Organoid chip capable of 3D culture and cell culture method using same

Abstract

A method for culturing cells on the basis of an organoid chip, according to an embodiment of the present application, comprises the steps of: performing first seeding on a first surface of a second layer; inverting the second layer; and performing second seeding on a second surface of the second layer, wherein the organoid chip comprises a first layer, a second layer, and a third layer.

Description

3D cultureable organoid chip and cell culture method using the same

[0001] The present invention provides an organoid chip capable of 2D and 3D culture, and a cell culture method using the organoid chip.

[0002]

[0003] Cell culture technology plays a crucial role in modern life science research and new drug development. Cell culture is primarily classified into 2D and 3D culture methods, with each method being used selectively depending on the purpose of specific research or experiments. 2D culture is a traditional method of culturing cells in a single layer, offering the advantages of simple setup and high reproducibility, while 3D culture is a method of culturing cells to form a three-dimensional structure, which mimics an environment similar to actual tissues and is advantageous for studying complex physiological responses.

[0004] With the recent emphasis on the need for precision medicine and personalized treatment in life science research, the importance of 3D culture technology is becoming increasingly prominent. 3D culture offers the advantage of being highly useful for new drug development and toxicity assessment because it can simulate cell-to-cell interactions, tissue-specific functions, and drug responses more realistically. However, since 2D culture is still widely used in laboratories, there is a need to develop technology that can flexibly utilize both culture methods.

[0005] Against this backdrop, the development of a chip that supports both 2D and 2D culture is crucial for maximizing research efficiency and meeting diverse research needs. In particular, if both culture methods can be implemented on the same device, it has the advantage of significantly improving the productivity of research and development by allowing various experiments to be performed without equipment replacement or additional costs.

[0006] Conventional chip technology has limitations in that it is difficult to support both culture methods simultaneously because 2D culture and 3D culture are designed with clear distinctions. To overcome these limitations, there is a need to develop technology that can support both 2D culture and 3D culture on the same chip.

[0007]

[0008] One objective of the present invention is to provide a cell culture method and an apparatus for performing the same, which enables both 2D culture and 3D culture methods by providing an organoid chip including a middle layer having a constant thickness.

[0009] The problems that the present invention aims to solve are not limited to those described above, and problems not mentioned will be clearly understood by those skilled in the art from this specification and the attached drawings.

[0010]

[0011] According to one embodiment, a method for culturing cells based on an organoid chip according to one embodiment comprises the steps of performing a first seeding on a first surface of a second layer, flipping the second layer, and performing a second seeding on a second surface of the second layer, wherein the organoid chip comprises a first layer, a second layer, and a third layer.

[0012] The means for solving the problem of the present invention are not limited to the means for solving the problem described above, and unmentioned means for solving the problem will be clearly understood by those skilled in the art from this specification and the attached drawings.

[0013]

[0014] According to one embodiment, by providing an integrated chip that supports both 2D culture and 3D culture in a single device, experimental efficiency and flexibility can be significantly improved, and the device can be utilized for various research purposes while maintaining consistency in the experimental environment.

[0015] According to one embodiment, the present invention can achieve excellent performance in both 2D and 3D cultures by controlling the thickness of the middle layer of the chip, thereby allowing researchers to freely select a culture method according to specific experimental purposes and enabling more precise cell physiological research and drug testing.

[0016] The effects of the present invention are not limited to the effects described above, and unmentioned effects will be clearly understood by those skilled in the art from this specification and the accompanying drawings.

[0017]

[0018] FIG. 1 is a diagram illustrating the structure of an organoid chip according to one embodiment.

[0019] FIGS. 2 and 3 are drawings for explaining the structure of a plurality of layers constituting an organoid chip according to one embodiment and the supply and recovery path of cell culture medium.

[0020] FIGS. 4 to 7 are drawings for explaining experimental data using an organoid chip according to one embodiment.

