A culture vessel and culture system

Abstract

The application discloses a culture container and a culture system, and relates to the technical field of cell culture containers. The culture container comprises a container body, a culture structure and a limiting component. The container body has a sealed culture cavity. The culture structure is arranged in the culture cavity and comprises a plurality of culture units for accommodating to-be-cultured substances. The limiting component is movably arranged in the culture cavity and can be switched between a first position and a second position. When the limiting component is located at the first position, the limiting component at least partially covers or blocks the culture units, so as to limit the to-be-cultured substances from leaving the culture units and allow fluids and non-target substances to flow between the culture units and the culture cavity. When the limiting component is located at the second position, the culture units are at least partially open, so as to allow the to-be-cultured substances to enter or move out of the culture units. The culture container can realize in-situ, safe, non-destructive and efficient culture of cell spheres or organoids in a sealed culture container.

Description

Technical Field

[0001] This application relates to the field of cell culture container technology, and in particular to a culture container and culture system. Background Technology

[0002] Cell spheroid or organoid culture techniques are widely used in stem cell differentiation, drug screening, tissue engineering, and cell therapy. Current culture methods typically employ microplates, microflasks, culture bags, or low-adsorption culture containers, allowing cells to aggregate in a microporous or low-adsorption environment to form cell spheroids or organoids. However, during the culture process, dead cells, metabolic waste, and old culture medium need to be periodically removed and replenished with fresh culture medium to maintain a suitable culture microenvironment.

[0003] Current microwell culture containers typically rely on manual aspiration, syringe manipulation, manual shaking, culture transfer, or continuous shaking for medium changes. On the one hand, directly changing the medium within the original container can easily lead to cell spheres or organoids escaping from the microwells or cavities, or even being discharged with waste liquid, due to medium flow or changes in container orientation. On the other hand, using culture transfer or repeated agitation and shaking to change the medium and remove dead cells can easily generate shear forces or mechanical impacts on the cell spheres or organoids, affecting their structural integrity, viability, and subsequent differentiation quality. Furthermore, as cell sphere and organoid culture moves towards large-scale, standardized, and automated processes, open medium changes or methods relying on precise manual manipulation are insufficient to meet the requirements of closed culture systems, increasing the risk of contamination and batch-to-batch variability.

[0004] Therefore, there is an urgent need for a culture container and culture system that can allow the culture medium, dead cells and metabolic waste to flow out during the medium exchange process in a closed culture container, while preventing cell spheres or organoids from escaping from the culture unit, so as to achieve in situ, safe, non-destructive and efficient culture of cell spheres or organoids. Summary of the Invention

[0005] The purpose of this application is to provide a culture container and culture system that, in a closed culture container, allows the culture medium, dead cells and metabolic waste to flow out during medium exchange, while preventing cell spheres or organoids from escaping from the culture unit, so as to achieve in situ, safe, non-destructive and efficient culture of cell spheres or organoids.

[0006] To achieve the above objectives, this application provides a culture container, comprising:

[0007] The container body has a sealed culture chamber;

[0008] A culture structure is set inside a culture chamber, and the culture structure includes multiple culture units for accommodating the cultured organisms.

[0009] The limiting component is movably disposed within the culture chamber and can switch between a first position and a second position;

[0010] When the limiting member is in the first position, the limiting member at least partially covers or blocks the culture unit to restrict the cultured organism from leaving the culture unit and allows fluid and non-target substances to flow between the culture unit and the culture chamber. When the limiting member is in the second position, the culture unit is at least partially open to allow the cultured organism to enter the culture unit or be removed from the culture unit.

[0011] In some embodiments, the limiting member has a communication portion corresponding to at least a portion of the culture unit. The communication portion is used to keep the culture unit in communication with the culture chamber when the limiting member is in a first position, and to prevent the cultured organism from leaving the culture unit through the communication portion.

[0012] In some embodiments, the connecting portion is one or more of an opening, slit, notch, gap, and porous region.

[0013] In some embodiments, a positioning space is provided between the culture structure and the inner wall of the container body. When the limiting member is in the first position, at least part of the limiting member is located in the positioning space so that the limiting member is positioned relative to the culture structure and covers or blocks multiple culture units.

[0014] In some embodiments, the culture container further includes a drive positioning structure for driving or positioning a limiting member while the container body remains sealed, so as to switch the limiting member between a first position and a second position.

[0015] In some embodiments, the drive positioning structure includes a first mating member disposed on the limiting member and a second mating member disposed on the outside of the container body. The first mating member and the second mating member can interact with each other across the container body to drive or fix the limiting member.

[0016] In some embodiments, a plurality of culture units are arranged in an array, and at least some of the culture units have a receiving space that contracts toward the bottom so that the cultured material entering the culture unit gathers toward the bottom of the culture unit.

[0017] In some embodiments, the container body is provided with a fluid communication structure, which is connected to the culture chamber. The fluid communication structure includes an inlet channel, an outlet channel, a collection channel, and a gas communication channel. The inlet channel, outlet channel, and collection channel extend to predetermined positions within the culture chamber. The gas communication channel is used to maintain pressure balance inside and outside the container body and is also used to communicate with the interior of the container body so that the interior of the container body maintains a gaseous environment suitable for the culture of the organism.

[0018] In some embodiments, a flow guiding structure is provided in the culture chamber. The flow guiding structure is used to reduce the impact of the fluid input through the fluid communication structure on the culture structure and to guide the fluid discharged through the fluid communication structure to the liquid outlet channel.

[0019] This application provides a cultivation system, comprising:

[0020] Culture containers as described above; and

[0021] Centrifuge device, used to centrifuge culture containers while they are in a closed state;

[0022] The centrifugation device includes a rotary drive mechanism, a container holding mechanism, and a container adapter mechanism. The container adapter mechanism is located within the container holding mechanism and is used to position and support the culture container. The rotary drive mechanism drives the container holding mechanism to move the culture container so that the biological material inside the culture container enters the culture unit under centrifugal action.

