Organoid cell culture device

Abstract

The application provides an organoid cell culture device, which comprises a device bottom layer, a first culture medium conveying layer, a cell culture layer, a second culture medium conveying layer and a cover layer which are sequentially stacked, the layer structure is sequentially penetrated and fixed by a fixing member, the first culture medium conveying layer is provided with a penetrating culture medium conveying section, the second culture medium conveying layer comprises a second culture medium inlet and a second culture medium outlet, the cover layer is provided with a first culture medium opening and a second culture medium opening, the cover layer cooperates with the second culture medium conveying layer to form another culture medium conveying section, and the cell culture layer is located above the first culture medium conveying layer and covers the first culture medium opening, so that the culture medium can be contacted on both sides. The dynamic culture medium flow environment of the upper and lower sides of the cell culture layer is constructed through the first culture medium conveying layer and the second culture medium conveying layer, and the influence of uneven distribution of local culture medium components and accumulation of metabolic products in the culture process is reduced.

Description

Technical Field

[0001] This application belongs to the field of cell culture technology, and in particular relates to an organoid cell culture device. Background Technology

[0002] Organoid culture technology, as an important research tool for simulating in vivo tissue development and physiological functions, has been widely applied in fields such as cochlear cell development, disease mechanism research, and drug screening. Currently, cochlear organoids or cochlear cells are usually cultured in static culture dishes, that is, cells are seeded in culture dishes and the culture medium is manually changed periodically to maintain cell growth.

[0003] However, in the aforementioned static culture methods, the transfer of culture medium components within the system relies primarily on diffusion. Nutrients, oxygen, and cell-secreted signaling molecules tend to form uncontrollable concentration distributions within the culture area, resulting in differences in the microenvironment of cells at different locations. Simultaneously, metabolic waste is difficult to remove in a timely manner, and the local microenvironment continuously changes over time, leading to poor stability of culture conditions.

[0004] Under the above culture conditions, the growth, proliferation and differentiation of cells are easily affected by the heterogeneity of the microenvironment. The cell cluster structure formed during the culture process has large individual differences, which is not conducive to maintaining the consistency of organoid structure and function, thus affecting the stability and reproducibility of culture results and making it difficult to meet the requirements of cochlear organoid culture.

[0005] It should be noted that the above content is not necessarily prior art, nor is it intended to limit the scope of patent protection of this application. Summary of the Invention

[0006] This application provides an organoid cell culture device to solve or alleviate one or more technical problems in the prior art.

[0007] The first aspect of this application provides an organoid cell culture apparatus, comprising: The bottom layer of the device; A first culture medium delivery layer is located above the bottom layer of the device. The first culture medium delivery layer has a through culture medium delivery section, and the surface of the first culture medium delivery layer also has a first culture medium flow port that communicates with the culture medium delivery section. A cell culture layer is located above the first culture medium delivery layer. The cell culture layer includes a porous membrane on which cells to be cultured are placed. The porous membrane covers the first culture medium flow port. The second culture medium delivery layer is located above the cell culture layer. The second culture medium delivery layer is provided with a second culture medium flow port, which is correspondingly arranged with the porous membrane. The second culture medium delivery layer also includes a second culture medium inlet and a second culture medium outlet. The second culture medium inlet is connected to the second culture medium flow port through a second culture medium inlet trough, and the second culture medium outlet is connected to the second culture medium flow port through a second culture medium outlet trough. A capping layer is located above the second culture medium delivery layer. The capping layer is provided with a first culture medium inlet and a second culture medium inlet. The first culture medium inlet is corresponding to the second culture medium inlet, and the second culture medium inlet is corresponding to the second culture medium outlet. The first culture medium inlet and the second culture medium inlet are used to communicate with an external culture medium supply device through a Luer connector. The organoid cell culture device further includes a fixing component, which passes through the capping layer, the second culture medium delivery layer, and the first culture medium delivery layer in sequence and is fixedly connected to the bottom layer of the device.

