Culture device, lid, container

Abstract

The application aims to provide a culture device, a cover and a container. The culture device comprises a first culture element, the first culture element comprises a cover and a container, the cover and the container form a closed space, the container is provided with a forming cavity and a first channel, the first channel is communicated with the forming cavity, so that the forming material can flow from the first channel to the forming cavity, the forming cavity can define the forming shape provided by the forming material, the cover provides a gas exchange channel for the communication between the outside and the culture space, at least one of the cover and the container is provided with a second channel, the second channel provides liquid exchange between the culture process and the outside, and a liquid spraying port, the liquid spraying direction of the liquid spraying port is towards one side of the forming cavity, so that the liquid sprayed from the liquid spraying port can reach the forming cavity.

Description

Technical Field

[0001] This application relates to a culture device, a cover, and a container. Background Technology

[0002] The extracellular matrix (ECM) is a three-dimensional network of proteins and carbohydrates secreted by cells. It not only provides support for tissues but also regulates cell fate, influencing development, repair, and disease progression. Decellularized extracellular matrix (dECM) is a core biomaterial in tissue engineering and regenerative medicine. By removing cellular components from allogeneic or xenogeneic tissues, it maximizes the preservation of the natural structure, mechanical properties, and bioactive components of the extracellular matrix. dECM possesses excellent biocompatibility and biomimetic microenvironment characteristics, supporting cell adhesion, proliferation, and differentiation, and effectively inducing in-situ tissue regeneration. It has been widely applied in various tissue repair fields, including cartilage repair, skin regeneration, and tissue repair in blood vessels, nerves, and the heart, demonstrating significant clinical translational potential.

[0003] Traditional animal-derived dECM still poses risks of disease transmission, residual immunogenicity, and significant batch-to-batch variability. In contrast, cell-derived ECM (cdECM) is prepared by in vitro secretion of human cells, contains no animal-derived components, has a clear origin, high safety, stronger process controllability, and is easier to standardize, making it particularly suitable for clinical-grade regenerative medicine applications.

[0004] Generally speaking, the cdECM fabrication process can be summarized into three main stages:

[0005] First, cells and biodegradable hydrogels are co-cultured in a three-dimensional environment, and the cells actively secrete their own ECM proteins to form a "cell-hydrogel" complex.

[0006] Subsequently, the artificial hydrogel scaffold was specifically degraded through enzymatic hydrolysis or ion chelation to obtain a “cell-native ECM” construct that is entirely maintained by cell-secreted ECM.

[0007] Finally, a decellularization process was used to remove cellular components, ultimately yielding pure cdECM material.

[0008] Currently, the process for cultivating cdECM materials has a long production cycle (even reaching several weeks or months) and heavily relies on open, manual operations, resulting in high overall process costs and low efficiency. Specifically, the long cultivation time not only significantly increases direct costs such as equipment occupancy, energy consumption, and culture medium consumption, but also places extremely high demands on the aseptic control of the production environment. Furthermore, the current predominantly manual, open operations (such as medium changes, transfers, and monitoring) further limit the reproducibility and scalability of the process, introducing not only contamination risks and batch-to-batch variability, but also making it difficult to increase production capacity per unit time, thus failing to meet the core requirements of economic efficiency and scalability for commercial applications.

[0009] To address at least one of the problems mentioned above, the applicant has proposed a culture device, a cover, or a container. Utility Model Content

[0010] A culture apparatus according to a first aspect of this application includes a first culture element, the first culture element comprising: a cover; a container, the cover and the container forming a closed space; the container having a forming chamber and a first channel, the first channel communicating with the forming chamber to allow forming material to flow from the first channel to the forming chamber, the forming chamber defining a forming shape provided by the forming material, the cover providing a gas exchange channel communicating with the outside world and the culture space; at least one of the cover and the container having: a second channel providing liquid exchange between the culture process and the outside world; and a spray nozzle, the spray direction of the spray nozzle facing one side of the forming chamber, such that liquid sprayed from the spray nozzle can reach the forming chamber.

[0011] In one or more embodiments of the culture device, the spray nozzle is disposed on a cover, the cover including a semi-permeable membrane providing a gas exchange channel between the outside and the space; preferably, the cover further includes a support that fixes the semi-permeable membrane; preferably, the support includes a first support and a second support, the first and second supports clamping and fixing the semi-permeable membrane, the first and second supports having perforated portions, allowing gas to exchange with the space through the perforated portions and the semi-permeable membrane; preferably, the dimensional boundary of the support provides the dimensional boundary of the cover; the first and second supports are spoke-shaped, the space between adjacent spokes providing the perforated portion; preferably, the spray nozzle is disposed in the middle of the support; preferably, the spray nozzle is provided with an atomizing element; preferably, a sealing element is disposed on the periphery of the cover, so that the cover seals against the side wall of the container.