[0023] Compared to the aforementioned background technology, the culture container provided in this application includes a container body, a culture structure, and a limiting member. The container body has a sealed culture chamber; the culture structure is disposed within the culture chamber and includes multiple culture units for accommodating the cultured organism; the limiting member is movably disposed within the culture chamber and can switch between a first position and a second position. When the limiting member is in the first position, it at least partially covers or blocks the culture units to prevent the cultured organism from detaching from the culture units and allows fluid and non-target substances to flow between the culture units and the culture chamber. When the limiting member is in the second position, the culture units are at least partially open to allow the cultured organism to enter or leave the culture units.

[0024] The beneficial effects of this type of culture container mainly include:

[0025] By incorporating a culture structure within a sealed culture chamber, comprising multiple culture units for containing the cultured organisms, the cultured organisms are individually confined within their respective culture units, thus providing a stable space for in-situ culture. Simultaneously, by providing a limiting member within the culture chamber that can switch between a first position and a second position, ensuring that the limiting member at least partially covers or obstructs the culture units in the first position, the cultured organisms are less likely to detach from the culture units during media exchange, even if fluid flow occurs within the culture chamber or the container's orientation changes. This reduces the risk of the cultured organisms being discharged with waste liquid or moving disorderly within the culture chamber. Furthermore, while restricting the detachment of the cultured organisms from detaching from the culture units, the limiting member still allows fluid and non-target substances to flow between the culture units and the culture chamber. Therefore, culture medium, dead cells, and metabolic waste can be discharged from the culture units and out of the culture chamber via the fluid-connecting structure, without the need to transfer the cultured organisms from the culture units or to exchange the media through repeated blowing or vigorous shaking. Therefore, this application enables the effective removal of culture medium, dead cells and metabolic waste in a closed culture container, while preventing the cultured organism from escaping from the culture unit, thereby achieving in-situ, safe, non-destructive and efficient culture of cell spheres or organoids. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the overall structure of the culture container in the embodiments of this application.

[0028] Figure 2 for Figure 1 The front view of the culture container shown.

[0029] Figure 3 for Figure 1 The right view of the culture container shown.

[0030] Figure 4 for Figure 1 The top view of the culture container shown.

[0031] Figure 5 for Figure 4 Enlarged view of part A in the middle.

[0032] Figure 6 for Figure 1 A schematic diagram of part of the culture structure in the culture container shown.

[0033] Figure 7 for Figure 1 Front view of the limiting component in the culture container shown.

[0034] Figure 8 for Figure 1 Right view of the limiting component in the culture container shown.

[0035] Figure 9 for Figure 1 Top view of the limiting component in the culture container shown.

[0036] Figure 10 for Figure 9 The diagram shows the opening in the limiting component.

[0037] Figure 11 for Figure 1 The front view of the outer magnetic plate in the culture container shown.

[0038] Figure 12 for Figure 1 The right view of the outer magnetic plate in the culture container shown.

[0039] Figure 13 for Figure 1 Top view of the outer magnetic plate in the culture container shown.

[0040] Figure 14 This is a schematic diagram of a container lid.

[0041] Figure 15 This is a schematic diagram of the centrifuge device in an embodiment of this application.

[0042] Figure 16 for Figure 15 Top view.

[0043] in:

[0044] 1-Cultivation container;

[0045] 10-Container body; 11-Cultivation chamber;

[0046] 20 - Culture structure; 21 - Culture unit;

[0047] 30 - Limiting component; 31 - Connecting part; 311 - Opening; 312 - Notch;

[0048] 40 - Positioning space;

[0049] 50 - Drive positioning structure; 51 - First mating part; 52 - Second mating part; 521 - Outer magnetic plate; 522 - Outer magnet;

[0050] 60 - Fluid-connected structure;

[0051] 70 - Container lid;

[0052] 80-Flow guiding structure;

[0053] 90-trapezoidal connection area;

[0054] 100 - Container neck;

[0055] 2-Centrifuge device;

[0056] 201-Rotary drive mechanism;

[0057] 202 - Container holding mechanism;

[0058] 203 - Container adapter mechanism;

[0059] 204 - Centrifuge chamber;

[0060] 205 - Vibration damping base. Detailed Implementation

[0061] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0062] To enable those skilled in the art to better understand the present application, the present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0063] Please refer to Figures 1 to 6 This application provides a culture container, which includes a container body 10, a culture structure 20, and a limiting member 30.

[0064] The container body 10 has a culture chamber 11 for containing culture medium and the cultured material. The container body 10 can be a rigid bottle or a flexible bag. When the container body 10 is a rigid bottle, it may include a bottom wall, side walls, a top wall, a trapezoidal connecting area 90, and a container neck 100. The container neck 100 is used to install a container cap 70 or to connect to external piping. For example, a cap with a filter membrane can be configured on the container neck 100 for small-batch manual processes, or a cap with piping that can be connected to a closed piping system can be configured for large-batch automated processes.

[0065] The culture structure 20 is disposed within the culture chamber 11 and may be located in the bottom region of the container body 10. The culture structure 20 includes multiple culture units 21, which are used to respectively contain the culture medium, so that the culture medium can be formed, remain or continue to be cultured in multiple relatively independent culture units 21.

[0066] The limiting member 30 is movably disposed within the culture chamber 11 and can switch between a first position and a second position. The limiting member 30 is generally a cover plate structure. When the limiting member 30 is in the first position, the limiting member 30 at least partially covers or blocks the culture unit 21 to restrict the cultured material from leaving the culture unit 21 and allows fluid and non-target substances to flow between the culture unit 21 and the culture chamber 11. When the limiting member 30 is in the second position, the culture unit 21 is at least partially open to allow the cultured material to enter or leave the culture unit 21.