[0008] Optionally, the cell culture layer further includes a first gel film layer and a second gel film layer. The first gel film layer is located below the second culture medium delivery layer, and the porous membrane is located between the first gel film layer and the second gel film layer. The first gel film layer has a first perforation, and the second gel film layer has a second perforation. The first perforation, the second perforation, and the second culture medium flow port are correspondingly arranged.

[0009] Optionally, the first adhesive film layer is provided with conductive wires, and the porous membrane is disposed on top of the conductive wires.

[0010] Optionally, a conductive filament is provided below the second adhesive film layer, and the porous membrane is located below the conductive filament.

[0011] Optionally, the conductive wire includes a first conductive wire and a second conductive wire, the first conductive wire extending along a first direction and the second conductive wire extending along a second direction, the first direction and the second direction being located on the same horizontal plane, and the first direction and the second direction being parallel or perpendicular to each other.

[0012] Optionally, the conductive wire is used to provide electrical stimulation during cell culture, and the electrical stimulation parameters include: a voltage of 5.5V, a current of 1mA, and a discharge frequency of 1kHz.

[0013] Optionally, the cells to be cultured are coated with a conductive layer.

[0014] Optionally, there are five first culture medium outlets, and the five first culture medium outlets are evenly distributed at intervals. There are five porous membranes, and each porous membrane is provided with a corresponding first culture medium flow port; The second culture medium has five flow ports, and each second culture medium flow port is set to correspond to one of the first culture medium flow ports.

[0015] Optionally, the first culture medium outlet is circular, and the porous membrane is rectangular, wherein the length of any side of the porous membrane is greater than the diameter of the first culture medium outlet.

[0016] The embodiments of this application employing the above-described technical solution may have the following advantages: This application constructs a dynamic culture medium flow environment on both sides of the cell culture layer through a first culture medium delivery layer and a second culture medium delivery layer. This helps to reduce the impact of uneven distribution of local culture medium components and accumulation of metabolites during the culture process, improves the stability and uniformity of the culture microenvironment, and thus improves the consistency and reproducibility of organoid culture results, providing more stable culture conditions for organoid culture and related research.

[0017] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0018] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0019] Figure 1 This is a schematic diagram of the exploded structure of the organoid cell culture device provided in the embodiments of this application.

[0020] Explanation of reference numerals in the attached figures: The device consists of: bottom layer 10; first culture medium delivery layer 20; culture medium delivery section 201; first culture medium flow port 202; porous membrane 303; first adhesive film layer 301; second adhesive film layer 302; conductive wire 305; second culture medium delivery layer 40; second culture medium flow port 405; second culture medium inlet 401; second culture medium outlet 402; second culture medium inlet tank 403; second culture medium outlet tank 404; sealing layer 50; first culture medium inlet 501; second culture medium inlet 502. Detailed Implementation

[0021] The embodiments of this application are described in detail below, examples of which are illustrated in the accompanying drawings. In the drawings, for clarity, the dimensions of layers, regions, elements, and their relative dimensions may be exaggerated. The same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] It should be understood that when an element or layer is referred to as "on," "adjacent to," "connected to," or "coupled to" other elements or layers, it may be directly on, adjacent to, connected to, or coupled to other elements or layers, or there may be intervening elements or layers. Conversely, when an element is referred to as "directly on," "directly adjacent to," "directly connected to," or "directly coupled to" other elements or layers, there are no intervening elements or layers. It should be understood that although the terms first, second, third, etc., may be used to describe various elements, components, areas, layers, and / or portions, these elements, components, areas, layers, and / or portions should not be limited by these terms. These terms are only used to distinguish one element, component, area, layer, or portion from another element, component, area, layer, or portion. Therefore, without departing from the teachings of this application, the first element, component, area, layer, or portion discussed below may be referred to as a second element, component, area, layer, or portion. And the discussion of a second element, component, area, layer, or portion does not imply that the first element, component, area, layer, or portion necessarily exists in this application.

[0023] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0024] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that the embodiments of this application described herein can be implemented, for example, in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0025] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0026] Exemplary embodiments according to this application will now be described in more detail with reference to the accompanying drawings. It should be understood that these exemplary embodiments may be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein.

