquid to a plurality of culture cavities below respectively, modular culture unit, modular culture unit includes a plurality of culture cavities, and the vertical fluid passage for liquid through is arranged below every culture cavity and extends along the vertical direction, modular culture unit has stackable configuration, and the four corners of every modular culture unit are provided with limit cooperation structure and are used for aligning and positioning with adjacent unit, the upper surface or lower surface of modular culture unit is equipped with annular sealing groove, and the O type sealing ring is embedded in sealing groove and is used for forming sealed contact under the stacked state, base structure, and the vertical fluid passage export of the lowermost layer modular culture unit is communicated with base structure, and the inside is provided with the confluence cavity and liquid outlet.

Description

TECHNICAL FIELD

[0001] The utility model relates to the field of three -dimensional cell culture in vitro, especially a kind of automatic perfusion culture device of organoid. BACKGROUND

[0002] With the wide application of organoid technology in the fields of disease model, individualized drug screening and regenerative medicine, higher requirements are put forward for the stability and controllability of its in-vitro culture conditions. Organoids are usually derived from stem cells or primary cells, which grow in a three-dimensional microenvironment and have a high degree of dependence on nutrient supply and metabolite removal. The existing common culture method mostly adopts a static liquid replacement mode, for example, using a well plate structure to regularly add or replace culture solution. However, in the face of long-term culture, high-density organoid generation and continuous experimental conditions, the static mode is difficult to maintain the steady state of the culture environment, which easily leads to problems such as metabolic disorder and structural degradation, thereby affecting the experimental repeatability and organoid functional expression.

[0003] To improve the material exchange efficiency, some studies introduce perfusion culture strategies, for example, using a peristaltic pump to drive the continuous flow of culture solution, simulating the in-vivo liquid microcirculation environment. Although this method improves the uniformity of nutrient distribution to some extent, the existing perfusion culture device still has many structural problems. For example, the fluid path in the multi-well culture system is complex, and the liquid flow is not evenly distributed, resulting in significant differences in perfusion efficiency between different culture wells; the device is designed as a whole, lacks modular structure, and is not conducive to flexible expansion and multi-layer configuration; the structure has high sealing performance but cannot be disassembled, making it inconvenient to extract samples and observe in real time; in addition, under conditions such as high-temperature sterilization, ethanol disinfection or ultraviolet treatment, the device is prone to structural deformation, sealing failure and other problems, affecting the stability and safety of the experiment.

[0004] In summary, the existing organoid perfusion culture device has technical deficiencies in liquid distribution uniformity, structural flexibility, operational convenience and long-term sealing reliability. To meet the stable culture needs of organoids under various experimental conditions, it is urgent to provide an automatic perfusion organoid culture device with modular structure, excellent sealing performance and convenient operation and maintenance, to realize accurate regulation and process protection of the culture environment. SUMMARY

[0005] The utility model aims at at least one of the technical problems existing in the prior art. To this end, one purpose of the utility model is to provide an automatic perfusion culture device for organoids, which comprises:

[0006] An upper cover structure is provided with a liquid inlet and a plurality of shunt channels for guiding perfusion liquid to the lower plurality of culture cavities, respectively;

[0007] The utility model provides modular culture unit, the modular culture unit includes a plurality of culture cavities, each culture cavity is equipped with the vertical fluid passage for liquid through under the vertical direction extension, the modular culture unit has the stackable configuration, and the four corners of each modular culture unit is equipped with the limit cooperation structure for with adjacent unit alignment positioning, the upper surface or lower surface of modular culture unit is equipped with annular sealing groove, and the O type sealing ring is embedded in the sealing groove, is used for forming the sealed contact under the stacked state,

[0008] The base structure is communicated with the vertical fluid passage export of the lowermost layer modular culture unit, and the inside is provided with the confluence cavity and the liquid outlet interface.

[0009] In some examples of the utility model, the shunt channel includes an annular main shunt groove provided on the inner surface of the upper cover structure, and a plurality of shunt grooves radially extending from the main shunt groove, the plurality of shunt grooves are uniformly distributed in the horizontal direction.

[0010] In some examples of the utility model, the vertical fluid passage includes an annular liquid receiving groove provided at the bottom of the culture cavity, a central passage provided at the center of the liquid receiving groove, and a lower through hole penetrating the thickness of the modular culture unit plate, which are sequentially communicated from top to bottom to form a continuous liquid perfusion path.

[0011] In some examples of the utility model, the limit cooperation structure includes a plug-in column structure and a matched plug-in groove structure, the cross section of the sealing groove is arc-shaped, and the O type sealing ring is made of an elastic material.

[0012] In some examples of the utility model, the upper cover structure is provided with a compression assembly for applying vertical pressure to compress the stacked modular culture units, the compression assembly includes a knob and a compression plate matched with the knob, and the knob drives the compression plate to move in the vertical direction.

[0013] In some examples of the utility model, the modular culture unit is made of an optically transparent material, and the material includes polycarbonate, polymethyl methacrylate or polydimethylsiloxane, which is used to support microscopic observation during the culture process.

[0014] In some examples of the utility model, the device supports the vertical stacking of multiple modular culture units, the plurality of shunt channels in the upper cover structure are respectively communicated with the plurality of culture cavities of the uppermost layer of modular culture units, the vertical fluid passages between the modular culture units are aligned and communicated to form a through perfusion path from top to bottom.

[0015] In some examples of the utility model, the liquid inlet is connected with an external peristaltic pump through a Luer joint, the peristaltic pump has programmable control function, supports setting continuous, intermittent or reverse perfusion mode, and flow rate is adjusted in the range of microliter per minute.