[0067] In this application, the culture medium can be cell spheres, organoids, micro-tissues, or other biological cultures that need to be cultured in a liquid culture environment; the fluid can be culture medium, washing solution, or buffer solution; and the non-target substance can be dead cells, cell debris, metabolites, or other substances that need to be discharged from the culture unit 21.

[0068] Considering that the culture container provided in this application can also be used to dissociate cell spheres and collect single-cell suspensions at the end of cell sphere culture, the fluid can also be a digestive fluid. In this way, cell spheres or organoids can be dissociated after washing and digestion, and finally single-cell suspensions can be output for other research.

[0069] Therefore, the culture structure 20 provides a container 21 for the cultured organisms. The movable limiting member 30 covers or shields the culture unit 21 during operations such as medium changes, allowing the culture medium, dead cells, and metabolic waste to drain out, while preventing the cultured organisms from escaping from the culture unit 21. Since the above process can be completed within the culture chamber 11, there is no need to transfer the cultured organisms to other containers or to change the medium through repeated blowing or vigorous shaking. This reduces the risk of mechanical damage and contamination, and improves the consistency and safety of the culture process.

[0070] To ensure the culture structure 20 can stably accommodate the cultured organisms and improve the uniformity of their distribution, in some embodiments, multiple culture units 21 are arranged in an array. The culture unit 21 can be a microcavity, groove, cup-shaped structure, or other spatial structure capable of accommodating the cultured organisms. Preferably, multiple culture units 21 are arranged in a regular matrix at the bottom of the container body 10, with adjacent culture units 21 arranged tangentially or separated by thin partition walls to increase the number of culture units 21 per unit area and reduce the amount of culture medium used. Of course, the cylindrical structure on the culture unit 21 can also be reduced or removed, retaining only the bottom core culture portion when removed, thereby reducing the amount of culture medium used.

[0071] Uniform dispersion of cell spheres (e.g., in situ inoculation and medium replacement of only one cell sphere or organoid in each microcavity) and stable suspension are achieved through the internal structure of the bottle (e.g., a matrix-arranged microcavity structure), reducing the need for manual operation.

[0072] In some embodiments, at least a portion of the culture unit 21 has a receiving space that tapers towards the bottom. For example, the longitudinal section of the culture unit 21 may be V-shaped, arc-bottomed, funnel-shaped, or other converging shapes. In this way, when the cell suspension enters the culture chamber 11, the cells or cell clusters can aggregate towards the bottom of the culture unit 21 under the action of gravity or centrifugal force, which is conducive to the formation of cell spheres or organoids with more uniform morphology and more compact structure.

[0073] Thus, multiple culture units 21 can provide relatively independent and stable culture carriers for multiple cultured organisms; the array arrangement of culture units 21 is conducive to batch culture and standardized operation; the structure of culture units 21 that shrinks towards the bottom can guide biological materials to concentrate at the bottom, reduce uneven sphericity caused by random cell dispersion, and improve the consistency of cultured organism formation and culture.

[0074] In order to maintain material exchange even when the limiting member 30 covers the culture unit 21, in some embodiments, please refer to Figures 7 to 10 The limiting member 30 is provided with a communicating portion 31. The communicating portion 31 may correspond to at least a portion of the culture units 21, or it may cover the area where multiple culture units 21 are located. The communicating portion 31 is used to keep the culture units 21 in communication with the culture chamber 11 when the limiting member 30 is in the first position, and to prevent the cultured material from leaving the culture units 21 through the communicating portion 31. Specifically, the communicating portion 31 may be an opening 311, a slit, a notch 312, a gap, a porous area, or a combination thereof.

[0075] For example, the connecting portion 31 can be a combination of an opening 311, a notch 312, and a gap. The opening 311 can be located in the limiting member 30 at a position corresponding to the center or top of the culture unit 21; the notch 312 can be located in the edge region or side of the limiting member 30 to improve the liquid flow efficiency on both sides of the limiting member 30. For example, a rectangular notch 312 can be provided on each of the four sides of the limiting member 30 to improve liquid flow efficiency and dead cell removal efficiency; simultaneously, a certain gap can be maintained between the limiting member 30 and the top of the culture unit 21. The size of the connecting portion 31 can be configured to be smaller than the external dimensions of the cultured organism, or the passage space formed between the connecting portion 31 and the culture unit 21 can be smaller than the external dimensions of the cultured organism, allowing fluid, dead cells, cell debris, and metabolites to pass through, while making it difficult for the cultured organism to pass through.

[0076] In some specific examples, when the cultured organism is a cell sphere or organoid with a diameter of approximately 500 to 1000 micrometers, the aperture of the opening 311 can be selected to be less than 50% of the average diameter of the cultured organism, for example, 100 to 300 micrometers. A certain gap, for example, 0 to 300 micrometers, can be maintained between the limiting member 30 and the top of the culture unit 21 to ensure gas communication between the inside and outside of the limiting member 30 and to prevent the cell sphere or organoid from lateral escape, thus balancing liquid flow, gas exchange, and the limiting of the cell sphere or organoid. The above values ​​are only examples and can be adjusted according to the size of the cultured organism and the size of the culture unit 21.

[0077] Thus, the connecting portion 31 and the limiting member 30 together form a flowable but impenetrable interception structure. On the one hand, culture medium, dead cells, and metabolic waste can be discharged from the culture unit 21 through the connecting portion 31, thereby preventing dead cells and waste from remaining in the culture unit 21 for a long time. On the other hand, the cultured organism, due to its large size, is jointly restricted by the limiting member 30 and the connecting portion 31, making it difficult for it to detach from the culture unit 21 with changes in liquid flow or container orientation, thereby achieving in-situ liquid exchange and low-damage culture. In this way, it can ensure the smooth outflow of dead cells and the smooth flow of gas-liquid exchange, while preventing cell spheres / organoids from escaping from the microcavity.