[0027] Please see Figure 1 This application provides an organoid cell culture device, which includes a bottom layer 10, a first culture medium delivery layer 20, a cell culture layer, a second culture medium delivery layer 40, a capping layer 50, and a fixing component. The following is a detailed description: The bottom layer 10 of the device is used to support the entire organoid cell culture device and serves as the mounting base for each layer of the structure. Preferably, the bottom layer 10 of the device can be made of materials with good mechanical strength and biocompatibility, such as glass, polycarbonate (PC), polymethyl methacrylate (PMMA), and polydimethylsiloxane (PDMS), or other materials that can meet the requirements of cell culture. This application does not limit the choice of materials.

[0028] The first culture medium delivery layer 20 is located above the bottom layer 10 of the device. The first culture medium delivery layer 20 has a through culture medium delivery section 201, and the surface of the first culture medium delivery layer 20 also has a first culture medium flow port 202 that communicates with the culture medium delivery section 201.

[0029] The culture medium delivery section 201 forms a culture medium flow channel. Both ends of this channel can be connected to an external culture medium supply device, allowing the culture medium to flow through the first culture medium delivery layer 20 under the drive of the external device. Preferably, the culture medium delivery section 201 can be a straight, serpentine, grid-like, annular, or other channel structure suitable for fluid transport to meet different culture requirements. The first culture medium flow port 202 enables fluid communication between the culture medium delivery section 201 and the cell culture layer, allowing the culture medium to exchange substances with the porous membrane 303 via the first culture medium flow port 202.

[0030] A cell culture layer is located above the first culture medium delivery layer 20. The cell culture layer includes a porous membrane 303, on which cells to be cultured are placed. The porous membrane 303 covers the first culture medium flow port 202.

[0031] In this embodiment, the porous membrane 303 provides a culture interface for the attachment, growth, and differentiation of the cultured cells, and allows the exchange of nutrients, oxygen, metabolites, and other culture factors in the culture medium through the membrane pores. Preferably, the porous membrane 303 can be made of polycarbonate membrane, polyethylene terephthalate membrane, polyester membrane, or other porous membrane materials with good biocompatibility. Its pore size, porosity, and thickness can be selected according to the cultured object, and this application does not limit them.

[0032] The second culture medium delivery layer 40 is located above the cell culture layer. The second culture medium delivery layer 40 is provided with a second culture medium flow port 405, which is correspondingly arranged with the porous membrane 303. The second culture medium delivery layer 40 also includes a second culture medium inlet 401 and a second culture medium outlet 402. The second culture medium inlet 401 is connected to the second culture medium flow port 405 through a second culture medium inlet trough 403, and the second culture medium outlet 402 is connected to the second culture medium flow port 405 through a second culture medium outlet trough 404.

[0033] The second culture medium delivery layer 40 is used to construct a culture medium flow path above the cell culture layer, allowing the culture medium to flow over the upper side of the cell culture layer, forming a culture medium flow environment on both the upper and lower sides of the cell culture layer together with the first culture medium delivery layer 20. Preferably, the second culture medium inlet channel 403 and the second culture medium outlet channel 404 can be designed with different channel sizes, flow paths, or flow rates according to actual needs to meet the requirements of culture medium delivery conditions at different culture stages. In this application, the number of each flow channel can also be multiple, thereby improving the uniformity of culture medium delivery within the culture area.

[0034] A capping layer 50 is located above the second culture medium delivery layer 40. The capping layer 50 is provided with a first culture medium inlet 501 and a second culture medium inlet 502. The first culture medium inlet 501 is correspondingly provided with the second culture medium inlet 401, and the second culture medium inlet 502 is correspondingly provided with the second culture medium outlet 402. The first culture medium inlet 501 and the second culture medium inlet 502 are connected to an external culture medium supply device through a Luer connector.