[0012] In one or more embodiments of the culture apparatus, the container has a first channel on its sidewall, the side on which the cover is installed is defined as the top, the opposite side as the bottom, and the direction extending from the top to the bottom is defined as the height direction. The bottom of the container has the molding chamber. Preferably, the bottom of the container includes a body and a recess recessed from the body, the recess providing the molding chamber, and the first channel communicating with the recess. Preferably, the container also has a second channel on its sidewall, the second channel communicating with the surface of the body. The second channel provides liquid exchange between the culture process and the outside environment. The body can provide a culture position for the cultured object after molding in the molding chamber, such that the first culture piece provides the molding position and culture position of the culture apparatus.

[0013] In one or more embodiments of the culture device, the bottom of the container includes a body and a bottom cover. The bottom cover and the body are separately disposed. The body is integrally formed and connected to the side wall of the container. The body is provided with a through hole extending through the height of the body. The bottom cover includes a bottom cover body and a protrusion extending from the bottom cover body. The protrusion and the through hole correspond to and cooperate with each other. The protrusion and the through hole can form the forming chamber.

[0014] Preferably, the bottom cover and the body are movable relative to each other in the height direction, and the bottom cover and the body have a first relative position and a second relative position, the second relative position being higher than the first relative position, and the bottom cover can move relative to the body from the first relative position to the second relative position; the top surface of the protrusion at the first relative position and the hole wall of the through hole form the molding chamber; preferably, the bottom cover and the body are integrally formed and connected, and the bottom cover and the body are not movable relative to each other in the height direction; preferably, the protrusion is provided with a sealing element in the circumferential direction, so that the protrusion seals with the hole wall of the through hole; preferably, the shape of the bottom cover body is a cone shape that decreases in size from top to bottom, and the cone surface of the bottom cover body is used to cooperate with the external bottom support; preferably, the top of the side wall of the container has a flange that protrudes radially outward from the side wall, and the flange is used to cooperate with the external top support; preferably, the top support and the bottom support are snap-fit ​​connected.

[0015] In one or more embodiments of the culture apparatus, a second culture element is further included; the second culture element includes: a cover; a container, the cover and the container forming a closed space; the container is provided with a second channel communicating with the space, allowing culture-related liquid to flow from the second channel into the space; the cover provides a gas exchange channel communicating with the outside world and the culture space; the first culture element provides the forming position of the culture apparatus, and the second culture element provides the culture position of the culture apparatus.

[0016] In one or more embodiments of the culture device, the cover does not have the liquid spraying port, the container does not have the first channel, and the molding chamber is not provided.

[0017] In one or more embodiments of the culture apparatus, the sidewalls and / or bottom of the container, and / or the cover, are made of a transparent material.

[0018] The beneficial effects of using the culture device described above include, but are not limited to, by designing the structure of the culture device, providing the container and cover with a molding chamber, a first channel, a second channel and a gas exchange channel, the culture process of cdECM materials is automated or semi-automated, as well as closed culture, avoiding the shortcomings of the existing technology of open operation (such as liquid change, transfer and monitoring) which has poor repeatability and scalability potential.

[0019] A cover according to a second aspect of this application includes: a spray nozzle for spraying liquid; a semi-permeable membrane providing a gas exchange channel for the cover; the cover further includes a support for fixing the semi-permeable membrane; preferably, the support includes a first support and a second support, the first and second supports clamping and fixing the semi-permeable membrane, the first and second supports having perforated portions allowing gas to pass through the perforated portions and the semi-permeable membrane for gas exchange; preferably, the dimensional boundaries of the support provide the dimensional boundaries of the cover; the first and second supports are spoke-shaped, with the space between adjacent spokes providing the perforated portions; preferably, the spray nozzle is disposed in the middle of the support; preferably, the spray nozzle is provided with an atomizing element; preferably, a sealing element is disposed on the periphery of the cover to seal the cover against the side wall of the container.

[0020] A container according to a third aspect of this application includes: a molding chamber and a first channel, the first channel communicating with the molding chamber to allow molding material to flow from the first channel to the molding chamber, the molding chamber defining a molding shape provided by the molding material; preferably, the first channel is provided on the side wall of the container, and the molding chamber is provided on the bottom of the container; preferably, the bottom of the container includes a body and a recess recessed from the body, the recess providing the molding chamber, the first channel communicating with the recess; preferably, the bottom of the container includes a body and a bottom cover, the bottom cover being separately disposed from the body, the body being integrally formed and connected to the side wall of the container, the body having a through hole extending through the height of the body, the bottom cover including a bottom cover body and a protrusion extending from the bottom cover body, the protrusion correspondingly engaging with the through hole, the protrusion and the through hole forming a molding shape. The molding chamber is described above; preferably, the bottom cover and the body are movable relative to each other in the height direction, and the bottom cover and the body have a first relative position and a second relative position, the second relative position being higher than the first relative position, and the bottom cover is movable relative to the body from the first relative position to the second relative position; the top surface of the protrusion at the first relative position and the hole wall of the through hole constitute the molding chamber; preferably, the bottom cover and the body are integrally molded and connected, and the bottom cover and the body are not movable relative to each other in the height direction; preferably, the protrusion is provided with a sealing element in the circumferential direction, so that the protrusion and the hole wall of the through hole are sealed; preferably, the shape of the bottom cover body is a cone shape that decreases in size from top to bottom, and the cone surface of the bottom cover body is used to cooperate with an external bottom support; preferably, the top of the side wall of the container has a flange that protrudes radially outward from the side wall, and the flange is used to cooperate with an external top support.