[0078] To improve the positioning stability of the limiting member 30 when it is in the first position, in some embodiments, please refer to... Figure 4 and Figure 5 A positioning space 40 is provided between the culture structure 20 and the inner wall of the container body 10. The positioning space 40 can be set in the outer area of ​​the culture structure 20, or it can be set on one or more sides of the culture structure 20. When the limiting member 30 is in the first position, at least part of the limiting member 30 is located within the positioning space 40, so that the limiting member 30 is positioned relative to the culture structure 20 and covers or blocks multiple culture units 21.

[0079] The positioning space 40 can be understood as an annular, frame-shaped, or local gap structure formed between the periphery of the culture structure 20 and the inner wall of the container body 10; when the limiting member 30 moves above the culture structure 20, the edge of the limiting member 30 can enter or abut against the positioning space 40, thereby limiting the lateral displacement of the limiting member 30.

[0080] In some specific examples, the culture structure 20 includes a microcavity array, with a moat-like gap formed between the outer periphery of the microcavity array and the sidewall of the container body 10. The edge of the limiting member 30 can be inserted into this gap. After the limiting member 30 is inserted into the positioning space 40, it covers the top of the culture unit 21, and the opening 311 corresponds to or substantially corresponds to the culture unit 21. The width and depth of the positioning space 40 can be set according to the edge dimensions of the limiting member 30, as long as reliable positioning of the limiting member 30 can be achieved.

[0081] Therefore, the positioning space 40 provides a mechanical positioning basis for the limiting member 30, making it less likely for the limiting member 30 to shift significantly relative to the culture structure 20 during liquid replacement, slight tapping, tilting, or rotation. Since the limiting member 30 can stably cover the culture unit 21, the risk of escape of the cultured organism during liquid replacement is further reduced. At the same time, the correspondence between the opening 311 and the culture unit 21 is more stable, which is beneficial for improving liquid replacement efficiency and culture consistency.

[0082] In order to adjust the position of the limiting member 30 while the container body 10 remains sealed, in some embodiments, please refer to... Figures 1 to 3 as well as Figures 11 to 13 The culture container 1 also includes a drive positioning structure 50. The drive positioning structure 50 is used to drive or position the limiting member 30 while the container body 10 remains sealed, so that the limiting member 30 switches between a first position and a second position. The drive positioning structure 50 may include a first mating member 51 disposed on the limiting member 30, and a second mating member 52 disposed on the outside of the container body 10. The first mating member 51 and the second mating member 52 can interact across the container body 10 to drive or fix the limiting member 30.

[0083] In some specific examples, the first mating component 51 includes a magnetic element disposed on the limiting member 30, and the second mating component 52 includes an outer magnetic plate 521 and / or an outer magnet 522 disposed on the outside of the container body 10. The outer magnetic plate 521 can be installed on the upper or lower part of the container body 10 to attract and fix the limiting member 30 to a second position or a first position. For example, nine small magnetic blocks are disposed through the outer magnetic plate 521, corresponding to nine small magnetic blocks embedded in the top of the limiting member 30; the outer magnet 522 can abut against the side wall of the container body 10 and move along the side wall to assist the limiting member 30 in moving within the culture chamber 11. The outer side of the container body 10 may also be provided with grooves, protrusions, positioning ribs or other positioning structures so that the second mating component 52 can reliably engage with the upper or lower part of the container body 10. The first mating component 51 and the second mating component 52 are not limited to magnetic mating components, but can also be diaphragm transmission structures, mechanical traction structures, negative pressure adsorption structures or combinations thereof, as long as they can move or position the limiting component 30 without destroying the sealed state.

[0084] Therefore, the drive positioning structure 50 allows operators or automated equipment to control the position of the limiting member 30 without opening the container body 10. During different stages such as inoculation, culture, medium change, and collection, the limiting member 30 can be switched to different positions, ensuring the airtightness of the culture chamber 11 while reducing the risk of contamination from opening the lid. Simultaneously, magnetic or other non-contact driving methods cause minimal disturbance to the fluid within the culture chamber 11, which helps reduce mechanical impact on the cultured organisms.

[0085] It is important to emphasize that by combining the fluid dynamics design of the closed system and using magnetic plates to prevent cell spheres or organoids from escaping, low shear force and high-efficiency fluid exchange can be achieved, avoiding mechanical damage to cell spheres or organoids.

[0086] In order to enable culture container 1 to be adapted for closed-loop media exchange and collection, in some embodiments, please refer to Figure 14The container body 10 is provided with a fluid communication structure 60. The fluid communication structure 60 communicates with the culture chamber 11 and is used to input or output fluid into or out of the culture chamber 11 while the container body 10 remains sealed. The fluid communication structure 60 can be located on the container lid 70, or on the side wall, top wall, neck 100, or other suitable location of the container body 10. The fluid communication structure 60 may include an inlet channel, an outlet channel, a collection channel, and a gas communication channel. The inlet channel is used to input culture medium, cell suspension, washing solution, or other liquids into the culture chamber 11; the outlet channel is used to discharge old culture medium, supernatant containing dead cells, or other waste liquids; the collection channel is used to collect the cultured material; the gas communication channel is used to connect to an air filter or a sterile gas channel to maintain pressure balance inside and outside the container body 10. The gas communication channel also communicates with the interior of the container body 10 to maintain a suitable gaseous environment for culturing the cultured material (cell spheres or organoids). The gas flow rate and ratio inside the container body 10 can be automatically controlled through a closed piping system.