[0035] The capping layer 50 is used to seal the culture device and provides inlet and outlet interfaces for the culture medium. It can be easily connected to syringes, peristaltic pumps, injection pumps, reservoirs, or other culture medium supply equipment via a Luer connector to achieve continuous or cyclic delivery of the culture medium. Furthermore, the Luer connector, when tightened, also tightens the second culture medium delivery layer 40 and the capping layer 50. In some embodiments, the capping layer 50 may also be provided with auxiliary structures such as vent holes, sampling holes, pressure balancing holes, or sealing plugs to meet different usage requirements during the culture process; this application does not limit the specific details.

[0036] The fastener passes sequentially through the capping layer 50, the second culture medium delivery layer 40, and the first culture medium delivery layer 20, and is fixedly connected to the bottom layer 10 of the device. It can be understood that the capping layer 50, the second culture medium delivery layer 40, the first culture medium delivery layer 20, and the bottom layer 10 of the device are all provided with threaded holes to facilitate the fastener's passage and connection.

[0037] Fasteners are used to press and fix the various layers of the structure, maintaining a stable positional relationship between the layers and improving the overall sealing performance and structural stability of the culture device. Preferably, the fasteners can be screws, bolts, locating pins, clips, or other structures that can achieve a fixed connection. The number of fasteners can be set according to the size of the culture device, for example, they can be set at the four corners of the device or at multiple positions around the circumference to ensure uniform stress on the device and reduce the risk of culture medium leakage.

[0038] The layers can be stacked and assembled to form an integrated culture device. The layers can be sealed together by a sealing structure to prevent culture medium leakage and ensure the stability of the culture environment. The layers can be detachably connected or permanently fixedly connected according to actual processing needs, and this application does not limit this.

[0039] In this embodiment, the use of the organoid cell culture device provided in this application is described using cochlear organoid culture as an example.

[0040] First, the first culture medium delivery layer 20 is installed on the bottom layer 10 of the device, and the culture medium delivery section 201 in the first culture medium delivery layer 20 is positioned in a preset position. Then, the cell culture layer is placed on top of the first culture medium delivery layer 20, so that the porous membrane 303 covers the first culture medium flow port 202. Next, the second culture medium delivery layer 40 is placed on top of the cell culture layer, so that the second culture medium flow port 405 is correspondingly positioned with the porous membrane 303. Then, the cochlear organoids to be cultured are inoculated onto the surface of the porous membrane 303. A preset standing time can be selected to allow the cells to be cultured to fully adhere to the porous membrane 303.

[0041] The capping layer 50 is installed above the second culture medium delivery layer 40, and is fixedly connected to the bottom layer 10 of the device by means of fasteners passing through the capping layer 50, the second culture medium delivery layer 40 and the first culture medium delivery layer 20 in sequence, thereby completing the assembly of the entire organoid cell culture device.

[0042] After assembly, the external culture medium supply device is connected via the first culture medium inlet 501 and the second culture medium inlet 502 on the capping layer 50. Driven by the culture medium supply device, the culture medium enters the second culture medium inlet 401 through the Luer connector along the first culture medium inlet 501, flows along the second culture medium inlet 403 to the second culture medium outlet 405 to contact the cells to be cultured, then flows along the second culture medium outlet 404 to the second culture medium outlet 402, and flows out through the second culture medium inlet 502 to form a loop.

[0043] Simultaneously, the culture medium supply device can also supply culture medium to the first culture medium delivery layer 20. The culture medium enters the culture medium delivery section 201 in the first culture medium delivery layer 20 and reaches the area below the porous membrane 303 through the first culture medium flow port 202, thereby creating a culture medium flow environment on both the upper and lower sides of the porous membrane 303. During the culture process, the culture medium can be continuously delivered or circulated according to the needs of different stages of cochlear organoid culture to continuously provide the cells with the nutrients, oxygen, and culture factors required for culture, and to promptly remove waste products generated by cell metabolism.