[0021] FIGS. 8 to 12 are drawings illustrating cell viability in a dynamic culture environment according to one embodiment.

[0022] FIGS. 13 and FIGS. 15 are drawings illustrating a method of seeding cells into an organoid chip according to one embodiment.

[0023] FIGS. 16 and FIGS. 18 are drawings illustrating a method of seeding cells into an organoid chip according to another embodiment.

[0024]

[0025] The aforementioned objectives, features, and advantages of the present application will become more apparent from the following detailed description in conjunction with the accompanying drawings. However, as the present application is subject to various modifications and may have various embodiments, specific embodiments are illustrated in the drawings and described in detail below.

[0026] Throughout the specification, identical reference numbers generally represent identical components. Additionally, components with identical functions within the same scope of concept appearing in the drawings of each embodiment are described using the same reference numeral, and redundant descriptions thereof are omitted.

[0027] If it is determined that a detailed description of known functions or configurations related to this application could unnecessarily obscure the essence of this application, such detailed description is omitted. Furthermore, numbers used in the description of this specification (e.g., First, Second, etc.) are merely identifiers to distinguish one component from another.

[0028] Furthermore, the suffixes "module" and "part" for components used in the following embodiments are assigned or used interchangeably solely for the ease of drafting the specification, and do not inherently possess distinct meanings or roles.

[0029] In the following examples, singular expressions include plural expressions unless the context clearly indicates otherwise.

[0030] In the following embodiments, terms such as "include" or "have" mean that the features or components described in the specification are present, and do not preclude the possibility that one or more other features or components may be added.

[0031] In the drawings, the size of components may be exaggerated or reduced for convenience of explanation. For example, the size and thickness of each component shown in the drawings are arbitrarily depicted for convenience of explanation, and the present invention is not necessarily limited to what is illustrated.

[0032] Where an embodiment can be implemented differently, the order of a particular process may be performed differently from the order described. For example, two processes described consecutively may be performed substantially simultaneously or proceed in the reverse order of the description.

[0033] In the following embodiments, when components are described as being connected, the case includes not only instances where the components are directly connected but also instances where components are indirectly connected by interposing them in between.

[0034] For example, when it is stated in this specification that components, etc. are electrically connected, it includes not only cases where the components, etc. are directly electrically connected, but also cases where components, etc. are interposed in between and are indirectly electrically connected.

[0035]

[0036] Hereinafter, a method for generating digital code-based art using a robotic device and a device for performing the same will be described with reference to the drawings.

[0037] FIG. 1 is a diagram illustrating the structure of an organoid chip according to one embodiment. Referring to FIG. 1 (a), the organoid chip (100) according to one embodiment may include at least one layer.

[0038] The organoid chip (100) may include a first layer (110), a second layer (120), and a third layer (130). The organoid chip (100) may be in a form in which the first layer (110), the second layer (120), and the third layer (130) are stacked sequentially. The first layer (110) may be referred to as the top layer, the second layer (120) as the middle layer, and the third layer (130) as the bottom layer.

[0039] The second layer (120) may include a cell culture section (121), and the cell culture section (121) may exist in an open form in the center of the second layer (120). Although the shape of the cell culture section (121) is depicted as circular in the drawing, it is not limited thereto and may be provided in various shapes having a predetermined length.

[0040] According to one embodiment, depending on the culture method, epithelial cells may be attached to the upper surface of the second layer (120), endothelial cells may be attached to the back surface, and an extracellular matrix (ECM) may be placed between the epithelial cells and the endothelial cells.

[0041] The organoid chip (100) disclosed in the present invention may be used with the same or corresponding meaning as various known terms that are used interchangeably in life science research and new drug development processes. For example, the term organoid chip may be used with the same or corresponding meaning as organ-on-a-chip, biochip, biochip, microfluidic chip, human-on-a-chip, MPS (Microphysiological System), etc. Hereinafter, the term organoid chip (100) will be used uniformly, but the term organoid chip (100) may refer to the aforementioned terms.