[0021] In one or more embodiments of the container, the sidewall of the container is further provided with a second channel, the second channel communicating with the surface of the body; the second channel provides liquid exchange between the culture process and the outside world; the body can provide a culture position for the cultured object after it has been formed in the forming chamber, so that the container provides both a forming position and a culture position.

[0022] A method for manufacturing a cell-derived ECM according to a fourth aspect of this application, using the culture apparatus described in the first aspect, includes the following steps:

[0023] The molding material is added to the molding chamber through the first channel, and the molding reaction liquid is sprayed onto the surface of the molding material through the spray nozzle to form a molded composite. Then, the cleaning liquid is injected through the second channel until the molding reaction liquid is removed, and the cleaning liquid is discharged through the second channel.

[0024] The complex is cultured by adding culture medium through the second channel;

[0025] A degradation substance is added through a second channel to degrade part of the molded material;

[0026] The scavenging solution is added through the second channel to remove cellular components. Attached Figure Description

[0027] The above and other features, properties and advantages of this application will become more apparent from the following description taken in conjunction with the accompanying drawings and embodiments, wherein:

[0028] Figure 1 This is a schematic diagram of the structure of a culture device from an external perspective, according to one embodiment.

[0029] Figure 2 This is a cross-sectional structural schematic diagram of a culture device according to an embodiment.

[0030] Figure 3 This is an exploded structural diagram of a culture device according to an embodiment.

[0031] Figure 4 This is a structural schematic diagram of the first culture element of a culture device according to an embodiment, viewed from the outside.

[0032] Figure 5 This is a cross-sectional structural schematic diagram of the first culture element of a culture device according to an embodiment.

[0033] Figure 6 This is an exploded structural diagram of the first culture element of a culture device according to an embodiment.

[0034] Figure 7 This is a schematic diagram of the structure of the second culture element of a culture device according to an embodiment, viewed from the outside.

[0035] Figure 8 This is a cross-sectional schematic diagram of the second culture element of a culture device according to one embodiment.

[0036] Figure 9 This is an exploded structural diagram of the second culture element of a culture device according to one embodiment.

[0037] Figure 10 This is a schematic diagram of the structure of a culture device and a support assembly according to one embodiment.

[0038] Figure 11 This is a structural schematic diagram of the cover from an external perspective of one embodiment.

[0039] Figure 12 This is a structural schematic diagram of the bottom cover of a container from an external perspective, according to one embodiment.

[0040] Figure 13This is a schematic flowchart of a cell culture method according to one embodiment.

[0041] Figure label:

[0042] 100 - Cultivation device; 101 - First culture element; 102 - Second culture element; 10 - Space.

[0043] 1-Cover, 11-Spray nozzle, 111-Atomizing element, 12-Semi-permeable membrane, 13-Support, 130-Hollow section, 131-First support, 132-Second support, 1301-Spoke, 14-Sealing element;

[0044] 2-Container, 20-Molding chamber, 201-First relative position, 202-Second relative position, 21-First channel, 22-Body, 221-Through hole, 23-Second channel, 24-Bottom cover, 241-Bottom cover body, 242-Protrusion, 243-Sealing element, 25-Recess, 26-Flange;

[0045] 31-Bottom support, 32-Top support. Detailed Implementation

[0046] The following discloses various implementations or embodiments of the described subject matter. To simplify the disclosure, specific examples of elements and arrangements are described below. These are merely examples and are not intended to limit the scope of protection of the invention. For instance, a first feature formed above or on a second feature, as described later in the specification, can include implementations where the first and second features are formed in a direct connection, or implementations where an additional feature is formed between the first and second features, thus the first and second features may not be directly connected. Furthermore, reference numerals and / or letters may be repeated in different examples in these disclosures. This repetition is for brevity and clarity and does not in itself indicate a relationship between the various implementations and / or structures to be discussed. Further, when the first element is described in connection with or combined with a second element, the description includes implementations where the first and second elements are directly connected or combined with each other, as well as implementations where one or more other intervening elements are added to indirectly connect or combine the first and second elements with each other.

[0047] It is understood that the following flowcharts are used to illustrate the steps performed by the cultivation method according to embodiments of this application. It should be understood that, depending on the actual situation, the preceding or following steps may not necessarily be performed precisely in order. Other steps may be added to these processes, or one or more steps may be removed from them.

[0048] like Figures 1 to 6As shown, the culture device 100, such as the culture device 100 for cdECM, includes a first culture element 101, which includes a cover 1 and a container 2, the cover 1 and the container 2 forming a closed space 10.

[0049] The container 2 is provided with a molding chamber 20 and a first channel 21. The first channel 21 is connected to the molding chamber 20, so that the molding material can flow from the first channel 21 to the molding chamber 20. The molding chamber 20 can define the molding shape provided by the molding material.