[0044] Because the porous membrane 303 has good permeability, the culture medium flowing on both sides of the porous membrane 303 can exchange substances through the membrane pores, keeping the cultured cells in a continuously renewed culture microenvironment. This improves the transport conditions of nutrients, oxygen, and other culture factors within the culture area and promotes the timely removal of metabolic waste. Compared with traditional static culture methods, this application constructs a dynamic culture medium flow environment on both sides of the cell culture layer through the first culture medium delivery layer 20 and the second culture medium delivery layer 40. This helps to reduce the impact of uneven distribution of local culture medium components and accumulation of metabolic products during the culture process, improves the stability and uniformity of the culture microenvironment, and thus improves the consistency and reproducibility of organoid culture results, providing more stable culture conditions for cochlear organoid culture and related research.

[0045] In this embodiment, the culture medium can be selectively configured according to different developmental stages of cochlear organoids. For example, during the cell attachment and early culture stages, a basal culture medium can be used and supplemented with cell growth factors, nutrients, and necessary serum substitutes to promote cell survival and adhesion; during the organoid induction and differentiation stages, Wnt signaling pathway-related regulatory factors, Notch signaling regulators, or other inducing components that promote cochlear cell differentiation can be further added to regulate the differentiation process of cochlear hair cells and supporting cells.

[0046] During the cultivation process, the culture medium can be continuously or intermittently delivered to the first culture medium delivery layer 20 and the second culture medium delivery layer 40 through an external culture medium supply device to achieve dynamic renewal of the culture medium. Preferably, a culture medium circulation and renewal cycle can be set to periodically remove metabolic waste and replenish fresh culture medium, thereby maintaining the relative stability of the concentration of nutrients and signal molecules in the culture system.

[0047] In addition, at different culture stages, the culture medium composition or delivery parameters of the first culture medium delivery layer 20 and the second culture medium delivery layer 40 can be adjusted to form different or the same microenvironmental conditions on the upper and lower sides of the cell culture layer, so as to achieve gradient regulation or homogenization regulation of the local microenvironment of cochlear organs, thereby adapting to the needs of cochlear cells for microenvironmental conditions at different developmental stages.

[0048] Once the cell culture is complete, the fixation between the layers can be released by removing the fixing devices, thereby separating the capping layer 50, the second culture medium delivery layer 40, and the first culture medium delivery layer 20 in sequence, thus exposing the cell culture layer. Subsequently, the capping layer 50 can be opened under sterile conditions to obtain the cultured cells on the porous membrane 303.

[0049] After the culture is completed, each component can be individually cleaned, sterilized, or replaced, thereby enabling the device to be reused, improving overall efficiency and reducing experimental costs.

[0050] In an optional embodiment, the cell culture layer further includes a first gel film layer 301 and a second gel film layer 302, and the porous membrane 303 is located between the first gel film layer 301 and the second gel film layer 302. The first gel film layer 301 has a first perforation, and the second gel film layer 302 has a second perforation. The first perforation, the second perforation, and the second culture medium flow port 405 are correspondingly arranged.

[0051] The first and second adhesive film layers 301 and 302 are used to clamp and fix the porous membrane 303 from top to bottom, thereby improving the stability of the porous membrane 303 under the flow of culture medium and preventing displacement or wrinkling of the porous membrane 303. The first and second perforations are designed to form liquid passages corresponding to the second culture medium flow port 405, so that the culture medium can flow stably and be evenly distributed above the cell culture layer.

[0052] Preferably, the first membrane layer 301 and the second membrane layer 302 can be made of biocompatible elastic or breathable materials to ensure structural support performance without affecting gas exchange and culture medium mass transfer efficiency. The porous membrane 303 can be selected with different pore sizes and thicknesses according to different culture requirements of cochlear organs to adjust cell adhesion strength and material exchange rate.

[0053] In cochlear organoid culture applications, this structure enables cochlear organoids to be stably distributed on the surface of the porous membrane 303 and maintain a relatively uniform microenvironment under the action of the flowing culture medium, which is conducive to the orderly spatial distribution and differentiation of cochlear hair cells and supporting cells, thereby improving the integrity of the organoid structure.

