Culture device and culture method for preparing separated combined organoids

Abstract

The invention discloses a culture device for preparing separated combined organoid. The culture device comprises a first culture chamber, a second culture chamber and at least one combined organoid culture unit, wherein the combined organ culture unit at least comprises a first cell culture cavity, a second cell culture cavity and one or more directional channels; wherein the first cell culture cavity and the second cell culture cavity are configured to culture first cells and second cells; the directional channel is filled with a directional induced growth culture medium, so that the first cells and / or the second cells are allowed to generate directional axon induced growth in the directional induced growth culture medium; the first culture cavity and the second culture cavity are respectively configured to contain a first culture medium suitable for growth of first cells and a second culture medium suitable for growth of second cells, and the first culture medium and the second culture medium are not mixed. The combined organ built by utilizing the culture device disclosed by the invention not only can be used for carrying out fundamental research on growth, projection and synaptic formation of various human neurons, but also can provide a research model for human nerve diseases and neuronal axon regeneration and myelination.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a culture device for preparing separate combined organoids, and a culture method for separate combined organoids. Background Art

[0002] Organoids are cultures obtained in vitro through 3D induction methods that have similar cell composition and structure to in vivo organs. Compared with 2D cells, organoids are more similar to in vivo tissues, especially human organoids, which fill the gap of differences in model animal species and have very important application value. For brain organoids, only organoids of a single brain region can be obtained at present, and projection and functional research between brain regions cannot be carried out.

[0003] Assembloid is an emerging concept in organoid culture, which is to co-culture different types of organoids, which can be used to study the interactions between different organoids. For example, thalamic organoids and telencephalic organoids are co-cultured to study the projection and function between the thalamus and telencephalon. Assembloids in existing technologies all adopt a fusion approach, in which two or more organoids are directly contacted and cultured with the same culture medium. A major disadvantage of culturing assembloids by direct fusion is that different types of organoids must be cultured in the same culture medium, which is not conducive to the survival, differentiated differentiation and maturation of organoids. Summary of the invention

[0004] The present application discloses a culture device for preparing separated combined organoids, and a culture method for separated combined organoids, which enables any homologous or heterologous organoids to form separated combined organoids through long-range axon connections, and heterologous organoids can grow in their respective most suitable culture media.

[0005] The present application discloses a culture device for preparing separated combined organoids, the culture device comprising:

[0006] at least one combined organoid culture unit and its corresponding culture chamber;

[0007] Wherein, the combined organoid culture unit comprises at least a first cell culture chamber, a second cell culture chamber, and one or more directional channels for connecting the cell culture chambers in the combined organoid culture unit;

[0008] Wherein, the first cell culture chamber is configured to culture a first cell, and the first cell is a human or animal cell;

[0009] The second cell culture chamber is configured to culture second cells, wherein the second cells are human or animal cells;

[0010] The directional channel is filled with a directional growth induction culture medium, thereby allowing the first cell and / or the second cell to produce directional axon induced growth in the directional growth induction culture medium;

[0011] The first cell culture chamber is located in the first culture chamber, and the second cell culture chamber is located in the second culture chamber; the first culture chamber is configured to contain a first culture medium suitable for the growth of first cells, and the second culture chamber is configured to contain a second culture medium suitable for the growth of second cells; and the first culture medium and the second culture medium are configured not to mix with each other.

[0012] In a preferred embodiment, the first cell and the second cell are the same or different.

[0013] In a preferred embodiment, the first cell is selected from any one of the following:

[0014] Neurons, brain organoids, retinal organoids, spinal cord organoids; or

[0015] Glial cells, muscle organoids, heart, pancreas, small intestine and other visceral organoids, tumor organoids.

[0016] In a preferred embodiment, the second cell is selected from the group consisting of neurons, brain organoids, retinal organoids, and spinal cord organoids; or

[0017] Glial cells, muscle organoids, cardiac organoids, pancreatic organoids, intestinal organoids, tumor organoids.

[0018] In a preferred embodiment, the directional growth induction culture medium is an axon directional growth induction culture medium.

[0019] In a preferred embodiment, the directed growth induction culture medium is Matrigel.

[0020] In a preferred embodiment, the cell origin is any one of the following: mammals, rodents, non-human primates, and humans.

[0021] In a preferred embodiment, after culturing, the first cell culture formed by the first cells and the second cell culture formed by the second cells form a combined state in the directional channel, thereby forming a separated combined organoid;

[0022] In another preferred embodiment, the first cells form a first cell culture, and / or the second cells form a second cell culture; wherein,

[0023] The first cell culture and / or the second cell culture is an organoid.