[0016] In some examples of the utility model, the modular culture unit or base structure is provided with an interface or slot for mounting a sensor, the sensor includes a temperature sensor, a pH sensor, a dissolved oxygen sensor or a flow sensor, and is connected with a data acquisition system.

[0017] In some examples of the utility model, the device is of detachable structure, and any one of buckle structure, threaded connection structure or knob pressing structure is used in the sealed connection part, and the closed structure does not deform or fail in sealing under high temperature, ethanol treatment and ultraviolet irradiation conditions

[0018] Additional aspects and advantages of the utility model will be partially given in the following description, some will become obvious from the following description, or the following beneficial effects will be understood by the practice of the utility model:

[0019] The utility model optimizes the organ perfusion culture device at the structural level, solves the problems of uneven liquid distribution, poor module expandability, insufficient sealing performance and unstable perfusion path in the prior art, and has good technical adaptability and practical value.

[0020] Firstly, the upper cover structure is provided with a liquid inlet and a plurality of shunt channels, which can realize uniform distribution after the liquid enters the system, provide a stable inlet for the perfusion path, and effectively improve the flow rate consistency between the culture holes. The modular culture unit adopts a stacked structure, each layer is provided with a plurality of culture cavities, and a vertical communication fluid passage is arranged below, so that the perfusion liquid can flow from top to bottom, which improves the liquid exchange efficiency and enhances the expandability of the overall structure, and meets the multi-layer culture demand.

[0021] The limiting cooperation mechanism arranged in the stacked structure ensures the accurate alignment between the modules, effectively prevents the deviation of the passage caused by the layer misalignment, and improves the assembly stability and reusability of the device. The contact surface of each module is provided with an annular sealing groove, and an O-shaped sealing ring is embedded, which forms reliable sealing after the assembly is stacked, avoids liquid leakage or cross contamination during operation, and improves the reliability and safety of the system sealing.

[0022] In addition, the shell structure adopts a detachable design, and in combination with the cooperation of the sealing groove and the O-shaped ring, good stress buffering and heat adaptation capability are achieved at the structural connection part, so that the device can still maintain stable structural form and non-failure sealing performance under the sterile processing conditions such as high-temperature sterilization, alcohol immersion or ultraviolet irradiation, and meet the long-term use requirements in the sterile experimental environment.

[0023] The base structure is provided with a flow collection cavity and a liquid outlet interface for collecting perfusion liquid, can uniformly guide the waste liquid in each layer of culture cavity out, is convenient for subsequent analysis or recycling, and is helpful to improve the integration and cleaning management efficiency of the system.

[0024] In summary, the organoid automatic perfusion culture device provided by the utility model realizes uniform flow distribution, flexible stacking combination, stable sealing performance, wide environmental adaptation and other technical advantages, and improves the overall performance and application reliability of the organoid perfusion culture system. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical scheme in the embodiments of the utility model or the prior art, the drawings needed to be used in the embodiment or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the utility model, and those skilled in the art can also obtain other drawings according to these drawings without creating creative labor.

[0026] Figure 1 The organoid automatic perfusion culture device structure schematic view provided by the utility model embodiment is shown in the figure.

[0027] Figure 2 The overall explosion structure schematic view of the organoid automatic perfusion culture device of the utility model is shown in the figure.

[0028] Figure 3 The detailed explosion structure schematic view of each component of the utility model is shown in the figure.

[0029] Figure 4 The top view structure schematic view of the modular culture unit structure is shown in the figure.

[0030] Explanation of reference signs:

[0031] 100, organoid automatic perfusion culture device;

[0032] 200, upper cover structure;210, liquid inlet;220, shunt channel;

[0033] 300, modular culture unit;310, culture cavity;320, annular liquid receiving groove;330, central channel;340, lower through hole;350, limiting cooperation structure;360, sealing groove;370, O-shaped sealing ring;

[0034] 400, base structure; 410, confluence cavity; 420, liquid outlet interface;

[0035] 500, Luer fitting;

[0036] 600, sensor interface. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0038] In the description of the present application, it should be understood that the orientations or positional relationships indicated by the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the features defined as "first" and "second" can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0039] In the description of the present application, it should be noted that, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0040] The embodiments of the present application will be described in detail below, and examples of the embodiments are shown in the drawings, in which the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by reference to the drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0041] Figure 1 The organ-like automatic perfusion culture device provided by the embodiment of the utility model provides a structure schematic diagram; Figure 2 The utility model organ-like automatic perfusion culture device's whole explosion structure schematic diagram is provided. Figure 3 The utility model each component exploded detailed explosion structure schematic diagram is provided. Figure 4 The utility model provides a structure schematic diagram of the top view of the modular culture unit.

[0042] Please refer to Figures 1-3 In a possible implementation, the organ-like automatic perfusion culture device 100 includes an upper cover structure 200, a modular culture unit 300 and a base structure 400. The upper cover structure 200 is provided with a liquid inlet interface 210, and a plurality of shunt channels 220 are formed on the inner surface thereof, each shunt channel 220 being used to guide the perfusion liquid introduced from the liquid inlet interface 210 to the plurality of culture cavities 310 below respectively, wherein the shunt channels 220 specifically include liquid flow channels arranged along the surface of the upper cover structure 200 to realize uniform distribution of the liquid. The modular culture unit 300 includes a plurality of culture cavities 310, and each culture cavity 310 is provided with a vertical fluid passage extending in the vertical direction at the bottom, which is used to pass through the liquid flow, and the vertical fluid passage structure penetrates the module unit to the lower module or the base from the bottom of the culture cavity 310. The modular culture unit 300 is designed to be stackable, and is provided with a limiting matching structure 350 at the four corners of the unit, so as to realize the positioning and alignment of the adjacent module units in the stacking through the mechanical matching mode, and prevent the misplacement in the horizontal direction and the rotational direction. The upper surface or the lower surface of the modular culture unit 300 is processed with an annular sealing groove 360, and an O-shaped sealing ring 370 made of elastic material is arranged in the sealing groove 360, so as to realize the sealing contact by pressing the O-shaped sealing ring 370 in the unit stacking, and ensure that the liquid only flows in the predetermined channel.