[0050] The cover 1 provides a gas exchange channel connecting the outside world with the culture space 10.

[0051] At least one of the cover 1 and the container 2 is provided with: a second channel 23, which provides liquid exchange between the culture process and the outside environment; and a spray nozzle 11, the spray direction of which is directed toward one side of the forming chamber 20, so that the liquid sprayed from the spray nozzle 11 can reach the forming chamber 20.

[0052] Specifically, the substance transported in the first channel 21 is a molding material. Taking the cdECM culture device as an example, the molding material is a cell-hydrogel premix formed by mixing cells with crosslinkable matrix materials. Since the molding material is generally attached to the first channel 21, the first channel 21 is not used as a material exchange channel for culture medium, waste liquid and / or gas, etc., which need to exchange substances during the culture process, but is generally only used for the input of the molding material.

[0053] The shape of the molding chamber 20 can be defined by the molding shape provided by the molding material. Taking the cdECM culture device as an example, the cdECM material can be a patch, i.e., a disc, and the molding chamber can correspond to a cylindrical hole or groove structure.

[0054] The cover 1 provides a gas exchange channel connecting the outside world and the culture space 10, such as providing the gases required for culture and discharging the waste gases generated.

[0055] The second channel 23 provides for the exchange of liquids between the culture process and the external environment, such as the discharge of waste liquid, cleaning solution, and other liquids.

[0056] Taking the cdECM culture device as an example, during the scaffold degradation stage, an appropriate amount of scaffold degradation solution can be injected into the space 10 through the second channel 23, immersing the cell-hydrogel complex in the scaffold degradation solution to promote the specific degradation of the scaffold material. After the reaction is complete, the degradation waste liquid is discharged through the second channel 23. This process can be repeated until the degradable scaffold components in the cell scaffold tissue are completely decomposed and removed. During the decellularization stage, an appropriate amount of decellularization buffer is injected through the second channel 23, and under the control of the external control console, the space 10 is periodically shaken at a preset intensity and time to gently lyse the cells and promote the separation and detachment of cell components from the extracellular matrix. The decellularization is observed under a microscope, and DNA content detection and other methods are used to confirm whether the cells and their residual components have been completely removed to ensure that the obtained extracellular matrix meets the requirements for subsequent applications.

[0057] The structure of the spray nozzle 11, with the spray direction of the nozzle 11 facing one side of the molding chamber 20, enables precise control over the molding process. As shown in the figure, the second channel 23 can be located in the container 2, while the spray nozzle 11 is located in the cover, which can be directly opposite the opening of the molding chamber 20. This structure is simple and compact. However, this is not a limitation; for example, the second channel 23 can also be located in the cover 1, while the spray nozzle 11 can be located on the side wall of the container 2.

[0058] For the cultivation of cdECM, the molding material can be a cell-hydrogel premix formed by mixing cells with crosslinkable matrix material. A crosslinking agent is added through the spray port 11 located in the cover body 1. The crosslinking agent can be uniformly sprayed through the atomizing element 111 and covered on the surface of the premix, thereby initiating a crosslinking reaction. Finally, the cell-hydrogel complex is formed in the molding chamber 20.

[0059] Continue to refer to Figures 1 to 6 as well as Figure 11 As shown, in some embodiments, the cover 1 may include a semi-permeable membrane 12, which provides a gas exchange channel connecting the outside world to the space 10. The semi-permeable membrane 12 is a material with selective permeability, such as polytetrafluoroethylene (PTFE), achieving gas permeability without liquid permeability, ensuring efficient gas exchange while maintaining the system's airtightness and sterility. It can be understood that the semi-permeable membrane 12 can also be other materials, such as polyvinylidene fluoride (PVDF), etc.

[0060] Preferably, in some embodiments, the mounting and fixing structure for the semi-permeable membrane 12 may include a cover 1 further comprising a support 13, which fixes the semi-permeable membrane 12. Preferably, in some embodiments, the specific structure of the support 13 may include a first support 131 and a second support 132, which clamp and fix the semi-permeable membrane 12. The first support 131 and the second support 132 have perforated portions 130, allowing gas to exchange with the space 10 through the perforated portions 130 and the semi-permeable membrane 12. This results in a larger gas exchange area for the semi-permeable membrane 12, and the fixed structure is stable and reliable, which can better adapt to the high gas flow rate and vibration environment of the shaker during the cultivation process. Preferably, in some embodiments, a sealing element 14 is provided on the periphery of the cover 1 so that the cover 1 and the side wall of the container 2 are sealed. The structure of the sealing element 14 can be, for example, the form of a common sealing ring. This allows the cover 1 and the container 2 to achieve a tight fit through the friction generated by the sealing ring, further ensuring that the system is kept sealed and sterile in the space 10 while ensuring efficient gas exchange.