[0024] In a second aspect, the present application discloses a method for culturing a separated combined organoid, wherein the method is configured to construct a combined organoid in a separated manner; the culturing method comprises:

[0025] - Using a culture device, by occupying the channel of the culture device and applying Matrigel, the directional growth of axons in three-dimensional space is achieved;

[0026] - Place isolated homologous or xenogeneic organoids in different chambers on both sides;

[0027] -The organoids in different chambers on both sides are connected by axons to form combined organoids;

[0028] In a preferred embodiment, when the organoids in the two side chambers need to be grown in different culture media, a baffle is set in the middle of the two side chambers to separate the two side chambers, so that the heterogeneous organoids can grow in their respective most suitable culture media.

[0029] In another preferred embodiment, the culture device is configured with a plurality of different chambers and the chambers are connected by pipes.

[0030] In a third aspect, the present application further discloses a method for culturing separate combined organoids, wherein the method cultures neural cells in multiple culture regions, and the neural cells in the culture regions perform neural projection between the multiple culture regions.

[0031] In a preferred example, the plurality of culture areas are configured with corresponding culture media according to the nerve cells in the culture areas, and the culture media are not mixed with each other.

[0032] In a preferred example, a directional channel is arranged between the culture areas, and a directional induction growth culture medium is arranged in the directional channel. The nerve cells produce directional axon induced growth through the directional induction growth culture medium, thereby forming neural projection.

[0033] In a preferred embodiment, the directed growth induction culture medium is Matrigel.

[0034] In a fourth aspect, the present application further discloses a separate combined organ, which includes a plurality of nerve aggregation regions, wherein projections are formed between the plurality of nerve aggregation regions through directional growth of axons.

[0035] In a preferred embodiment, the separated combined organoids are human retinal organoids and the human retinal organoids form projections through three-dimensionally directed growth of long-range axons and thalamic organoids.

[0036] In a fourth aspect, the present application further discloses an axon regeneration model, which is constructed using the culture device as described above.

[0037] In a fifth aspect, the present application also discloses an axon myelination model, which is constructed using the culture device described above.

[0038] This application has at least the following beneficial technical effects:

[0039] 1) Using patient-derived induced pluripotent stem cells to differentiate organoids, composite organoids are constructed on this device to construct disease models to study diseases related to axon growth, projection, and synapse formation.

[0040] 2) Unlike fusion-type combined organoids, where the neuron axons are inside the organoids, the combined organoids constructed using this device are separated, and the axons grow in the tubes connecting the organoids. For example, the projections between the optic nerve and the thalamus, the projections between different brain regions, the connection between motor neurons and muscles, etc.

[0041] 3) The device described in this application can be used as a structural unit to achieve not only unidirectional and bidirectional projection, but also to expand to one-to-many, many-to-one, and multi-level combination culture of more than three types of organoids. In addition to neuronal projection, this device can also be used in theory for vascular organoids to induce vascular directional growth.

[0042] The specification of the present application records a large number of technical features, which are distributed in various technical solutions. If all possible combinations of technical features of the present application (i.e., technical solutions) are to be listed, the specification will be too long. In order to avoid this problem, the various technical features disclosed in the above-mentioned invention content of the present application, the various technical features disclosed in the various embodiments and examples below, and the various technical features disclosed in the accompanying drawings can be freely combined with each other to form various new technical solutions (these technical solutions should all be regarded as having been recorded in this specification), unless the combination of such technical features is technically infeasible. For example, in one example, feature A+B+C is disclosed, and in another example, feature A+B+D+E is disclosed, and features C and D are equivalent technical means that play the same role. Technically, only one can be used, and it is impossible to use them at the same time. Feature E can be combined with feature C technically. Then, the solution of A+B+C+D should not be regarded as having been recorded because it is technically infeasible, and the solution of A+B+C+E should be regarded as having been recorded. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a model diagram and a physical diagram of a mold used to make the culture device of the present application;

[0044] Figure 2It is a physical picture of the culture device of the present application prepared by using a mold and agarose;

[0045] Figure 3 is a schematic diagram of implantation and culture of a first organoid according to the culture method of the present application;

[0046] Figure 4 is a schematic diagram of axons of the first organoid sample obtained according to the culture method of the present application, which grows in a 3D directional manner in a channel;