[0043] The base structure 400 is arranged below the lowermost modular culture unit 300, and the upper surface of the base is communicated with the outlet of the vertical fluid passage of the lowermost modular culture unit 300. A confluence cavity 410 is formed in the base, which receives the liquid discharged from all the vertical fluid passages, and discharges the liquid through the liquid outlet interface 420 arranged in the base. The device structure realizes the introduction of the liquid from the upper cover, the entry of the liquid into the plurality of culture cavities 310 through the shunt channels 220, the downward penetration of the liquid through each culture cavity 310 and the corresponding vertical fluid passage, and finally the collection and discharge of the liquid in the base, so as to form a complete and continuous perfusion path.

[0044] Through the above structural design, the device can realize the synchronous perfusion of multiple culture cavities 310, ensure the uniformity of the liquid receiving conditions of each culture cavity 310, and improve the overall consistency and controllability of organoid culture. The stacking mode of the modular culture unit 300 allows the culture scale to be flexibly expanded, and the limiting cooperation structure 350 ensures the stable alignment between each unit, improving the reliability of system assembly. The sealing structure of the annular sealing groove 360 and the O-shaped sealing ring 370 forms an effective seal between the module units, preventing liquid leakage and ensuring the stable operation of continuous perfusion culture. The manifold cavity 410 and the liquid outlet interface 420 provided on the base effectively guide the liquid outflow, avoid liquid retention, and reduce the risk of contamination.

[0045] It is worth noting that the liquid inlet interface 210 can adopt a buckle joint, a threaded joint or a Luer joint 500 to adapt to different types of external liquid supply systems, and ensure liquid tightness and stable liquid supply under different connection modes. The number of culture cavities 310 of the modular culture unit 300 can be adjusted to four cavities, eight cavities or sixteen cavities according to application requirements, but each culture cavity 310 needs to have a corresponding vertical fluid passage, and the limiting cooperation and sealing structure of each unit remains unchanged, thereby ensuring the stability of the stacked structure and the continuity of the liquid passage. In addition to the plug-in column structure, the limiting cooperation structure 350 can also use magnetic attraction cooperation structure or flange fitting structure, and various alternative structures can realize reliable positioning of the module unit. In addition to the O-shaped sealing ring 370, the sealing structure can also use silicone sealing gasket or fluororubber sealing ring to improve corrosion resistance and high temperature resistance performance on the basis of ensuring long-term sealing performance. The liquid outlet interface 420 of the base structure 400 can be provided with a one-way check valve as needed to prevent backflow from interfering with the upper culture environment, thereby further improving the reliability of system operation.

[0046] Please refer to Figure 3 In one possible implementation, like the aforementioned organoid automatic perfusion culture device 100, the shunt channel 220 includes an annular main shunt groove provided on the inner surface of the upper cover structure 200, and a plurality of shunt grooves extending radially from the main shunt groove, and the plurality of shunt grooves are uniformly distributed along the horizontal direction. The upper cover structure 200 is formed by integral molding or assembly, and the liquid inlet interface 210 is arranged at the top or side of the upper cover structure 200, directly communicating with the upper side or one side of the main shunt groove, ensuring that the perfusion liquid is quickly distributed into the annular groove when introduced.

[0047] The main distribution channel is designed as a closed or open annular channel. Its shape can be a standard circle, or it can be adjusted to an ellipse or polygon according to the layout requirements of the modular culture unit 300. After the perfusion liquid is injected through the inlet port 210, it first flows around the main distribution channel, forming a pressure balance through the annular layout and avoiding uneven liquid supply caused by the inlet position. On the inner wall or bottom surface of the main distribution channel, multiple radially extending distribution channels are evenly formed, each distribution channel corresponding to an inlet of a culture chamber 310 in the modular culture unit 300 below. These distribution channels extend radially outward from the main distribution channel, and their cross-sections can be rectangular, semi-circular, trapezoidal, or other shapes that facilitate perfusion, in order to reduce the flow resistance and retention of liquid in the channel and improve flow efficiency.

[0048] The number of distribution channels strictly corresponds to the number of culture chambers 310 set on the modular culture unit 300, and they are evenly distributed in the horizontal direction. For example, in an eight-chamber unit, each distribution channel has an outlet every 45 degrees along the circumference to ensure that each culture chamber 310 receives the same volume of perfusion liquid. During the design process, the cross-sectional area of ​​the main distribution channel and the distribution channels can be adjusted according to the target flow rate and pressure gradient set by the system, thereby controlling the distribution ratio of liquid in each branch channel and further improving the uniformity and stability of fluid distribution.

[0049] To further enhance the reliability of system operation, the top cover structure 200 is preferably made of a material with high dimensional stability, such as polycarbonate (PC), polymethyl methacrylate (PMMA), or polydimethylsiloxane (PDMS). These materials can maintain long-term mechanical strength while having good chemical resistance and transparency, facilitating visual monitoring during perfusion. Furthermore, to improve the smoothness of liquid flow, micron-level polishing treatment can be applied to the surfaces of the main distribution tank and each distribution tank to reduce frictional resistance during liquid flow, prevent bubble accumulation, and ensure continuous and stable liquid supply to the culture chamber 310.