[0042]

[0043] FIGS. 2 and 3 are drawings for explaining the structure of a plurality of layers constituting an organoid chip according to one embodiment and the supply and recovery path of cell culture medium.

[0044] Referring to FIGS. 2 and FIGS. 3, the organoid chip (100) may include a path for supplying and recovering cell culture medium, and the path for supplying and recovering (or circulating) the cell culture medium may include a first channel (c1) and a second channel (c2).

[0045] More specifically, a path capable of guiding the flow of fluid may be formed in each of the first layer (110), the second layer (120), and the third layer (130), and the first layer (110) to the third layer (130) may be arranged in close contact, and the first plate (210) and the second plate (220) may be arranged so that the fluid does not leak to the outside, thereby forming the first channel (c1) and the second channel (c2) by each path. Here, the first channel (c1) may be referred to as the upper channel, and the second channel (c2) may be referred to as the lower channel.

[0046] The cell culture medium can circulate inside the organoid chip (100) through the first channel (c1) and affect the upper surface of the cell culture portion (121) of the second layer (120), and can circulate inside the organoid chip (100) through the second channel (c2) and affect the back surface of the cell culture portion (121).

[0047] Each of the first layer (110), the second layer (120), and the third layer (130) may have a predetermined thickness. The first layer (110) may have a first thickness (t1), the second layer (120) may have a second thickness (t2), and the third layer (130) may have a third thickness (t3). For example, the first thickness (t1) may be 1.1 mm, the second thickness (t2) may be 0.6 mm, and the third thickness (t3) may be 0.6 mm. Meanwhile, the diameter of the cell culture portion (121) of the second layer (120) may be formed with a predetermined radius (d1), and for example, the radius may be 6 mm.

[0048] However, the thickness of the first layer (110) to the third layer (130) described above is exemplary and may be set differently depending on the experimental subject and conditions. For example, the second thickness (t2) of the second layer (120) may be formed to correspond to the size of the organoid being tested. The organoid may vary in size from 0.2 mm to 1 mm, and the second thickness (t2) may be set to correspond to this. The organoid cultured in the cell culture section (1210) must maintain a static state while receiving appropriate shear stress. If the second thickness (t2) is too small, the shear stress transmitted to the organoid becomes too strong, making it difficult to maintain a static state, and the organoid may be swept away by the fluid flow. On the other hand, if the second thickness (t2) is too large, the applied shear stress may become too weak; therefore, it is important that the second thickness (t2) has an appropriate height. Preferably, the second thickness (t2) can be formed between 0.5 mm and 1 mm.

[0049] Meanwhile, fluid flow through the first channel (c1) and the second channel (c2) may occur according to predetermined conditions and may circulate at a speed according to the predetermined conditions. At this time, the speed according to the predetermined conditions may be determined by considering the size of the organoid and the thickness of the second layer (120) so that an appropriate shear stress is transmitted while maintaining a static state in the cell.

[0050] Although not shown in the drawing, the organoid chip (100) may include a reservoir, and the reservoir may include an internal space capable of storing and keeping cell culture medium. The cell culture medium stored in the reservoir may circulate inside the organoid chip (100) through the first channel (c1) and the second channel (c2) according to a predetermined driving method.

[0051]

[0052] FIGS. 4 to 7 are drawings for explaining experimental data using an organoid chip according to one embodiment. Referring to FIGS. 4 to 7, experimental results of cell culture performed using the organoid chip (100) of the present invention can be confirmed.

[0053] Referring to Figure 4, the experiment was performed through a coating step, a cell seeding step, a fabrication and chip loading step, an overlay step, a dynamic culture step, and a staining and imaging step.

[0054] For the coating step (duration: 1 day), the chip was coated with an ECM (Extracellular Matrix) solution, which contained Collagen (0.4 mg / mL) and Fibronectin (0.1 mg / mL). This created an environment where cells could adhere.