[0047] Figure 5 is a schematic diagram of implantation and culture of a second organoid according to the culture method of the present application;

[0048] Figure 6 is a schematic diagram of a separated retinal-thalamic combined organoid connected by long-range axons obtained according to the culture method of the present application;

[0049] Figure 7 is a schematic diagram of the representation of synapses formed in the retinal-thalamic combined organoid obtained according to the culture method of the present application;

[0050] Figure 8 It is a schematic diagram of constructing a human optic nerve regeneration model based on the culture device and culture method of the present application. DETAILED DESCRIPTION

[0051] After extensive research, the inventors of this application have proposed a separate combined organoid culture device and a culture method thereof. By utilizing the characteristics that axons of certain types of neurons (such as retinal ganglion cells, cross-brain projection neurons, etc.) can be projected over long distances, the separate combined organoid culture device of this application not only realizes the 3D directional growth of neuronal axons, but also allows different types of organoids to differentiate and mature in their most suitable culture medium, and project and connect through axons. The combined organoids cultured using this culture device can study the axon growth, projection and synapse formation mechanisms of various types of human neurons, and then screen regulatory factors that affect neuronal neural projection and synapse formation. In addition, by using patient-derived induced pluripotent stem cells to differentiate organoids, building combined organoids on this device, and constructing disease models, diseases related to axon growth, projection and synapse formation can be studied. At the same time, unlike the fusion-type combined organoids, where the neuronal axons are inside the organoids, the combined organoids constructed using this device are separated, and the axons grow in the pipes connecting the organoids. Therefore, it is very convenient to study the regeneration and myelination of neuronal axons and explore the conditions for the regeneration and myelination of axons of various types of human neurons. In summary, the combined organoids constructed using this device can not only conduct basic research on the growth, projection and synapse formation of various types of human neuronal axons, but also provide a research model for human neurological diseases and neuronal axon regeneration, and have potential clinical application value.

[0052] In the following description, many technical details are provided to help readers better understand the present application. However, those skilled in the art can understand that the technical solution claimed in the present application can be implemented even without these technical details and various changes and modifications based on the following embodiments.

[0053] the term

[0054] As used herein, "separate combined organoid culture device", "combined organoid culture device", "culture device", and "device" are used interchangeably and all refer to the culture device as described in the present application.

[0055] The following is a brief description of some innovative aspects of the implementation methods of this application:

[0056] The culture device of the present application is used to induce 3D directional growth of neuronal axons in organoids and form projections with another organoid, thereby constructing separate combined organoids. The organoids connected by axons can be homologous or heterologous, and heterologous organs can be cultured in their most suitable culture medium.

[0057] The culture device includes two parts: the device body and the baffle. The device body is provided with a plurality of independent, open chambers, which are used to accommodate organoids. Connected pipes are provided between the chambers. These pipes are combined with the physical spacers and the application of Matrigel to realize the 3D directional induced growth of neuronal axons in organoids. The pipes between different chambers can form a one-to-one connection, and / or a one-to-many connection, and / or a many-to-many connection. The baffle is used to separate the chambers on both sides, block the connection of the culture medium on both sides of the culture vessel, and realize the differentiated culture of the organoids on both sides.

[0058] by Figure 2 Taking as an example, an embodiment of the culture device of the present application is described: the culture device comprises two chambers, a pipe connecting the two chambers, and a baffle set up in the middle of the two chambers. Different types of organoids are placed in the chambers on both sides for separate culture, and Matrigel is injected into the middle pipe connecting the two chambers as a matrix for inducing axon growth, and the culture medium on both sides is separated by a baffle. The organoid grows axons in the chamber on one side and grows in a 3D form in the pipe toward the chamber on the other side, and finally forms a projection with the organoid on the other side. In the embodiments of the present application, retinal organoids and thalamic organoids are used to demonstrate the device. Any different organoids connected by long-range axons can be used for combined organoid culture using this device, and it can be expanded to more than 3 types of organoids for combined culture.

[0059] The separated combined organoid culture device of the present application includes a device body and a baffle. The device body includes a groove-shaped culture unit. In a favorable embodiment, there are multiple culture units on the device body. In the direction in which the culture unit extends, a first chamber and a second chamber are provided. The first chamber and the second chamber are in a concave spherical shape and have a closed shape at the opening. In an optional embodiment, the diameter of the first chamber is 1.5 mm, and the diameter of the second chamber is 1.8 mm.