[0050] In the assembled state, the main distribution channel and each distribution channel are located on the lower surface of the upper cover structure 200, tightly fitting with the upper surface of the modular culture unit 300. The sealing contact formed during stacking prevents liquid leakage. During stacking, the synergistic action of the limiting fit structure 350, the annular sealing groove 360, and the O-ring seal 370 ensures accurate and sealed alignment between the main distribution channel and the inlet of each culture chamber 310, thereby maintaining the integrity of the perfusion system. The entire liquid path remains continuous and unobstructed from the inlet port 210 to the inlet of the culture chamber 310, enabling liquid distribution without additional pressurization, effectively avoiding system complexity and improving overall ease of use.

[0051] Please see Figure 3 , 4In one possible implementation, as described in the aforementioned automated organoid perfusion culture device 100, the vertical fluid pathway includes an annular liquid-receiving trough 320 disposed at the bottom of the culture chamber 310, a central channel 330 disposed at the center of the liquid-receiving trough, and a lower through hole 340 penetrating the thickness of the modular culture unit 300 plate. The three are connected sequentially from top to bottom to form a continuous liquid perfusion path.

[0052] Specifically, an annular liquid-receiving trough 320 is provided at the bottom region of each culture chamber 310. This trough is arranged around the center of the culture chamber 310 and has a closed or semi-closed annular structure to collect the perfusion liquid flowing out of the culture chamber 310. The cross-section of the liquid-receiving trough can be rectangular, semi-circular, or trapezoidal. Its depth and width can be adjusted according to different perfusion requirements. Typically, the trough width is set to 10% to 30% of the diameter of the culture chamber 310, and the trough depth is set between 0.5 mm and 2 mm to ensure sufficient liquid collection capacity while avoiding excessive liquid retention.

[0053] A central channel 330 is provided in the central area of ​​the liquid receiving tank. The central channel 330 extends downward from the bottom of the liquid receiving tank to the lower surface of the modular culture unit 300 plate, forming a path for vertical liquid flow. The diameter of the central channel 330 is usually smaller than the inner diameter of the liquid receiving tank. Its design must consider the balance between liquid flow velocity and resistance to ensure that the liquid can flow in stably without sedimentation due to sudden drops in local flow velocity. The central channel 330 adopts a cylindrical structure and the channel wall surface is smoothed to reduce flow resistance and prevent turbulence during liquid flow.

[0054] The central channel 330 connects to the lower through-hole 340, which extends through the entire thickness of the modular culture unit 300 plate, ensuring that the perfusion liquid can flow from top to bottom to the manifold 410 of the next module or base structure 400. The size and position of the lower through-hole 340 correspond to those of the central channel 330, ensuring the consistency and continuity of the liquid path. Through the continuous arrangement of the liquid receiving tank, the central channel 330, and the lower through-hole 340, a vertically continuous flow path is constructed from the culture chamber 310 to the base, effectively achieving the orderly discharge of the perfusion liquid.

[0055] During perfusion, the liquid first flows naturally from the bottom of the culture chamber 310 into the annular receiving trough 320, forming a local temporary storage within the trough. Then, it is guided vertically downwards through the central channel 330 and discharged through the lower through-hole 340 into the confluence chamber 410 below the modular culture unit 300 or inside the base structure 400. The annular receiving trough 320 not only collects the liquid but also buffers the flow. Even with slight fluctuations in the perfusion rate, local buffering is achieved within the receiving trough, ensuring the stability of the liquid flow velocity at the outlet of the central channel 330. This fluid management mechanism effectively reduces disturbances to the culture environment caused by unstable flow rates and improves the stability of the liquid supply during organoid culture.

[0056] To further enhance liquid flow performance, the modular culture unit 300 is preferably made of a polymer material with high transparency, corrosion resistance, and easy processing, such as polycarbonate (PC), polymethyl methacrylate (PMMA), or polydimethylsiloxane (PDMS). These materials not only ensure the precision of the fine channel structure molding but also facilitate microscopic observation during the culture process. In terms of manufacturing processes, high-precision injection molding, micromilling, or laser engraving can be employed to achieve high-precision machining of the microstructure while ensuring structural strength, avoiding burrs or impurities on the inner surface of the channels, thereby further ensuring smooth liquid flow.

[0057] This device achieves consistent liquid management from the outlet of the culture chamber 310 to the bottom of the system by setting up a continuous liquid perfusion path formed by an annular liquid receiving tank 320, a central channel 330, and a lower through hole 340. This ensures that the entire perfusion system can maintain the continuity and stability of liquid flow even when multiple modules are stacked. This structural design effectively avoids problems such as liquid stagnation, local deposition, and uneven perfusion that are common in traditional culture systems, improving the consistency and controllability of the cell growth environment during culture, which is of great significance for maintaining the functional state of organoids.

[0058] Please see Figure 3 , 4 In one possible implementation, as described in the aforementioned automated organoid perfusion culture device 100, the limiting and fitting structure 350 includes a post structure and a mating slot structure, and the sealing groove 360 ​​has an arc-shaped cross-section, and the O-ring 370 is made of an elastic material.

[0059] The modular culture unit 300 has insert structures and matching slot structures at its four corners. Each unit has an insert and a slot on a pair of adjacent sides, allowing the insert of the upper unit to accurately insert into the slot of the lower unit during multi-unit stacking, thus achieving precise positioning in both horizontal and rotational directions. The inserts are cylindrical, frustum-shaped, or have guide ramps. Their diameter and height are optimized based on the thickness of the modular unit and stacking tolerances, typically with a diameter between 2 mm and 5 mm and a height between 3 mm and 10 mm, ensuring sufficient positioning strength without adding excessive structural complexity.