[0055] In the case of the cell seeding step (duration: 1 day), the cell is seeded onto the chip using a seeding kit, and the cell is seeded onto a layer constituting the organoid chip (100) and stabilization is performed.

[0056] In the case of the chip fabrication and loading step (duration: 1 hour), the layer in which cell culture is completed is loaded onto the organoid chip (100), and after the loading is completed, cell culture begins in earnest.

[0057] For the overlay step (duration: 3 hours), an additional layer is formed on top of the cells using an ECM solution, which helps the cells grow more stably in a 3D environment.

[0058] In the case of the dynamic culture stage (duration: 2 days), the organoid chip (100) is mounted on a platform (e.g., a biological tissue chip driving device) and then cell growth can be induced through dynamic culture under conditions that mimic a real biological environment. The platform is a device manufactured in a form to which the organoid chip (100) can be coupled, and is a device that drives fluid flow.

[0059] The staining and imaging step (duration: 1-4 days) is a step in which cultured cells are stained to visually identify specific markers or structures, and through this step, the morphology, location, activity, etc. of the cells can be analyzed. The experimental data obtained through this is described later with reference to FIGS. 5 to 7.

[0060] Figure 5(a) shows the experimental results for HepaRG, Figure 5(b) shows the experimental results for 2D culture of Hepatic Organoid (HO), and Figure 5(c) shows the experimental results for 3D culture of Hepatic Organoid (HO).

[0061] The aforementioned HepaRG cells are a cell line derived from human liver cells that mimics the major functions of liver cells, possesses high metabolic activity, and can express enzymes specific to liver cells. Because HepaRG cells effectively mimic the drug metabolism processes of the human liver, they are used as an important model in drug metabolism research and play a crucial role in evaluating drug interactions and toxicity during the new drug development process.

[0062] The aforementioned hepatic organoid (HO) refers to a model related to liver tissue designed to possess a structure and function similar to the actual liver through the self-organization of various cell types, including hepatocytes. Hepatic organoids (HO) can reproduce the complex cellular composition of the liver by including hepatocytes, biliary cells, and stellate cells, and are used to study the physiology of liver diseases (hepatitis, cirrhosis, liver cancer, etc.).

[0063] Referring to Figures 5 (a) to (c), after 48 hours had passed since the cell culture, the cell viability was measured through staining (live cell, dead cell staining), and it was confirmed that the cell viability was well maintained.

[0064] More specifically, as can be seen in the diagram, the green fluorescence labeled "Live" indicates living cells, the red fluorescence labeled "Dead" indicates dead cells, the "Merge" panel shows the combined locations of living and dead cells, and the "Hoechst33342" stain visualizes cell nuclei, allowing for clearer observation of cell density and tissue structure.

[0065] As can be seen in Figures 5 (a) to (c), the green fluorescence is widely spread, indicating that most of the cells are alive, and in the HO-2D culture and HO-3D culture, the cells are evenly distributed in a planar manner, and the cells are clustered to form a spherical structure.

[0066]

[0067] Figure 6 is a diagram comparing the viability and specific physiological functions of cells under three different culture conditions, and Figures 6 (a) to (c) show the results of measuring the functional activity of cells under different culture conditions.

[0068] In the case of Figure 6 (a), the amount of albumin produced by the cells was compared, and albumin levels exceeding a certain amount were detected in all three culture conditions. More specifically, it can be confirmed that higher levels were observed in the HO-2D and HO-3D culture environments than in HepaRG, and that higher levels were observed in the HO-3D culture environment than in the HO-2D culture environment.

[0069] In the case of Figure 6 (b), the amount of urea produced by the cells was compared, and since all three culture conditions showed similar values, it can be said that liver cell function is well maintained under all conditions.

[0070] Figure 6 (c) shows the alanine (Alanine Aminotransferase) activity in cells, which is higher than that of HepaRG in HO-2D and HO-3D culture environments.