[0054] In an optional embodiment, the perforation shape of the first film layer 301 and the second film layer 302 can be set to be circular, strip-shaped or grid-shaped to adapt to different fluid flow patterns; or multiple independent through-hole areas can be set according to the needs of the culture area to achieve zonal control of cell culture conditions in different areas.

[0055] In an optional embodiment, the first adhesive film layer 301 is provided with conductive wires 305, and the porous membrane 303 is disposed on top of the conductive wires 305.

[0056] The conductive wire 305 is used to apply periodic or non-periodic electrical stimulation to cells on the porous membrane 303 during cell culture to simulate the electrical signal environment under the intrinsic physiological state of the cochlea, thereby providing cells with stimulation conditions close to the in vivo microenvironment. The electrical signal of the conductive wire 305 may include constant current, pulsed current, or alternating electrical signal, and its parameters can be adjusted according to different culture stages to achieve dynamic regulation of the cell development process.

[0057] The conductive wire 305 can be electrically connected to an external signal generating device. Through a preset control program, the frequency, amplitude, and duration of electrical stimulation can be controlled and adjusted to simulate physiological stimulation environments of different intensities, so as to adapt to the needs of cochlear organs at different developmental stages.

[0058] In an optional embodiment, conductive wires are also provided below the second adhesive film layer 302, and the porous membrane 303 is located below the conductive wires.

[0059] In this embodiment, the conductive wires disposed below the second adhesive film layer 302 and the conductive wires above the first adhesive film layer 301 can cooperate to form an upper and lower double-sided electrical stimulation structure, so that the cells on the porous membrane 303 are in a more uniform electric field or gradient electric field environment, thereby enhancing the spatial consistency of electrical signal stimulation.

[0060] The above structure can simulate the electrophysiological response and liquid microenvironment disturbance caused by acoustic stimulation in the cochlear tissue during the culture process, making the hair-like cells closer to the state of real cochlear hair cells in vivo in terms of structure, morphology, signal response capability and electrophysiological characteristics, thereby improving the functional maturity and experimental research value of cochlear organoids.

[0061] In an alternative embodiment, the upper and lower sets of conductive wires can be controlled independently or synchronously to form electrical stimulation modes of different intensities, such as unilateral stimulation mode, symmetrical stimulation mode, or gradient stimulation mode.

[0062] Furthermore, the conductive wire includes a first conductive wire and a second conductive wire, the first conductive wire extends along a first direction, the second conductive wire extends along a second direction, the first direction and the second direction are located on the same horizontal plane, and the first direction and the second direction are parallel or perpendicular to each other.

[0063] Taking the first and second directions as an example of being perpendicular to each other, the first and second conductive wires are arranged in a crisscross pattern, thereby forming a two-dimensional electrical stimulation network structure in the region where the porous membrane 303 is located. This allows the electrical stimulation signal to be distributed in multiple directions within the culture area, improving the spatial uniformity of the electrical stimulation effect. This crisscross structure avoids the problem of uneven local stimulation caused by unidirectional electrical stimulation, improving the consistency of cell stimulation.

[0064] Preferably, the first conductive wire and the second conductive wire can be electrically connected to an independent control unit to achieve independent on / off control or parameter adjustment of the conductive wires in different directions, thereby forming different electrical stimulation modes, such as unidirectional stimulation, bidirectional synchronous stimulation or alternating stimulation mode, to adapt to the different needs of cochlear organs for electrical stimulation intensity and spatial distribution at different stages of cultivation.

[0065] In an optional embodiment, the conductive wire is used to provide electrical stimulation during cell culture, and the electrical stimulation parameters include: a voltage of 5.5V, a current of 1mA, and a discharge frequency of 1kHz.

[0066] The above-described electrical stimulation parameters are only a preferred embodiment, used to simulate the electrical signal environment of cochlear tissue under specific physiological conditions. In practical applications, the voltage, current, and discharge frequency can be adjusted according to different culture stages and cell types of cochlear organoids to adapt to the stimulation requirements of different processes such as cell proliferation, differentiation, or functional maturation.