[0060] The mating slot structures are created at corresponding positions. The slot hole diameter is slightly larger than the pin diameter, typically controlled within a gap tolerance range of 0.05 mm to 0.2 mm, to balance smooth insertion and positioning accuracy. The slot depth is usually slightly larger than the pin height to ensure full engagement during stacking and increase structural stability. Both the pins and slots are integrally molded at the edge of the module unit or fixed to the module unit structure through ultrasonic welding, hot pressing, or other methods, ensuring connection reliability under long-term use.

[0061] An annular sealing groove 360 ​​is machined on the upper or lower surface of the module unit. The sealing groove 360 ​​is arranged around the liquid supply or drainage passage of each culture chamber 310 for embedding O-rings 370. The cross-section of the sealing groove 360 ​​is designed as a standard arc shape, that is, a curved structure with a consistent radius and smooth curvature, to ensure that the O-ring 370 can generate uniform contact pressure under compression, thereby forming an effective seal and preventing liquid leakage between stacked units. The width and depth of the sealing groove 360 ​​are determined according to the specifications of the selected O-ring 370. Typically, the width is 1.1 to 1.3 times the wire diameter of the sealing ring, and the depth is approximately 0.8 to 1 times the wire diameter, ensuring that the O-ring is within the optimal deformation range under compression, which guarantees the sealing effect while avoiding damage to the sealing ring due to excessive compression.

[0062] The O-rings 370 used for sealing are preferably made of elastic materials with good elasticity and chemical corrosion resistance, such as silicone rubber, fluororubber (FKM), and ethylene propylene diene monomer (EPDM). These materials can maintain good resilience and sealing performance under long-term flooding conditions and withstand a certain range of temperature changes, pH changes, and liquid composition changes. The specifications of the sealing rings can be selected according to the size of the modular unit and the stacking pressure requirements. The wire diameter is usually between 0.5 mm and 2 mm to balance sealing performance and ease of assembly.

[0063] During the module stacking process, the mechanical cooperation of the insert and slot structures quickly achieves precise alignment of the upper and lower units, avoiding problems such as poor sealing ring crimping caused by misalignment or tilting. Simultaneously, the annular sealing groove 360 ​​and the embedded O-ring 370 form an independent and reliable liquid sealing interface between each stacked unit, ensuring that the perfusion liquid flows along the predetermined flow path without side leakage or leakage, significantly improving the sealing performance and perfusion stability of the entire culture system.

[0064] Through the above design, this device can ensure rapid and accurate positioning and sealing connection between multiple modular units when stacking them, improving assembly efficiency and ease of operation, while ensuring the long-term reliability and safety of the system. During long-term use or frequent disassembly and assembly, the mechanical durability of the insert and slot structure and the elastic recovery performance of the O-ring 370 remain at a high level, thus ensuring stable sealing and stacking accuracy even after multiple cycles of operation, greatly improving the repeatability and reliability of the organoid culture process.

[0065] It is worth noting that the structure of the annular receiving tank 320 can be appropriately adjusted according to the perfusion requirements. For example, in high-flow-rate scenarios, the tank can be widened to increase buffer capacity, while in low-flow-rate scenarios, the tank size can be reduced to shorten the liquid residence time. The cross-section of the central channel 330 can also be designed in other shapes, such as elliptical or polygonal, to further optimize flow characteristics. The lower through-hole 340 can be equipped with auxiliary microporous structures for bubble removal or micro-liquid diversion, enhancing the stability of system operation. In special applications, a micron-level sieve structure can also be set inside the receiving tank or the central channel 330 to prevent culture debris or cell clumps from entering the downstream flow channel system, reducing the risk of system blockage and extending the service life of the device.

[0066] Therefore, by adopting a continuous liquid pathway structure combining an annular liquid-receiving trough 320, a central channel 330, and a lower through hole 340 in the modular culture unit 300, this invention not only ensures the efficiency and uniformity of liquid flow during organoid culture, but also effectively improves the overall scalability and reliability of the system. It is suitable for biological culture experiments of various scales and types, and has good application prospects and promotion value.

[0067] Please see Figure 3 In one possible implementation, as described in the aforementioned automated organoid perfusion culture device 100, the upper cover structure 200 is provided with a clamping assembly for applying vertical pressure to press the stacked modular culture units 300 together. The clamping assembly includes a knob and a mating plate therewith, the knob causing the plate to move vertically.

[0068] Specifically, a clamping assembly is provided above the upper cover structure 200, which consists of a knob and a pressure plate. The knob is installed at the top of the upper cover structure 200 and is fixed to the upper cover body by a threaded connection or a snap-fit ​​connection. A pressure plate is connected to the lower end of the knob, and the size of the pressure plate covers the entire top surface or most of the area of ​​the stacked modular culture unit 300 to ensure that pressure can be applied evenly.

[0069] The rotation of the knob drives the pressure plate to rise and fall vertically. In operation, by manually rotating the knob, the knob engages with the threaded mechanism, causing the pressure plate to slowly descend along the set guide mechanism. Once the pressure plate contacts the upper surface of the modular culture unit 300, continuing to rotate the knob gradually applies vertical pressure, pressing all the stacked modular units together. The knob structure preferably features a design with anti-slip texture or indicator marks to facilitate precise control of the clamping force.

[0070] The pressure plate is typically made of rigid and corrosion-resistant materials, such as aluminum alloy, stainless steel, or engineering plastics, to ensure that it does not deform significantly when vertical pressure is applied, thus guaranteeing uniform force application. A flexible gasket, such as a silicone or polyurethane gasket, can be placed on the side of the pressure plate that contacts the modular culture unit 300 to prevent direct metal contact from causing localized damage to the modular unit, while also improving the overall sealing and pressing effect.