[0071]

[0072] FIG. 7 is a diagram for comparing experimental data performed under different conditions. According to one embodiment, a first experiment was performed after culturing for 48 hours and a second experiment was performed after culturing for 8 days, and the experimental data described above through FIG. 4 and FIG. 6 is the experimental data for the first experiment.

[0073] In the case of the second experiment, the experiment was conducted in the same way as the first experiment within the allowable error range, but the difference is that the culture period was increased to 8 days.

[0074] Referring to Figure 7, the ALB, Urea, and ALT values ​​obtained through the second experiment can be seen, and it can be confirmed that the results are similar to the data from the first experiment to some extent. For reference, the data from the second experiment includes only data measured in the HO-2D and HO-3D culture environments.

[0075] More specifically, referring to Fig. 7(a), ALB levels were detected in the HO-2D and HO-3D culture environments, and it can be seen that higher levels were observed in the HO-3D culture environment than in the HO-2D culture environment. Referring to Fig. 7(b), Urea levels were detected in the HO-2D and HO-3D culture environments, and it can be seen that higher levels were observed in the HO-3D culture environment than in the HO-2D culture environment. Referring to Fig. 7(c), ALT levels were detected in the HO-2D and HO-3D culture environments, and it can be seen that higher levels were observed in the HO-2D culture environment.

[0076] As can be seen from the first and second experimental data, ALB and Urea levels tend to increase more in the HO-3D culture environment than in the HO-2D culture environment, while ALT levels tend to decrease in the HO-3D culture environment than in the HO-2D culture environment.

[0077]

[0078] FIGS. 8 to 12 are drawings illustrating cell viability in a dynamic culture environment according to one embodiment. Referring to FIGS. 8 to 12, it can be seen that when a dynamic culture environment is provided through a platform device according to one embodiment, cell viability is further increased.

[0079] FIG. 8(a) is experimental data performed under plate culture conditions, and FIG. 8(b) is experimental data performed under chip culture conditions. Here, the plate culture conditions refer to performing culture in a static environment, and the chip culture conditions refer to performing culture in a dynamic environment. More specifically, the dynamic environment may be provided through a platform device according to one embodiment, and the platform device may be a biological tissue chip driving device, configured in a form capable of mounting an organoid chip (100), and may be a device that induces cell growth under conditions simulating a real biological environment through dynamic culture by circulating fluid according to a predetermined method.

[0080] In the case of Figures 8 and 9, cell viability was evaluated through imaging methods (distinguishing between living and dead cells by performing imaging using a cell viability measurement kit provided by Thermo-Fisher) and luminescence measurement methods (measuring cell viability using luminescence via the CellTiter-Glo 3D cell viability measurement method provided by Promega). In addition, the morphology and number of cells were visually represented by staining the cell nuclei using a nuclear stain (Hoechst 33342).

[0081] More specifically, a concentration of DMSO (Dimethyl Sulfoxide) of 0.1% was used in the cell culture solution to serve as a control group, and the actual effects of other drugs (TG (Thapsigargin), PG (Prostaglandin)) were evaluated.

[0082] Referring to Figures 8 to 10, it can be seen that in hepatic organoid (HO) culture, when treated with TG (Thapsigargin) or PG (Prostaglandin), cell viability decreases more significantly under chip culture conditions than under plate culture conditions.

[0083] This suggests that the sensitivity of cells to TG and PG treatment increased under dynamic culture conditions (chip culture conditions) compared to static culture conditions (plate culture conditions), which may imply that dynamic culture conditions increase cellular metabolic activity or stress responses, thereby causing the toxic effects of the drug to be more pronounced. In other words, under dynamic culture conditions, cells are exposed to a continuous fluid flow, which can promote the distribution and absorption of the drug and increase cellular metabolic activity and stress responses. These results may indicate that chip culture conditions provide a physiological environment more similar to the human body, allowing for a more accurate simulation of the toxic effects of the drug.