[0087] In some specific examples, the inlet channel, outlet channel, and collection channel can each extend to a predetermined location within the culture chamber 11. For example, the outlet of the inlet channel can be positioned towards or adjacent to the flow guide structure 80, allowing the input liquid to be dispersed by the flow guide structure 80 first; the outlet channel can extend to a location close to the flow guide structure 80 or the liquid collection area within the container body 10, to fully discharge waste liquid; the collection channel can extend to the area where the side wall of the container body 10 intersects with the trapezoidal connection area 90, or to a location where the cultured material easily collects in its collection posture. The ends of the channels can be V-shaped (facilitating liquid drainage), beveled, porous, or other structures that facilitate drainage and prevent clogging. The container cap 70 can have multiple through holes, and the channels, after passing through the through holes, achieve a sealed connection through axial seals, sealant, welded parts, or an integrally molded structure.

[0088] For example, the inlet and outlet channels (also called inlet / outlet pipelines): the inside of the bottle has V-shaped ends that abut against the inner baffle to ensure complete liquid disposal; the collection channel (also called collection pipeline): the inside of the bottle has V-shaped ends that abut against the side wall at the trapezoidal connection area 90's side junction post; the gas communication channel on the bottle cap (also called gas pipeline): it is connected to an air filter to ensure pressure balance inside and outside the bottle, cooperating with a peristaltic pump and / or realizing liquid inlet and outlet, etc. Of course, an L-shaped quick connector can also be used to connect the two pipes to achieve pipeline bending.

[0089] Therefore, the fluid communication structure 60 enables the culture container 1 to perform liquid inlet, liquid outlet, pressure balancing, and collection operations in a closed state, reducing the risk of contamination caused by opening the lid. Since the liquid inlet channel, liquid outlet channel, and collection channel extend to predetermined positions within the culture chamber 11, liquid input, waste liquid discharge, and collection of cultured materials can be optimized according to the internal structure and operating posture of the culture container 1, thereby improving the thoroughness of liquid replacement and collection efficiency.

[0090] In this way, the design of this application embodiment supports integration with a closed pipeline system, which can realize automatic liquid replacement, avoid opening the cap and delicate manual operation (only in the early stage of liquid replacement is the bottom of the culture container tapped to remove dead cells), and improve the safety and efficiency of operation.

[0091] To reduce the impact of the liquid infusion process on the culture structure 20 and the cultured organisms, in some embodiments, please refer to... Figures 1 to 3 The culture chamber 11 is equipped with a flow guiding structure 80. The flow guiding structure 80 can be located between the trapezoidal connecting area 90 and the culture chamber 11, or near the outlet of the liquid inlet channel. The flow guiding structure 80 can be a baffle (used to slow down the flow rate, avoid or reduce bubble generation, and prevent liquid splashing; in manual processes, the pipette or automatic process piping can rest against the center of this baffle, allowing the liquid to slowly flow into the culture container to both sides during the inlet process), a guide plate, a partition, or other structures that can change the fluid direction and reduce the flow rate. When the fluid enters the culture chamber 11 through the liquid inlet channel, the fluid can first impact or contact the flow guiding structure 80, and then slowly flow into the culture chamber 11 along both sides or around the flow guiding structure 80, thereby avoiding direct impact of the liquid flow on the culture unit 21 on the culture structure 20. The flow guiding structure 80 can also guide the liquid in the culture chamber 11 to converge towards the area where the liquid outlet channel is located during drainage, thereby improving the efficiency of draining old culture medium and non-target substances.

[0092] Therefore, the flow guiding structure 80, in conjunction with the fluid communication structure 60, can reduce the disturbance caused by the liquid inlet to the culture medium and the culture structure 20, and reduce the generation of bubbles and liquid splashing. On the other hand, it can promote the flow of waste liquid, dead cells and metabolic waste to the outlet channel during liquid drainage, thereby improving the efficiency of liquid exchange and reducing the risk of escape or damage to the culture medium.

[0093] To further illustrate the use of culture container 1 in actual culture processes, the following explanation uses cell sphere or organoid culture as an example. It should be noted that this procedure is merely an example; the actual cultured organisms, culture medium types, centrifugation conditions, medium change frequency, and collection methods can all be adjusted according to specific cell types and process requirements.

[0094] Before use, the limiting member 30 can be held in the second position, for example, near the top of the container body 10, so that the culture unit 21 is at least partially open. If a magnetically driven positioning structure 50 is used, the first fitting member 51 can be attracted by the second fitting member 52 (outer magnetic plate 521) located on the upper outer side of the container body 10, thereby fixing the limiting member 30 in the second position. The culture container 1 can be used as a disposable sterile consumable, while external components such as the outer magnetic plate 521, the outer magnet 522, and the container adapter mechanism 203 can be reused. Holding the limiting member 30 in the second position when not unpacked or during transportation helps to prevent the limiting member 30 from impacting the culture structure 20 due to shaking.

[0095] During inoculation, the cell suspension can be introduced into the culture chamber 11 through the inlet channel in the fluid communication structure 60. After entering the culture chamber 11, the cell suspension can slowly flow into the container body 10 under the action of the guide structure 80, reducing the direct impact of the liquid flow on the culture structure 20. Subsequently, the culture container 1 can be placed in the centrifuge device 2 for centrifugation, allowing the biological material to enter the culture unit 21 and aggregate at the bottom under centrifugal force. The centrifugation speed, time, and centrifugal force can be adjusted according to the cell type, the size of the culture unit 21, and the size of the target cultured material. For example, in some specific processes, centrifugation conditions of 300 rpm to 1000 rpm and 1 minute to 10 minutes can be used. After centrifugation, the cells or cell clusters form preliminary aggregates in the culture unit 21 and can continue to be cultured in the culture chamber 11.