[0071] By setting up the aforementioned clamping components, after the modular culture units 300 are stacked, a moderately adjustable vertical pressure can be applied to ensure that the limiting fit structure 350 and the sealing structure (such as the annular sealing groove 360 ​​and the O-ring 370) between each unit are fully pressed into contact, thereby improving the sealing and stability between modules and preventing liquid leakage or system failure due to loose stacking or external vibration during the perfusion process. Compared with the traditional screw fixing method, the knob and pressure plate design has the advantages of simple operation, adjustable clamping force, and long service life.

[0072] It is worth noting that the knob can be replaced with a fine-tuning handwheel with graduated indicators for more precise control of the clamping force; the pressure plate structure can adopt a multi-point support pressure plate or an elastic pre-tightening pressure plate to further improve the uniformity of force application; a spring mechanism can also be added between the knob and the pressure plate to provide flexible buffering during pressure application and prevent excessive compression of the sealing ring, which could lead to seal failure. Furthermore, the pressure plate material can be selected based on the requirements of the culture environment, choosing materials that are resistant to high temperatures and sterilization, such as PEEK or anodized aluminum, to meet the long-term use requirements under different culture conditions.

[0073] Therefore, by setting a pressing component that drives the pressure plate to move vertically with a knob, this utility model can not only effectively achieve reliable pressing and sealing after the modular culture units 300 are stacked, but also improve the ease of operation, repeatability and long-term operational reliability of the device, and meet the actual needs of automated organoid perfusion culture for high sealing and high stability connection structure.

[0074] Please see Figure 2 , 3 In one possible implementation, such as the aforementioned automated organoid perfusion culture device 100, the modular culture unit 300 is made of an optically transparent material, including polycarbonate, polymethyl methacrylate, or polydimethylsiloxane, to support microscopic observation during the culture process.

[0075] The main body of the modular culture unit 300 is made of a highly transparent material to ensure real-time, intuitive microscopic observation during organoid culture. The selected optically transparent material must balance mechanical strength, chemical stability, and processability. Polycarbonate (PC) has good impact resistance and dimensional stability, making it suitable for withstanding the mechanical loads that may occur during perfusion; polymethyl methacrylate (PMMA) has excellent optical transmittance, providing near-glass visual clarity, suitable for scenarios requiring high-resolution imaging; and polydimethylsiloxane (PDMS) is suitable for flexible and deformable culture environments due to its excellent biocompatibility and flexible molding properties.

[0076] The modular culture unit 300 is fabricated using processes such as injection molding, soft template molding, or laser engraving micromachining to ensure high-precision molding of details such as the culture chamber 310, fluid pathways, and sealing structures. During unit molding, the surface finish of the material is carefully controlled, with a surface roughness preferably below 0.2 micrometers to reduce light scattering and improve microscopic imaging. For modular units using PMMA or PC materials, further polishing or surface coating techniques can enhance their scratch resistance and cleanability, extending their service life.

[0077] Made from optically transparent materials, the modular culture unit 300 supports multiple imaging modes, including bright-field microscopy, fluorescence microscopy, and confocal microscopy, in practical applications. Researchers can directly observe the growth, differentiation, morphological changes, and fluorescence signals of organoids through the transparent module during culture, eliminating the need for frequent disassembly or sampling, greatly improving operational convenience and real-time data acquisition. Furthermore, the low autofluorescence properties of the transparent material itself help improve the signal-to-noise ratio of fluorescence imaging, reduce background interference, and enhance data quality.

[0078] Through the above material selection and manufacturing process design, the modular culture unit 300 not only has good structural strength and durability, but also ensures the stability of the optical properties of the materials under long-term perfusion culture conditions, and does not deteriorate significantly due to liquid erosion, temperature changes or ultraviolet irradiation, thereby ensuring the need for continuous visual monitoring during the culture process.

[0079] It is worth noting that the modular culture unit 300 can also be equipped with other optically transparent materials, such as epoxy resin or highly transparent polyimide, to meet different culture requirements and adapt to long-term culture applications in higher temperatures or special chemical environments. Furthermore, a hydrophilic coating or anti-fouling coating can be added to the surface of the transparent material as needed to further improve liquid flow and reduce protein adsorption, thereby enhancing the stability and data consistency of the culture process. In specific applications, micro-optical elements (such as microlens arrays) can be integrated within the modular unit to enhance local imaging quality, achieving higher resolution or multi-focal imaging effects.

[0080] Please see Figure 2 , 3 In one possible implementation, as described above, the automated perfusion culture device 100 for organoids supports multiple modular culture units 300 stacked vertically. Multiple diversion channels 220 in the top cover structure 200 are respectively connected to multiple culture chambers 310 of the uppermost modular culture unit 300. The vertical fluid passages between the modular culture units 300 are aligned and connected to form a top-down through-flow perfusion path.

[0081] Specifically, the modular culture unit 300 is designed with standardized dimensions and has limiting and fitting structures 350 at the four corners to ensure that each unit can be quickly and accurately positioned during vertical stacking. The vertical fluid passage of each modular unit, including the annular liquid receiving groove 320, the central channel 330 and the lower through hole 340, is processed according to strict tolerance control to ensure precise docking of the fluid outlet and inlet between the upper and lower units, thereby forming a continuous and uninterrupted perfusion path.

[0082] In the assembled state, the uppermost modular culture unit 300 is directly connected to the lower surface of the upper cover structure 200. Multiple diversion channels 220 inside the upper cover structure 200 are evenly distributed horizontally, with the outlet of each diversion channel 220 aligned with the inlet of the corresponding culture chamber 310. After the perfusion liquid is input through the inlet port 210, it is first pre-distributed in the upper cover structure 200 via the main diversion channel, and then flows through each diversion channel 220 into the respective culture chambers 310 of the uppermost modular unit. Subsequently, the liquid flows sequentially from the uppermost modular unit into the next layer of modular units through vertical fluid pathways, layer by layer, finally converging into the manifold 410 of the base structure 400 and being discharged through the outlet port 420, completing the overall continuous perfusion process.