[0061] Preferably, in some embodiments, the size boundary of the support 13 provides the size boundary of the cover 1; the first support 131 and the second support 132 are spoke-shaped, and the space between adjacent spokes 1301 provides a hollow portion 130, and a liquid spraying port 11 is provided in the middle of the support 13; such a cover structure layout is more compact, which further expands the gas exchange area of ​​the semi-permeable membrane 12, and the first support 131 and the second support 132 can be made of highly transparent materials, such as transparent polycarbonate (transparent PC), so as to facilitate the observation of the cultivation process through a microscope.

[0062] For the container structure, please refer to [reference needed]. Figures 1 to 6As shown, in some embodiments, the sidewall of container 2 is provided with a first channel 21, defining the side where the cover 1 is installed as the top and the opposite side as the bottom, defining the direction extending from the top to the bottom as the height direction, and the bottom of container 2 is provided with a forming chamber 20. This structure facilitates the spraying of liquid from the spray nozzle 11 of the cover 1 to the corresponding position of the forming chamber 20. Preferably, in some embodiments, the bottom of the container includes a body 22 and a recess 25 recessed from the body 22, the recess providing the forming chamber 20, and the first channel 21 communicating with the recess 25. Preferably, the sidewall of container 2 is also provided with a second channel 23, the second channel 23 communicating with the surface of the body 22; the second channel 23 provides liquid exchange between the culture process and the outside world; the body 22 can provide the culture position of the cultured object after forming in the forming chamber 20, so that the first culture piece 101 provides the forming position and the culture position of the culture device 100. In this way, the forming and culture processes can be realized in the same culture piece, i.e., the first culture piece 101, to achieve full automation of the culture process, and the first culture piece 101 does not need to be opened during the culture process. The culture device has excellent airtightness. After the cell-hydrogel premix enters the molding chamber through the first channel and the cell-hydrogel complex is obtained, the complex located at the bottom can be automatically moved into the space 10 for programmed culture without any manual transfer, which greatly reduces the damage and contamination of the cell-hydrogel complex.

[0063] Continue to refer to Figures 1 to 6As shown, in some embodiments, the container structure may include a bottom body 22 and a bottom cover 24. The bottom cover 24 and the body 22 are separately disposed. The body 22 is integrally formed and connected to the side wall of the container 2. The body 22 is provided with a through hole 221 extending through the height of the body 22. The bottom cover 24 includes a bottom cover body 241 and a protrusion 242 extending from the bottom cover body 241. The protrusion 242 and the through hole 221 are correspondingly engaged, and the protrusion 242 and the through hole 221 can form a molded cavity 20. Preferably, the protrusion 242 is provided with a sealing element 243 in the circumferential direction, so that the protrusion 242 seals the hole wall of the through hole 221. The structure of the sealing element 243 can be, for example, a sealing ring, and a corresponding mounting groove 244 for the sealing element 243 can be provided in the protrusion 242. Preferably, the bottom cover 24 and the body 22 are movable relative to each other in the height direction, and the bottom cover 24 and the body 22 have a first relative position 201 and a second relative position 202, with the second relative position 202 being higher than the first relative position 201. The bottom cover 24 can move relative to the body 22 from the first relative position 201 to the second relative position 202. The top surface of the protrusion 242 at the first relative position 201 and the wall of the through hole 221 form a molding chamber 20. This movable bottom cover 24 structure is particularly suitable for cases where the cell-hydrogel complex structure is relatively soft and easily adheres to the inner surface of the through hole 221. Since the bottom cover 24 is movable, after molding, a rinsing buffer can be introduced through the second channel 23, and then the molded complex in the molding chamber 20 can be gently pushed into the space 10 for culture. Afterwards, the residual cross-linking agent is thoroughly cleaned by a shaker culture device 100, and the waste liquid is discharged through the second channel 23. This cleaning process is repeated until the cross-linking agent is completely removed, followed by three-dimensional culture. It can be understood that, as Figures 4 to 6 In some embodiments shown, the bottom cover 24 and the body 22 can also be integrally formed, that is, the bottom cover 24 and the body 22 can be fixedly connected rather than being relatively movable. Such a structure is simple and suitable for scenarios where the cell-hydrogel complex structure is relatively tough, and the operation efficiency is high. Specifically, after the complex is formed, a washing buffer is added through the second channel 23, and the complex is detached from the forming chamber 20 and enters the space 10 by shaking. Then, a cross-linking agent washing operation is performed, and the waste liquid is discharged through the second channel 23. The washing is repeated until the cross-linking agent is completely removed before entering the three-dimensional culture stage.