[0084] Referring to Figures 11 and 12, the levels of ALB, Urea, ALT, and AST under plate culture conditions and chip culture conditions can be observed. Referring to Figures 11 (a) and (b) and Figure 12 (b), it can be seen that the chip culture condition shows a tendency for ALB, Urea, and AST levels to increase more than the plate culture condition. Referring to Figure 12 (a), it can be seen that the ALT level shows a tendency to decrease more under chip culture conditions than under plate culture conditions.

[0085]

[0086] FIGS. 13 and 15 are drawings illustrating a method for seeding cells into an organoid chip according to one embodiment. An organoid chip (100) according to one embodiment includes a plurality of layers (first to third layers), and in particular, is characterized in that the second layer (120) is formed to have a constant thickness, and thus the method for seeding cells is also characterized.

[0087] Referring to FIG. 13, a cell seeding method according to one embodiment may include a step of performing a first seeding on a first surface of a middle layer (S110), a step of flipping the middle layer (S120), a step of performing a second seeding on a second surface of the middle layer (S130), a cell stabilization step (S140), and a chip assembly step (S150).

[0088] Referring to FIG. 14 (a), a first seeding may be performed on a first surface of the middle layer (120), and the first seeding may refer to a process of forming a base on which cells can attach by coating an extracellular matrix or a specific functional membrane on the surface of the middle layer (120) and attaching endothelial cells. The first surface may be the upper surface of the middle layer (120), and the first seeding may be performed with the first surface positioned to face opposite gravity.

[0089] Referring to FIG. 14 (b) and (c), the operation of flipping the middle layer (120) so that the second surface of the middle layer (120) faces opposite to gravity may be performed. Subsequently, a second seeding may be performed on the second surface of the middle layer (120). The second seeding may include organoid seeding and ECM seeding.

[0090] A cell stabilization step can be performed as shown in Fig. 15 (d), and then an assembly step can be performed as shown in Fig. 15 (e) and (f). More specifically, the first layer (110) can be placed on top of the second layer (120) and the third layer (130) can be placed on the bottom, and then the first plate (210) and the second plate (220) can be placed and assembled respectively to prevent leakage of the culture medium.

[0091]

[0092] FIGS. 16 and FIGS. 18 are drawings illustrating a method of seeding cells into an organoid chip according to another embodiment.

[0093] Referring to FIG. 16, according to another embodiment, a method for seeding cells into an organoid chip may include a step of performing a first seeding on a first surface of a middle layer (S210), a step of flipping the middle layer (S220), a chip assembly step (S230), a step of performing a second seeding on a second surface of the middle layer (S240), and a shielding and monitoring step (S250).

[0094] A seeding method according to another embodiment may be partially similar to the seeding method described with reference to FIGS. 13 to 15. For example, the first step (S210), second step (S220), third step (S230), and fourth step (S240) included in the seeding method according to another embodiment may correspond to the first step (S110), second step (S120), fifth step (S150), and third step (S130) included in the seeding method according to one embodiment, respectively, and since the description thereof has been described above, a redundant description is omitted.

[0095] A seeding method according to another embodiment is characterized in that, compared to a seeding method according to one embodiment, the chip assembly step (S230) is performed after the step of flipping the middle layer (S220). In other words, in a seeding method according to another embodiment, a second seeding can be performed on the second side of the middle layer after the chip assembly is performed. Afterward, when it is confirmed that the second seeding has been completed, a shielding and monitoring step (S250) can be performed.

[0096] More specifically, referring to FIG. 17(c), when a seeding method according to another embodiment is performed, at least one part of the first plate (210) may include an open area (e.g., a hole). The open area may be formed at a position corresponding to the cell culture portion (121) of the second layer (120). Meanwhile, the first plate (210) may be made of a transparent material. Since at least one part of the first plate (210) is open and the material is designed to be transparent, the cell culture process can be visually observed and monitored.