[0096] Therefore, by combining closed-loop liquid inoculation with centrifugal aggregation, inoculation and positioning of biological materials can be completed without opening the culture container 1. The contractile containment space of the culture unit 21, together with centrifugal force, makes it easier for cells to enter and aggregate at the bottom of the culture unit 21, thereby improving the efficiency and consistency of cell formation.

[0097] During medium replacement, the limiting member 30 can be switched from the second position to the first position by driving the positioning structure 50. For example, after removing the outer magnetic plate 521 located on the upper part of the container body 10, the limiting member 30 can move towards the area where the culture structure 20 is located under the action of gravity; then, the outer magnetic plate 521 is fixed to the lower part of the container body 10, so that the limiting member 30 stably covers or blocks the culture unit 21. If necessary, the position of the limiting member 30 can also be adjusted by moving the outer magnet 522 along the side wall of the container body 10. When the limiting member 30 is in the first position, the culture medium is confined within the culture unit 21, while the culture medium, dead cells, and metabolic waste can be discharged through the connecting part 31 or the gap between the limiting member 30 and the culture unit 21.

[0098] After the limiting member 30 is in the first position, the culture container 1 can be gently tapped, tilted, rotated, or reciprocated to release dead cells, cell debris, and metabolic waste from the culture unit 21 into the culture chamber 11. For example, the bottom of the container body 10 can be gently tapped to allow non-target substances to overflow from the culture unit 21 as much as possible; the culture container 1 can also be reciprocated by rotating or shaking along its long side (e.g., rotating 180 degrees clockwise and counterclockwise 3-5 times along the central axis of the long side) to promote the outflow of non-target substances from each culture unit 21; then, the culture container 1 is tilted near the flow guide structure 80, allowing the liquid to flow to the area where the outlet channel is located under gravity, and left to stand for a period of time to allow larger dead cell clumps or flocculent matter to collect with the liquid. Afterwards, the old culture medium containing non-target substances is discharged through the outlet channel, and new culture medium is added through the inlet channel. After the medium change is completed, the external magnetic plate 521 can be removed or adjusted so that the limiting member 30 moves away from the first position and returns to the second position, allowing the culture to continue to be cultured statically within the culture unit 21. Alternatively, the limiting member 30 can be kept in the first position to ensure stable culture of the culture within the culture unit 21.

[0099] Thus, during the liquid replacement process, the limiting component 30, the connecting part 31, the culture unit 21, the fluid communication structure 60, and the flow guiding structure 80 work together to allow the old culture medium and non-target substances to be discharged from the culture unit 21 and discharged from the culture chamber 11 through the liquid outlet channel; at the same time, the cultured material is not easy to escape due to the shielding of the limiting component 30, and does not need to be transferred to other containers, thereby achieving in-situ, safe, low-damage, and efficient liquid replacement.

[0100] During collection, the limiting member 30 can be switched from the first position or the culture position to the second position, so that the culture unit 21 is at least partially open. For example, an external magnetic plate 521 can be placed on the upper part of the container body 10, and the limiting member 30 can be attracted and moved to the top area of ​​the container body 10 by means of an external magnet 522, so that the culture unit 21 is opened. Subsequently, the culture container 1 can be tilted or rotated to a predetermined angle (rotated 100-150 degrees along the central axis of the wide side), and the container body 10 can be gently tapped to move the cultured material out of the culture unit 21 and gather in the predetermined collection area of ​​the culture chamber 11. After the cultured material gathers, it can be discharged from the culture chamber 11 through the collection channel. The inlet of the collection channel can be set at a position where the cultured material can easily gather in the tilted state to improve collection efficiency and reduce residue.

[0101] Therefore, during the collection stage, by moving the limiting component 30 away from the culture unit 21, the cultured material can be moved out of the culture unit 21; by adjusting the container posture and cooperating with the collection channel, the cultured material can be collected in a closed state, avoiding contamination and damage caused by opening the lid and repeated blowing, and improving the efficiency of batch collection.

[0102] To make the culture container 1 suitable for automated or large-scale culture, this application also provides a culture system. Please refer to... Figure 15 and Figure 16 In some embodiments, the culture system includes a culture container 1 and a centrifuge device 2. The centrifuge device 2 is used to centrifuge the culture container 1 while it is in a sealed state. The centrifuge device 2 includes a rotary drive mechanism 201, a container holding mechanism 202, and a container fitting mechanism 203. The rotary drive mechanism 201 may include a drive motor, a spindle, bearings, and control components to provide rotational power and control the centrifugation speed and time. The container holding mechanism 202 may include a gantry, a basket, or other structure for supporting the culture container 1. The gantry is used to suspend the basket, facilitating operation and positioning, and supporting the structural stability of the entire centrifugation system. The container fitting mechanism 203 is disposed on the container holding mechanism 202 and is used to position and support the culture container 1, so that the culture container 1 maintains a predetermined posture during centrifugation. The centrifuge device 2 may also include a centrifuge chamber 204 and a shock-absorbing base 205. The centrifuge chamber 204 is used to accommodate the container holding mechanism 202 and the culture container 1, providing a uniform centrifugal force field to ensure cell separation efficiency. The shock-absorbing base 205 is used to reduce vibration during the centrifugation process and improve the stability and safety of the centrifugation process.

[0103] In some specific examples, the container adapter mechanism 203 can be configured as a customized adapter according to the shape of the culture container 1 to support the bottom, side walls, and / or neck 100 of the culture container 1, preventing the culture container 1 from shaking or deforming during centrifugation. The fluid communication structure 60 of the culture container 1 can maintain a sealed connection before centrifugation, so that the culture container 1 can be directly centrifuged after inoculation without opening the cap. When the rotation drive mechanism 201 drives the container holding mechanism 202 to move, the biological material in the culture container 1 enters the culture unit 21 under centrifugal action and gathers at the bottom of the culture unit 21.