[0064] Continue to refer to Figures 1 to 6 ,as well as Figure 10 , Figure 12As shown, in some embodiments, the bottom cover body 241 is tapered from top to bottom, with the tapered surface of the bottom cover body 241 engaging with the external bottom support 31. Correspondingly, the top of the sidewall of the container 2 has a flange 26 extending radially outward from the sidewall, which engages with the external top support 32, and the top support 32 is snap-fitted to the bottom support 31. Before using the culture device 100, the flange 26 can be connected to the top support 32, and the bottom cover body 241 can be connected to the bottom support 31, closing the cover 1 and the container 2 to seal the space 10. The top support 32 and the bottom support 31 can be locked together, for example, by a lockable structure in the height direction, similar to a slide rail column structure 33, which is snap-fitted together. For structures where the bottom cover 24 and the body 22 are movable relative to each other in the height direction, the protrusion 242 of the bottom cover 24 is pushed into the recess 25 to a first predetermined depth, so that the sealing element 243 initially achieves a sealing state, and can be further advanced during the culture process; while for structures where the bottom cover 24 and the body 22 are not movable in the height direction, its protrusion 242 has been pre-push to the second depth of the recess 25. The assembled culture device is fixed to the shaker by the top support 32 and the bottom support 31, and each fluid interface is connected to the external pipeline. The bottom cover body 241 is a cone-shaped structure that decreases in size from top to bottom. Its advantage is that it can guide the cell-hydrogel complex to naturally gather in the central area, thereby significantly improving the convenience of microscopic observation. It not only facilitates the quality of the cell-hydrogel complex, but also ensures the complete removal of the crosslinking agent. It can be understood that in some embodiments, the structure of the bottom support 31 can also be provided with a hollowed-out 310, so as to facilitate microscopic observation.

[0065] refer to Figures 7 to 9 As shown, in some embodiments, besides the fully automated culture scheme where the first culture medium 101 serves as both the molding and culture positions as described in the above embodiments, the first culture medium 101 can also serve as the molding position, while the culture device 100 can be equipped with a second culture medium 102 as the culture position. This achieves semi-automatic culture, which has the advantage that, for scenarios with high impurity content requirements, the first culture medium 101, serving as the molding position, can be thoroughly washed, minimizing the risk of introducing impurities that may occur due to the molding and culture positions being located on the same culture medium. In other words, the molding and culture processes of the cell-hydrogel complex can be performed step-by-step, and by flexibly adjusting the complex formation conditions, it can adapt to the preparation needs of different types of cell-hydrogel complexes. During the transfer process, non-compliant complexes can be screened and excluded, thereby effectively improving the quality stability of the final product.

[0066] The second culture element 102 includes a cover 1a and a container 2a, which together form a closed space 10a. The container 2a is provided with a second channel 23, which communicates with the space 10a, allowing culture-related liquids to flow from the second channel 23 into the space 10a. The cover 1a provides a gas exchange channel connecting the outside world to the culture space 10a. The first culture element 101 provides the forming position for the culture device 100, and the second culture element 102 provides the culture position for the culture device 100. It can be understood that, unlike the first culture element 101, the structure of the second culture element 102 can be further simplified. The cover 1a does not have a spray nozzle 11, the container 2a does not have a first channel 21, and it does not have a forming chamber 20. In addition, whether it is the first culture medium 101 or the second culture medium 102, the side walls and / or bottom of the container 2 and / or the cover 1 are made of transparent material, so as to enable microscopic observation of the molding process and / or the culture process. The transparent material can be, for example, the transparent PC described above, which is low in cost, easy to process and mold, and also suitable for withstanding the temperature and pressure in the culture, but is not limited to this. For example, it can also be other materials such as polystyrene.

[0067] As introduced above, such as Figure 13 As shown, this application also provides a cultivation method using the cultivation apparatus 100 described in the above embodiments. The cultivation method includes the following steps:

[0068] S100. The molding material is added to the molding chamber 20 through the first channel 21, and the molding reaction liquid is sprayed onto the surface of the molding material through the spray nozzle 11 to obtain the molded composite. Then, the cleaning liquid is injected through the second channel 23 until the molding reaction liquid is removed, and the cleaning liquid is discharged through the second channel 23.

[0069] Specifically, cells (such as primary animal cartilage) can be mixed with a crosslinkable matrix material to form a cell-hydrogel premix, which is then added to the molding chamber 20 through the first channel 21. The crosslinking agent solution is atomized and sprayed onto the surface of the premix through the atomizing element 111 of the top spray nozzle 11 to induce crosslinking and form a cell-gel complex. Subsequently, a cleaning solution is injected through the second channel 23, and the mixture is cleaned under agitation conditions and the waste liquid is discharged. This cleaning operation is repeated to ensure the removal of the crosslinking agent.

[0070] S200. Add culture medium through the second channel 23 to culture the complex;

[0071] Specifically, culture medium can be added through the second channel 23, and three-dimensional culture can be carried out under shaking conditions until the predetermined time, during which the culture medium is replaced regularly.

[0072] S300. Add degradation material through the second channel 23 to degrade part of the molded material.

[0073] Specifically, a de-crosslinking agent solution can be added through the second channel 23 to soak the cell-gel complex to partially degrade the matrix material. After soaking, the waste liquid is discharged. This treatment can be repeated. The complex is then cultured under the same three-dimensional culture conditions.

[0074] After S400 culture is completed, add the scavenging solution through the second channel 23 to remove cell components.

[0075] Specifically, cell lysis buffer and alkaline treatment solution can be added sequentially through the second channel 23 for treatment. Buffer is used for washing between each treatment step, and cell components are removed through a programmed oscillation reaction to finally obtain cell-derived extracellular matrix.