[0067] In other embodiments, the electrical stimulation parameters can be adjusted according to conditions such as the type of cells to be cultured, such as voltage: 1V to 10V; current: 0.1mA to 2mA; frequency: 1Hz to 5kHz.

[0068] In one optional embodiment, the cells to be cultured are coated with a conductive layer. This conductive layer forms a uniform electrical signal conduction interface on the outer side of the cells, allowing electrical stimulation to act more directly and uniformly on the cells, thereby improving the transmission efficiency and consistency of the electrical stimulation signal. By providing a conductive layer, the attenuation and uneven distribution of electrical stimulation in the culture medium can be reduced, improving the overall synchronicity of cell stimulation.

[0069] Preferably, the conductive layer can be formed using biocompatible conductive materials, such as conductive polymers, carbon-based conductive materials, or metal nanostructures, and can be prepared as thin films, meshes, or coating layers to meet the needs of different cell sizes and culture morphologies.

[0070] In an optional embodiment, there are five first culture medium flow ports 202, which are evenly distributed at intervals. There are five porous membranes 303, each corresponding to one of the first culture medium flow ports 202. There are five second culture medium flow ports 405, each corresponding to one of the first culture medium flow ports 202.

[0071] By setting multiple evenly spaced first culture medium flow ports 202 and second culture medium flow ports 405, the culture area can be divided into multiple independent but cooperative culture units, thereby achieving spatial partitioning and uniform coverage of the culture medium and avoiding the problems of uneven local flow rate or nutrient distribution caused by a single large-area flow structure.

[0072] Preferably, each first culture medium flow port 202, the corresponding porous membrane 303, and the second culture medium flow port 405 constitute an independent culture unit, so that the culture conditions can be kept consistent between the culture units and the stability of the culture results can be guaranteed.

[0073] By using the above-mentioned multi-unit array structure, the uniformity of the distribution of the culture medium in the vertical direction of the cell culture layer can be improved, so that the cochlear organoid cells are in a relatively consistent nutrient supply and metabolic environment in different spatial locations, thereby reducing the problem of uneven cell differentiation caused by local microenvironment differences and improving the consistency of organoid structure formation.

[0074] In an optional embodiment, the first culture medium outlet 202 is circular, and the porous membrane 303 is rectangular, wherein the length of any side of the porous membrane 303 is greater than the diameter of the first culture medium outlet 202.

[0075] By setting the first culture medium flow port 202 to be circular and the porous membrane 303 to be rectangular, the coverage adaptability of the culture area can be improved while ensuring the ease of structural processing. The circular flow port is conducive to forming a relatively uniform fluid diffusion distribution, while the rectangular porous membrane 303 can provide a larger area for cell attachment, thereby improving the cell carrying capacity per unit culture area.

[0076] Preferably, the size of the porous membrane 303 is larger than the size of the first culture medium flow port 202, so that the culture medium can form a fluid distribution that gradually expands from the center to the periphery during the diffusion of the culture medium from the first culture medium flow port 202 to the porous membrane 303, thereby increasing the coverage of the culture medium on the surface of the porous membrane 303 and reducing the situation of insufficient local supply.

[0077] The above structural design allows the culture medium to form a relatively stable and uniformly diffused supply environment below the cell culture layer. This is beneficial for cochlear organoid cells to maintain relatively consistent nutrient supply and metabolic exchange conditions under large-area culture conditions, thereby improving the uniformity of cell growth and the consistency of organoid formation.

[0078] In one optional embodiment, the culture medium delivery method includes at least one of a unidirectional flow mode, a circulating flow mode, or an intermittent flow mode, wherein the culture medium is controlled by an external culture medium supply device to form a continuously updated or periodically updated flow state between the first culture medium delivery layer 20 and the second culture medium delivery layer 40.

[0079] The culture media in the first culture medium delivery layer 20 and the second culture medium delivery layer 40 can have the same or different compositions, so as to form the same or different microenvironment on the upper and lower sides of the cell culture layer, thereby realizing the regulation of the cell differentiation process.