[0104] Thus, the culture system can connect closed inoculation, centrifugation and aggregation, and subsequent culture processes. Since the centrifuge device 2 positions and supports the culture container 1 through the container adapter mechanism 203, the posture and force of the culture container 1 are more stable during the centrifugation process, which is conducive to the uniform entry of biological materials into multiple culture units 21; at the same time, the culture container 1 remains in a closed state, reducing the risk of contamination caused by post-inoculation transfer and opening operations.

[0105] To improve large-scale culture capabilities, in some embodiments, multiple culture containers 1 can be connected via tubing to form a culture assembly. The multiple culture containers 1 can be connected to a main tubing via sterile heat-sealed connectors or sterile fittings. The main tubing can be connected to a dispensing device, a medium exchange device, a peristaltic pump, an automated culture device, or a cell workstation for uniformly distributing cell suspension or culture medium to the multiple culture containers 1. The multiple culture containers 1 can be used as a set of consumables, for example, forming two-unit, four-unit, six-unit, or more-unit culture assemblies. The culture assembly can be integrally housed within the container holding mechanism 202 of the centrifuge device 2, and each culture container 1 is positioned separately via a container adapter mechanism 203.

[0106] Taking the six-piece consumable set as an example, each set consists of six cell culture flasks. This means that a maximum of six or more cell culture flasks / bags can be centrifuged at a time. All cell culture flasks are first evenly separated through a centripetal converging main tubing via a sterile heat-sealed connector (or connected by puncture or sterile connector), and then centrifuged to form cell spheres or organoids for subsequent culture.

[0107] Therefore, by connecting multiple culture containers 1 through pipelines, unified liquid feeding, separation, centrifugation, culture, and liquid exchange can be achieved, reducing the errors and contamination risks caused by individual operations. The culture components, in conjunction with the centrifuge device 2 and the automated culture device, are beneficial for improving the throughput, standardization, and commercial production adaptability of the culture process.

[0108] In other embodiments, the culture container 1 can be a culture bag. In this case, the container body 10 can be a flexible container body, the culture structure 20 is disposed within the flexible container body, and the limiting member 30 is also movably disposed within the culture chamber 11. The flexible container body can be supported and fixed by an external support structure. The external support structure may include clamps, frames, trays, buckles, limiting plates, or combinations thereof, for maintaining the shape and position of the culture bag during centrifugation, culture, medium replacement, and collection. For the flexible container body, the limiting member 30 can be magnetically attached to the top or bottom of the flexible container body, or its position can be fixed by snap-fits, clamping structures, or other positioning structures inside or outside the flexible container body.

[0109] Therefore, by setting the container body 10 as a flexible bag, it can be adapted to some disposable closed culture processes and flexible consumable systems; by supporting the flexible container body with an external support structure, the deformation of the flexible bag during centrifugation and liquid replacement can be reduced, thus reducing the impact on the fit between the culture unit 21 and the limiting component 30; by controlling the position of the limiting component 30 through magnetic force, buckles, or other means, it is still possible to achieve anti-escape liquid replacement and collection in a closed state.

[0110] In some embodiments, the inner walls of the culture structure 20 and the container body 10 may be provided with low-adsorption or ultra-low-adsorption surfaces. These low-adsorption or ultra-low-adsorption surfaces can be achieved through coatings, surface modification, or surface texturing. For example, the inner surface of the culture unit 21 and the inner wall of the container body 10 may have coatings that reduce cell adhesion, thereby reducing the likelihood of the cultured material adhering to the culture unit 21 or the inner wall of the container body 10. These coatings may include, but are not limited to, ultra-low-adsorption methacryloyloxyethylphosphorylcholine polymer coatings or other low-adsorption treatment layers.

[0111] Therefore, low-adsorption or ultra-low-adsorption surfaces can reduce undesirable adhesion between the cultured material and the inner wall of the container, allowing the cultured material to maintain a good morphology and activity during the culture process, and making it easier to remove and collect it from the culture unit 21 during the collection stage, thereby improving culture stability and collection efficiency.

[0112] The pore size and bottom geometry of traditional microporous culture flasks cannot support the stable attachment and growth of large-diameter cell spheres exceeding 300 micrometers, resulting in limited cell sphere size and making it difficult to meet the culture requirements of high-diameter cell spheres. In contrast, this embodiment combines the specially designed bottom structure and surface treatment technology mentioned above to improve the attachment ability of large-diameter (greater than 300 micrometers) cell spheres, preventing them from detaching or floating during medium changes.

[0113] As can be seen from the above, the focus of this application is to construct an efficient integrated system for interception, purification, medium exchange, and automation suitable for cell spheroid or organoid culture. This system, through its unique interception structure, effectively removes dead cells and secondary metabolites generated during culture while ensuring the efficiency of culture medium circulation and gas exchange, achieving continuous purification of the culture environment. Simultaneously, this application employs an in-situ medium exchange method, eliminating the need for direct contact with cell spheroids or organoids or transfer of the culture medium, thus achieving quantitative or even near-complete replacement of the culture medium. This avoids mechanical damage to cell spheroids or organoids during medium exchange, maintaining their structural integrity, physiological activity, and functional stability. Compared to traditional manual medium exchange or the delicate, manual medium exchange methods in micro-well culture flasks, this application achieves more efficient, stable, and non-destructive medium exchange operations, significantly improving the success rate and consistency of cell culture.