[0083] The modular stacking structure allows for flexible adjustment of the number of module units according to the experimental scale, generally supporting continuous stacking of 2 to 10 layers. To ensure overall stability and sealing after multi-layer stacking, in addition to the limiting fit structure 350, each module unit is fitted with adjacent units through O-rings 370 to form a sealed interface for independent liquid pathways, preventing liquid leakage or contamination of other culture chambers 310 during long-term perfusion. The vertical alignment accuracy between stacked units is typically controlled within ±0.1 mm to ensure a smooth and continuous fluid path without significant flow resistance or liquid stagnation.

[0084] Through the aforementioned stacking configuration, this device enables consistent perfusion culture of organoids under large-scale and diverse culture conditions, effectively improving the system's throughput and experimental flexibility. It is suitable for various applications such as high-throughput screening, long-term dynamic observation, and tissue engineering. The top-down liquid flow path design ensures the stability of the liquid supply and facilitates the overall cleaning and maintenance of the system.

[0085] Please see Figures 1-3 In one possible implementation, as described in the aforementioned automated organoid perfusion culture device 100, the inlet port 210 is connected to an external peristaltic pump via a Luer connector 500. The peristaltic pump has programmable control functionality, supports setting continuous, intermittent, or reverse perfusion modes, and the flow rate can be adjusted within the range of microliters per minute.

[0086] The inlet port 210 adopts a standardized Luer connector 500 design, which has good versatility and sealing performance, and can be easily connected to various models of external peristaltic pumps or fluid supply equipment. The Luer connector 500 typically uses a threaded tightening structure to prevent loosening or leakage at the connection point. The preferred materials are polypropylene (PP), polycarbonate (PC), or medical-grade stainless steel to ensure good sealing and mechanical strength during long-term use and repeated connection.

[0087] The externally connected peristaltic pump is a programmable control device that supports setting perfusion parameters via a control panel, external signals, or a remote communication interface. The peristaltic pump can be set to different perfusion modes according to experimental needs, including: Constant Flow mode to maintain a stable fluid supply environment; Pulsatile Flow mode to simulate a pulsating in vivo fluid flow environment through periodic start-stop cycles; and Reverse Flow mode to provide flow reversal functionality when flushing, removing deposits, or dynamically stimulating the culture system is required. Parameters for each mode, such as time intervals and reversal duration, can be precisely set.

[0088] To meet the requirements of micro-volume culture systems, the peristaltic pump's flow rate control range is set at the microliter per minute (μL / min) level, with a typical flow rate range covering 10 μL / min to 1000 μL / min. Through precise flow rate adjustment, optimal perfusion conditions can be flexibly set according to the different needs of organoid type, growth stage, and culture medium composition, ensuring that cells or tissues grow and differentiate under suitable shear stress conditions.

[0089] In actual operation, the peristaltic pump output is connected to the liquid inlet 210 of the upper cover structure 200 via a Luer connector 500. The liquid is delivered to the inside of the device through the pump tube and distributed to each culture chamber 310 via the upper cover distribution channel 220. During perfusion, the closed-loop design of the peristaltic pump effectively avoids direct contact between the liquid and the drive components, reducing the risk of contamination. Furthermore, by replacing the pump tube, it can adapt to different culture liquid requirements, improving system flexibility and maintainability.

[0090] The above configuration enables highly controllable and flexible adjustment of the perfusion culture process, meeting the refined requirements of different experimental designs for the dynamic characteristics of liquid supply, improving the consistency and reproducibility of culture results, and facilitating process control and automated management in complex culture processes.

[0091] It is worth noting that the inlet port 210 can also be a quick-connect fitting or a microfluidic connector to adapt to the requirements of different specifications or high-throughput automation systems. The peristaltic pump can also be replaced with other microfluidic drive devices such as gear pumps, syringe pumps, or piezoelectric pumps, depending on the specific flow control accuracy and application scenario. In some applications requiring extremely high stability or special flow patterns (such as ultra-slow circulating flow), the peristaltic pump system can be linked with external sensors (such as pressure sensors and flow meters) to achieve closed-loop control, further improving the accuracy and reliability of the fluid supply system.

[0092] Please see Figure 3 In one possible implementation, such as the aforementioned automated organoid perfusion culture device 100, the modular culture unit 300 or the base structure 400 is provided with a sensor interface 600 for mounting sensors, including temperature sensors, pH sensors, dissolved oxygen sensors or flow sensors, and connected to a data acquisition system.

[0093] To enable real-time monitoring and control of key parameters in the culture environment, the modular culture unit 300 or the base structure 400 is equipped with a dedicated sensor interface 600. The interface positions are rationally distributed according to the system layout to ensure that the sensor can directly or indirectly contact the fluid pathway for accurate measurement of the physicochemical properties of the perfusion liquid. The sensor interface 600 preferably adopts a standard threaded hole, snap-fit ​​groove, or custom slot form to facilitate quick installation and replacement of different types of sensors, while ensuring the sealing and mechanical stability after installation.

[0094] The types of sensors installed on the interface include temperature sensors, used to detect changes in the culture medium temperature in real time to ensure that the system is maintained within a suitable growth temperature range; pH sensors, used to monitor the acidity or alkalinity of the culture medium and provide early warning of possible metabolic imbalances or contamination; dissolved oxygen sensors, used to detect the dissolved oxygen concentration in the liquid, which is particularly suitable for studying aerobic metabolic organoid models; and flow sensors, used to monitor the perfusion rate in the system to ensure that the set flow rate conditions are strictly followed and to promptly detect problems such as pump control abnormalities or flow path blockages.