[0076] The advantages of this culture method lie in its ability to integrate and continuously operate the entire process of three-dimensional cell culture, cell-hydrogel formation and degradation, and decellularization through a dedicated fluid channel for changing the culture medium and corresponding solutions. This method not only significantly reduces the risk of introducing external contamination due to frequent opening of the culture chamber, but also effectively reduces product quality differences between different batches caused by operational fluctuations, thereby improving experimental repeatability and product consistency.

[0077] In summary, the beneficial effects of the culture device, cover, and container described above include, but are not limited to, the fact that, through the structural design of the culture device, the container, and the cover provide a molding chamber, a first channel, a second channel, and a gas exchange channel, the automating or semi-automating of the cdECM material culture process, as well as closed culture, is achieved. This avoids the shortcomings of existing technologies with predominantly manual open operations such as liquid changing, transfer, and monitoring, which have poor repeatability and scalability potential.

[0078] While this application discloses preferred embodiments as described above, it is not intended to limit the scope of this application. Any changes and modifications can be made by those skilled in the art without departing from the spirit and scope of this application. Therefore, any modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this application without departing from the content of the technical solution of this application shall fall within the protection scope defined by the claims of this application.

Claims

1. A culture device (100), characterized in that, Includes a first culture element (101), the first culture element (101) comprising: Cover (1); The container (2) and the cover (1) together form a closed space (10). The container (2) is provided with a molding chamber (20) and a first channel (21), the first channel (21) being connected to the molding chamber (20) so that molding material can flow from the first channel (21) to the molding chamber (20), the molding chamber (20) being able to define the molding shape provided by the molding material. The cover (1) provides a gas exchange channel connecting the outside world with the culture space (10); At least one of the cover (1) and the container (2) is provided with: The second channel (23) provides liquid exchange between the culture process and the external environment; The liquid spraying nozzle (11) is directed toward one side of the molding chamber (20) so that the liquid sprayed from the nozzle (11) can reach the molding chamber (20).

2. The culture apparatus (100) as described in claim 1, characterized in that, The spray nozzle (11) is disposed on the cover (1), and the cover (1) includes a semi-permeable membrane (12), which provides a gas exchange channel for communication between the outside and the space (10); Preferably, the cover (1) further includes a bracket (13) that fixes the semi-permeable membrane (12); Preferably, the support (13) includes a first support (131) and a second support (132), the first support (131) and the second support (132) clamping and fixing the semi-permeable membrane (12), the first support (131) and the second support (132) having a hollow portion (130) so that gas can pass through the hollow portion (130) and the semi-permeable membrane (12) to exchange gas with the space (10); Preferably, the size boundary of the bracket (13) provides the size boundary of the cover (1); the first bracket (131) and the second bracket (132) are spoke-shaped, and the space between adjacent spokes (1301) provides the hollow portion (130). Preferably, the spray nozzle (11) is provided in the middle of the support (13). Preferably, the spray nozzle (11) is provided with an atomizing element (111). Preferably, a sealing element (14) is provided on the periphery of the cover (1) so that the cover (1) is sealed to the side wall of the container (2).

3. The culture apparatus (100) as described in claim 1, characterized in that, The container (2) has the first channel (21) on its side wall. The side on which the cover (1) is installed is defined as the top and the opposite side as the bottom. The direction in which the top extends toward the bottom is defined as the height direction. The bottom of the container (2) has the molding chamber (20). Preferably, the bottom of the container includes a body (22) and a recess (25) recessed from the body (22), the recess providing the molding chamber (20), and the first channel (21) communicating with the recess (25). Preferably, the side wall of the container (2) is further provided with a second channel (23), the second channel (23) is connected to the surface of the body (22); the second channel (23) provides liquid exchange between the culture process and the outside world; the body (22) can provide the culture position of the cultured object after it is formed in the molding chamber (20), so that the first culture piece (101) provides the molding position and culture position of the culture device (100).

4. The culture apparatus (100) as described in claim 3, characterized in that, The bottom of the container includes a body (22) and a bottom cover (24). The bottom cover (24) and the body (22) are separately disposed. The body (22) is integrally formed and connected to the side wall of the container (2). The body (22) is provided with a through hole (221) extending through the height of the body (22). The bottom cover (24) includes a bottom cover body (241) and a protrusion (242) protruding from the bottom cover body (241). The protrusion (242) and the through hole (221) are correspondingly engaged. The protrusion (242) and the through hole (221) can form the molding chamber (20). Preferably, the bottom cover (24) and the body (22) are movable relative to each other in the height direction, and the bottom cover (24) and the body (22) have a first relative position (201) and a second relative position (202), the second relative position (202) being higher than the first relative position (201), and the bottom cover (24) being able to move relative to the body (22) from the first relative position (201) to the second relative position (202); the top surface of the protrusion (242) at the first relative position (201) and the hole wall of the through hole (221) constitute the molding chamber (20). Preferably, the bottom cover (24) is integrally formed and connected to the body (22), and the bottom cover (24) and the body (22) cannot move relative to each other in the height direction; Preferably, the protrusion (242) is provided with a sealing element (243) in the circumferential direction, so that the protrusion (242) seals against the wall of the through hole (221); Preferably, the bottom cover body (241) is cone-shaped, decreasing in size from top to bottom, and the cone surface of the bottom cover body (241) is used to cooperate with the external bottom support (31); Preferably, the top of the sidewall of the container (2) has a flange (26) that extends radially outward from the sidewall, the flange (26) being used to cooperate with an external top support (32); Preferably, the top bracket (32) is snapped together with the bottom bracket (31).