[0114] Furthermore, existing culture containers are often limited by their physical structure, making it difficult to accommodate different particle sizes, especially larger cell spheres or organoids. This can easily lead to problems such as uneven culture medium flow, low medium exchange efficiency, and cell sphere or organoid escape. To address these shortcomings, this application improves the adaptability of the closed culture container to cell spheres or organoids of different sizes through a synergistic structural design of inner and outer cover plates. It can meet the culture needs of larger particle sizes and can also be adapted to smaller particle sizes by adjusting the live cell inoculation concentration, thereby broadening the application range of closed containers in cell sphere or organoid culture. Simultaneously, this application utilizes an integrated closed medium exchange system, enabling medium exchange, collection, and cleaning operations to be completed within the same culture container. This eliminates the need for frequent container changes or dispersing of cell spheres and organoids, reducing human intervention and contamination risks, lowering labor intensity, and improving operational standardization, automation, and result reproducibility. This provides a solid technical foundation for high-throughput experiments, large-scale production, and commercial applications.

[0115] In summary, this application, through the cooperation of the container body 10, culture structure 20, limiting member 30, connecting part 31, positioning space 40, driving positioning structure 50, fluid communication structure 60, flow guiding structure 80, and centrifuge device 2, enables the culture medium to be stably formed and cultured within the culture unit 21. During fluid exchange, the limiting member 30 restricts the culture medium from detaching from the culture unit 21 while allowing the flow of fluid and non-target substances. During collection, the limiting member 30 switches to a second position, allowing the culture medium to be removed from the culture unit 21 and collected through the collection channel. Therefore, this application enables in-situ, safe, non-destructive, and efficient culture of cell spheres or organoids in a closed culture container, and has good prospects for automation and large-scale application.

[0116] It should be noted that in this specification, relational terms such as first and second are used only to distinguish one entity from several other entities, and do not necessarily require or imply any such actual relationship or order between these entities.

[0117] The culture container and culture system provided in this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are only for the purpose of helping to understand the solution and core ideas of this application. It should be noted that those skilled in the art can make several improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of this application.

Claims

1. A culture container, characterized in that, include: The container body (10) has a sealed culture chamber (11). A culture structure (20) is disposed within the culture chamber (11), and the culture structure (20) includes a plurality of culture units (21) for accommodating the cultured organism. The limiting member (30) is movably disposed within the culture chamber (11) and can switch between a first position and a second position; When the limiting member (30) is in the first position, the limiting member (30) at least partially covers or blocks the culture unit (21) to restrict the cultured organism from leaving the culture unit (21) and allows fluid and non-target substances to flow between the culture unit (21) and the culture chamber (11). When the limiting member (30) is in the second position, the culture unit (21) is at least partially open to allow the cultured organism to enter the culture unit (21) or move out of the culture unit (21).

2. The culture container as described in claim 1, characterized in that, The limiting member (30) is provided with a connecting portion (31) corresponding to at least a portion of the culture unit (21). The connecting portion (31) is used to keep the culture unit (21) connected to the culture chamber (11) when the limiting member (30) is in the first position, and to prevent the cultured material from leaving the culture unit (21) through the connecting portion (31).

3. The culture container as described in claim 2, characterized in that, The connecting part (31) is one or more of the following: opening (311), slit, notch (312), gap, and porous region.

4. The culture container as described in claim 1, characterized in that, A positioning space (40) is provided between the culture structure (20) and the inner wall of the container body (10). When the limiting member (30) is located in the first position, at least part of the limiting member (30) is located in the positioning space (40) so that the limiting member (30) is positioned relative to the culture structure (20) and covers or blocks multiple culture units (21).

5. The culture container as described in claim 1, characterized in that, The culture container (1) further includes a drive positioning structure (50), which is used to drive or position the limiting member (30) while the container body (10) is kept sealed, so that the limiting member (30) switches between the first position and the second position.

6. The culture container as described in claim 5, characterized in that, The drive positioning structure (50) includes a first mating member (51) disposed on the limiting member (30) and a second mating member (52) disposed on the outside of the container body (10). The first mating member (51) and the second mating member (52) can interact with each other across the container body (10) to drive or fix the limiting member (30).

7. The culture container as described in claim 1, characterized in that, The plurality of culture units (21) are arranged in an array, and at least some of the culture units (21) have a receiving space that contracts toward the bottom so that the cultured material entering the culture unit (21) gathers toward the bottom of the culture unit (21).

8. The culture container as described in claim 1, characterized in that, The container body (10) is provided with a fluid communication structure (60), which is connected to the culture chamber (11). The fluid communication structure (60) includes an inlet channel, an outlet channel, a collection channel and a gas communication channel. The inlet channel, the outlet channel and the collection channel extend to predetermined positions within the culture chamber (11). The gas communication channel is used to maintain the pressure balance inside and outside the container body (10). The gas communication channel is also used to communicate with the interior of the container body (10) so that the interior of the container body (10) maintains a gaseous environment suitable for the culture of the organism.

9. The culture container as described in claim 8, characterized in that, The culture chamber (11) is provided with a flow guiding structure (80), which is used to reduce the impact of the fluid input through the fluid communication structure (60) on the culture structure (20) and to guide the fluid discharged through the fluid communication structure (60) to the liquid outlet channel.

10. A culture system, characterized in that, include: The culture container (1) as described in any one of claims 1 to 9; and Centrifuge device (2) is used to centrifuge the culture container (1) while the culture container (1) is in a closed state; The centrifugation device (2) includes a rotation drive mechanism (201), a container holding mechanism (202), and a container adapter mechanism (203). The container adapter mechanism (203) is disposed on the container holding mechanism (202) and is used to position and support the culture container (1). The rotation drive mechanism (201) is used to drive the container holding mechanism (202) to move the culture container (1) so that the biological material in the culture container (1) enters the culture unit (21) under centrifugation.

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