[0080] It should be noted that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," etc., are used only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this application. The directional terms "inner" and "outer" refer to the inside or outside relative to the outline of the component itself. For example, if a device in the drawings is inverted, a device described as "above" or "on top of" other devices or structures will subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein are interpreted accordingly.

[0081] It should also be noted that the terms "one embodiment," "another embodiment," and "embodiment" used in this application refer to specific features, structures, or characteristics described in connection with that embodiment, which are included in at least one embodiment described in the general description of this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, the intention is to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this application.

[0082] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0083] It should also be noted that the above are merely preferred embodiments of this application and do not limit the scope of patent protection of this application. Any equivalent structural or procedural changes made using the content of this application’s specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of this application.

Claims

1. An organoid cell culture device, characterized in that, include: The bottom layer of the device; A first culture medium delivery layer is located above the bottom layer of the device. The first culture medium delivery layer has a through culture medium delivery section, and the surface of the first culture medium delivery layer also has a first culture medium flow port that communicates with the culture medium delivery section. A cell culture layer is located above the first culture medium delivery layer. The cell culture layer includes a porous membrane on which cells to be cultured are placed. The porous membrane covers the first culture medium flow port. The second culture medium delivery layer is located above the cell culture layer. The second culture medium delivery layer is provided with a second culture medium flow port, which is correspondingly arranged with the porous membrane. The second culture medium delivery layer also includes a second culture medium inlet and a second culture medium outlet. The second culture medium inlet is connected to the second culture medium flow port through a second culture medium inlet trough, and the second culture medium outlet is connected to the second culture medium flow port through a second culture medium outlet trough. A capping layer is located above the second culture medium delivery layer. The capping layer is provided with a first culture medium inlet and a second culture medium inlet. The first culture medium inlet is corresponding to the second culture medium inlet, and the second culture medium inlet is corresponding to the second culture medium outlet. The first culture medium inlet and the second culture medium inlet are used to communicate with an external culture medium supply device through a Luer connector. The organoid cell culture device further includes a fixing component, which passes through the capping layer, the second culture medium delivery layer, and the first culture medium delivery layer in sequence and is fixedly connected to the bottom layer of the device.

2. The organoid cell culture device according to claim 1, characterized in that, The cell culture layer further includes a first gel film layer and a second gel film layer. The first gel film layer is located below the second culture medium delivery layer. The porous membrane is located between the first gel film layer and the second gel film layer. The first gel film layer has a first perforation, and the second gel film layer has a second perforation. The first perforation, the second perforation, and the second culture medium flow port are correspondingly arranged.

3. The organoid cell culture device according to claim 2, characterized in that, The first adhesive film layer is provided with conductive wires, and the porous membrane is disposed on top of the conductive wires.

4. The organoid cell culture device according to claim 2, characterized in that, A conductive filament is provided below the second adhesive film layer, and the porous membrane is located below the conductive filament.

5. The organoid cell culture apparatus according to any one of claims 3 or 4, characterized in that, The conductive wire includes a first conductive wire and a second conductive wire. The first conductive wire extends along a first direction, and the second conductive wire extends along a second direction. The first direction and the second direction are located on the same horizontal plane, and the first direction and the second direction are parallel or perpendicular to each other.

6. The organoid cell culture device according to claim 3, characterized in that, The conductive wire is used to provide electrical stimulation during cell culture. The electrical stimulation parameters include: voltage of 5.5V, current of 1mA, and discharge frequency of 1kHz.

7. The organoid cell culture apparatus according to claim 6, characterized in that, The cells to be cultured are wrapped with a conductive layer.

8. The organoid cell culture device according to claim 1, characterized in that, The first culture medium has five flow ports, which are evenly distributed at intervals. There are five porous membranes, and each porous membrane is provided with a corresponding first culture medium flow port; The second culture medium has five flow ports, and each second culture medium flow port is set to correspond to one of the first culture medium flow ports.

9. The organoid cell culture device according to claim 1, characterized in that, The first culture medium outlet is circular, and the porous membrane is rectangular, wherein the length of any side of the porous membrane is greater than the diameter of the first culture medium outlet.

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