5. The culture apparatus (100) as described in claim 1, characterized in that, It also includes a second culture element (102); The second culture element (102) includes: Cover (1a); The container (2a) and the cover (1a) together form a closed space (10a). The container (2a) is provided with a second channel (23) which is connected to the space (10a) so that culture-related liquid can flow from the second channel (23) to the space (10a). The cover (1a) provides a gas exchange channel that connects the outside world with the culture space (10a); The first culture element (101) provides the forming position of the culture device (100), and the second culture element (102) provides the culture position of the culture device (100).

6. The culture apparatus (100) as described in claim 5, characterized in that, The cover (1a) does not have the spray nozzle (11), the container (2a) does not have the first channel (21), and the molding chamber (20) is not provided.

7. The culture apparatus (100) as described in claim 1, characterized in that, The sidewalls and / or bottom of the container (2) and / or the cover (1) are made of transparent material.

8. A cover (1), characterized in that, include: The spray nozzle (11) is used to spray liquid; A semi-permeable membrane (12) provides a gas exchange channel for the cover (1); The cover (1) also includes a bracket (13) that fixes the semi-permeable membrane (12); Preferably, the support (13) includes a first support (131) and a second support (132), the first support (131) and the second support (132) clamping and fixing the semi-permeable membrane (12), the first support (131) and the second support (132) having a hollow portion (130) so that gas can pass through the hollow portion (130) and the semi-permeable membrane (12) for gas exchange; Preferably, the size boundary of the bracket (13) provides the size boundary of the cover (1); the first bracket (131) and the second bracket (132) are spoke-shaped, and the space between adjacent spokes (1301) provides the hollow portion (130). Preferably, the spray nozzle (11) is provided in the middle of the support (13). Preferably, the spray nozzle (11) is provided with an atomizing element (111). Preferably, a sealing element (14) is provided on the periphery of the cover (1) so that the cover (1) is sealed to the side wall of the container (2).

9. A container (2), characterized in that, include: A molding chamber (20) and a first channel (21) are connected to the molding chamber (20) so that molding material can flow from the first channel (21) to the molding chamber (20), and the molding chamber (20) can define the molding shape provided by the molding material; Preferably, the container (2) has the first channel (21) on its side wall and the forming chamber (20) on its bottom. Preferably, the bottom of the container includes a body (22) and a recess (25) recessed from the body (22), the recess providing the molding chamber (20), and the first channel (21) communicating with the recess (25). Preferably, the bottom of the container includes a body (22) and a bottom cover (24). The bottom cover (24) and the body (22) are separately disposed. The body (22) is integrally formed and connected to the side wall of the container (2). The body (22) is provided with a through hole (221) extending through the height of the body (22). The bottom cover (24) includes a bottom cover body (241) and a protrusion (242) protruding from the bottom cover body (241). The protrusion (242) and the through hole (221) are correspondingly engaged. The protrusion (242) and the through hole (221) can form the molding chamber (20). Preferably, the bottom cover (24) and the body (22) are movable relative to each other in the height direction, and the bottom cover (24) and the body (22) have a first relative position (201) and a second relative position (202), the second relative position (202) being higher than the first relative position (201), and the bottom cover (24) being able to move relative to the body (22) from the first relative position (201) to the second relative position (202); the top surface of the protrusion (242) at the first relative position (201) and the hole wall of the through hole (221) constitute the molding chamber (20). Preferably, the bottom cover (24) is integrally formed and connected to the body (22), and the bottom cover (24) and the body (22) cannot move relative to each other in the height direction; Preferably, the protrusion (242) is provided with a sealing element (243) in the circumferential direction, so that the protrusion (242) seals against the wall of the through hole (221); Preferably, the bottom cover body (241) is cone-shaped, decreasing in size from top to bottom, and the cone surface of the bottom cover body (241) is used to cooperate with the external bottom support (31); Preferably, the top of the sidewall of the container (2) has a flange (26) that extends radially outward from the sidewall, the flange (26) being used to engage with an external top support (32).

10. The container (2) as claimed in claim 9, characterized in that, The side wall of the container (2) is also provided with a second channel (23), which is connected to the surface of the body (22); the second channel (23) provides liquid exchange between the culture process and the outside world; the body (22) can provide a culture position for the cultured object after it is formed in the molding chamber (20), so that the container (2) provides a molding position and a culture position.

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