[0097] Referring to Fig. 18 (d), a second seeding can be performed through the open area, and a second seeding can be performed with the organoid chip assembled through such a structure.

[0098] Afterwards, referring to FIG. 18 (e), an operation to shield the open area of ​​the first plate (210) can be performed using a shielding member (230). Here, the shielding member (230) may be provided in a shape corresponding to the open area of ​​the first plate (210) and may perform the function of preventing the culture medium moving between the first channel (c1) and the second channel (c2) from leaking out by being inserted into the open area.

[0099] Meanwhile, at least one sensor may be attached to the shielding member (230), and an additional operation of continuously monitoring the state of the culture medium and cells (e.g., including an electrochemical monitoring method) through the at least one sensor may be performed.

[0100] Meanwhile, according to one embodiment, the operation of seeding or culturing cells in the manner described above can be performed through a cell culture device. The cell culture device includes at least one processor and can perform a series of operations described above in this specification based on a control signal of at least one processor.

[0101]

[0102] The features, structures, effects, etc. described in the embodiments above are included in at least one embodiment of the present invention and are not necessarily limited to only one embodiment. Furthermore, the features, structures, effects, etc. exemplified in each embodiment may be combined or modified and implemented in other embodiments by a person skilled in the art to which the embodiments belong. Accordingly, details regarding such combinations and modifications should be interpreted as being included within the scope of the present invention.

[0103] Furthermore, although the embodiments have been described above, this is merely illustrative and does not limit the invention. Those skilled in the art will understand that various modifications and applications not exemplified above are possible within the scope of the essential characteristics of the embodiments. In other words, each component specifically shown in the embodiments may be modified and implemented. Differences related to such modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims.

Claims

1. A method for culturing cells based on an organoid chip comprising a first layer, a second layer and a third layer, wherein the method comprises: A step of performing a first seeding on the first surface of the second layer; The step of flipping the second layer above; and A step comprising: performing a second seeding on the second surface of the second layer; Cell culture method.

2. In Paragraph 1, The above organoid chip is in the form in which the first layer, the second layer, and the third layer are sequentially stacked and arranged. The first to third layers include a path capable of guiding fluid flow, and by stacking and arranging the first to third layers, at least one channel for circulating cell culture medium is formed. Cell culture method.

3. In Paragraph 2, The above at least one channel includes a first channel formed based on the first layer and a second channel formed based on the third layer, and The cell culture medium circulates inside the organoid chip through the first channel and the second channel, Cell culture method.

4. In Paragraph 1, A cell culture portion is formed in at least one region of the second layer, and The cell culture portion is present in an open form in the center of the second layer, Cell culture method.

5. In Paragraph 4, The first surface is the upper surface of the second layer, and the second surface is the back surface of the second layer, and The first seeding is performed with the first surface positioned so as to face opposite gravity, and the second seeding is performed with the second surface positioned so as to face opposite gravity. Cell culture method.

6. In Paragraph 4, The second layer is formed with a predetermined thickness, and the predetermined thickness is determined based on the size of the organoid seeded in the cell culture section. Cell culture method.

7. In Paragraph 6, The above-mentioned predetermined thickness is formed to be 0.5mm to 1mm, Cell culture method.

8. In Paragraph 7, The above-mentioned predetermined thickness is formed to be 0.6mm, Cell culture method.

9. In Paragraph 7, The thickness of the third layer is formed to be the same as the thickness of the second layer. Cell culture method.

10. In Paragraph 9, The thickness of the first layer is formed to be 1.1 mm, Cell culture method.

11. In Paragraph 8, The step of performing the first seeding includes the step of coating a membrane on the first surface of the second layer to form a base to which cells can attach, and The step of performing the second seeding above includes the step of performing organoid seeding on the second surface of the second layer, Cell culture method.

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