[0095] Each sensor is connected to an external data acquisition system via signal cables. The data acquisition system supports multi-channel input, real-time data recording, and remote monitoring. The system typically includes a user interface that displays real-time graphs and curves of various parameters. It also supports setting alarm thresholds, issuing alerts when temperature, pH, dissolved oxygen, or flow rate exceeds normal ranges, allowing operators to take timely intervention measures. The system can also be integrated with control devices such as peristaltic pumps to achieve closed-loop control, such as automatically adjusting flow rate or culture medium replenishment rate, further enhancing the automation and safety of the culture process.

[0096] The sensor materials and structural design must consider biocompatibility and corrosion resistance with the perfusion fluid. Commonly used materials include medical-grade stainless steel, polytetrafluoroethylene (PTFE) encapsulation, and UV-resistant polymer materials to ensure long-term stable operation without affecting the organoid culture environment. Simultaneously, sealing gaskets or miniature sealing rings are installed at the interface to ensure the overall sealing of the perfusion system is not affected after sensor installation, preventing fluid leakage or contamination.

[0097] With the above settings, the culture system can monitor multiple key parameters in the liquid environment in real time, greatly improving the controllability and stability of the organoid culture process, and providing technical support for dynamic control, data analysis and anomaly handling under different experimental conditions.

[0098] Please see Figure 2 , 3In one possible implementation, as described above, the automated organoid perfusion culture device 100 is a detachable structure, and its sealing connection adopts any one of a snap-fit ​​structure, a threaded connection structure, or a knob-pressing structure, and the sealing structure does not undergo structural deformation or sealing failure under high temperature, ethanol treatment, and ultraviolet irradiation conditions.

[0099] The device is designed as a modular, detachable unit, comprising a top cover, modular culture units 300, and a base. The components are reliably connected and quickly assembled / disassembled via standardized interfaces. Sealing connections utilize either snap-fit, threaded, or knob-operated tightening methods depending on the specific functional requirements.

[0100] Snap-fit ​​structures are generally used in connection interfaces that require frequent opening and closing, such as the connection between the top cover and the module unit. The snap-fit ​​consists of paired protruding hooks and slots. After the hooks are inserted into the slots, the material's elasticity enables quick locking, allowing operators to perform one-handed assembly and disassembly without tools, thus improving the ease of operation of the cultivation system.

[0101] Threaded connections are typically used for the fixed connection between the base and the modular culture unit 300. They employ precision-machined external and internal threads, generating axial clamping force during tightening to achieve a robust and reliable mechanical connection and excellent sealing. An O-ring 370 or a flat washer is preferably added to the threaded connection to further improve sealing performance.

[0102] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0103] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the claims and their equivalents.

Claims

1. An automated perfusion culture device for organoids, characterized in that, include: The upper cover structure is provided with a liquid inlet and multiple diversion channels for guiding the perfusion liquid to multiple culture chambers below. The diversion channel includes an annular main diversion groove disposed on the inner surface of the upper cover structure, and a plurality of diversion grooves extending radially from the annular main diversion groove, the plurality of diversion grooves being evenly distributed in the horizontal direction. A modular culture unit includes multiple culture chambers, each with a vertical fluid passage extending vertically for liquid flow. The vertical fluid passage includes an annular liquid-receiving groove at the bottom of the culture chamber, a central channel at the center of the liquid-receiving groove, and a lower through-hole penetrating the thickness of the modular culture unit plate. These three elements are connected sequentially from top to bottom, forming a continuous liquid flow path. The modular culture units are stackable, with limiting fit structures at the four corners of each unit for alignment and positioning with adjacent units. An annular sealing groove is provided on the upper or lower surface of each modular culture unit, with an O-ring embedded within the groove to form a sealed contact in the stacked state. The base structure is connected to the vertical fluid passage outlet of the lowest modular culture unit, and has a manifold cavity and a liquid outlet interface inside.

2. The automated perfusion culture device for organoids according to claim 1, characterized in that: The limiting and fitting structure includes a pin structure and a mating slot structure; the cross-section of the sealing groove is arc-shaped, and the O-ring is made of elastic material.

3. The automated perfusion culture device for organoids according to claim 1, characterized in that: The upper cover structure is provided with a clamping assembly for applying vertical pressure to press the stacked modular culture units together. The clamping assembly includes a knob and a pressure plate that cooperates with it. The knob drives the pressure plate to move in the vertical direction.

4. The automated perfusion culture device for organoids according to claim 1, characterized in that: The device supports multiple modular culture units stacked vertically. Multiple diversion channels in the upper cover structure are connected to multiple culture chambers of the uppermost modular culture unit. The vertical fluid pathways between the modular culture units are aligned and connected to form a top-to-bottom through-flow path.

5. The automated perfusion culture device for organoids according to claim 1, characterized in that: The inlet port is connected to an external peristaltic pump via a Luer connector. The peristaltic pump has a programmable control function, supports setting continuous, intermittent or reverse perfusion modes, and the flow rate can be adjusted in the range of microliters per minute.

6. The automated perfusion culture device for organoids according to claim 1, characterized in that: The modular culture unit or base structure is equipped with a sensor interface for installation. The sensor includes a temperature sensor, a pH sensor, a dissolved oxygen sensor, or a flow sensor, and is connected to a data acquisition system.

7. The automated perfusion culture device for organoids according to claim 1, characterized in that: The organoid automated perfusion culture device has a detachable structure, and its sealing connection part adopts any one of the following: snap-fit ​​structure, threaded connection structure or knob tightening structure. Moreover, the sealing connection part does not undergo structural deformation or sealing failure under high temperature, ethanol treatment and ultraviolet irradiation conditions.

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