[0060] The middle pipe between the first chamber and the second chamber connects the two chambers. The middle pipe is used for the 3D directional growth of the axons of the organoid. There are pipe segments on the side of the first chamber away from the middle pipe (i.e., the left side of the first chamber) and the side of the second chamber away from the middle pipe (i.e., the right side of the second chamber). On the one hand, the pipe segments on both sides provide support for the steel ball position, and on the other hand, after the steel balls are removed, the culture medium enters the pipe to provide nutrition and oxygen supply to the organoid. The dimensional parameters such as the length and cross-sectional diameter of the pipe are designed differently according to the cultured combined organoids. In an optional embodiment, the diameter of the pipe on the left side of the first chamber is 0.7mm, the diameter of the middle pipe is 1.0mm, and the diameter of the pipe on the right side of the second chamber is 1.0mm. In the embodiment of the present application, it is preferably designed to adapt to 3.5cm culture dishes, and in theory it can be designed to adapt to any container.

[0061] The baffle is separated from the device body. When in use, the baffle can be mounted on the central axis of the culture dish, and the two ends of the baffle have shapes that are mutually nested and matched with the device body. The baffle is configured to block fluid communication between different culture media.

[0062] The combined organoids are cultured with the culture device of the present application, and the types of the first organoid and the second organoid can be the same or different. The organoid can be a combination of any human-derived organoid that can form long-range projection neurons and other types of organoids. The types of organoids that can be cultured in the embodiments include, but are not limited to: various types of neurons, neural tissue organoids, such as retinal organoids, brain organoids, spinal cord organoids, etc.; they can also be cell or tissue organoids that interact with the nervous system, such as glial cells, muscle organoids, heart, pancreas, small intestine and other visceral organoids, tumor organoids, etc.

[0063] Mold design of culture device

[0064] Figure 1 The figure shows the modeling diagrams (Figures A, B, and C) using SOLIDWORKS and the physical diagrams (Figures D, E, and F) obtained by 3D printing. Figures A and B show the top view and bottom view of the mold used for forming the device body, respectively. Figure C is a 3D printed baffle. In an optional embodiment, the material of the mold used for forming the device body is selected from resin; in an optional embodiment, the material of the baffle is PC.

[0065] Fabrication of culture device

[0066] After the mold is prepared, the method for preparing the culture device of the present application using the mold is as follows: Figure 2 As shown, the following steps are included:

[0067] 1. Dissolve low melting point agarose in DPBS under heating conditions to prepare a 3% agarose solution and filter it with a 0.22um filter membrane for sterilization. Agarose not only has good biocompatibility, but also has a soft texture, which is convenient for later cutting and removing organoids.

[0068] 2. Pour 1.7 ml of 3% agarose solution into a 3.5 cm culture dish and press the mold into the agarose (such as Figure 2 (as shown in A).

[0069] 3. After the agarose solution is completely cooled, remove the mold and make the required chambers and channels in the agarose (such as Figure 2 (as shown in B). Figure 2 View B and Figure 2 View C shows the back of the culture device and the front of the chamber and tube under the stereomicroscope. To prevent the fillings in the tube and the organoids in the chamber from shaking in the culture medium and keep them fixed, the tube and chamber are designed with closed ends ( Figure 2 View D and 2 View E).

[0070] In order to better understand the technical solution of the present application, a specific embodiment is provided below for explanation. The details listed in this example are mainly for ease of understanding and are not intended to limit the scope of protection of the present application.

[0071] Example 1: Implantation and culture of the first organoid

[0072] 1. Insert a sterile steel ball into the channel on the left side of the first chamber. The purpose of placing the steel ball is to prevent Matrigel from flowing out of the channel on this side and to prevent the left side of the organoid from being wrapped by Matrigel ( Figure 3 At the same time, a sterile steel ball is placed at the right end of the middle channel between the two chambers, close to the second chamber, to prevent Matrigel from flowing out of the middle channel into the second chamber ( Figure 3 View A in .

[0073] 2. Use a Pasteur tube to suck up the organoid (here, retinal organoids are used as an example) and place it in the first chamber. Completely drain the culture medium in the chamber and the tube. At this time, the left side of the retinal organoid is completely attached to the steel ball in the left tube ( Figure 3 View in B, indicated by white arrow).

[0074] 3. Inject Matrigel into the first chamber where the organoid is located and into the middle channel on the right side. Figure 3 C), and place the culture device body in a cell culture incubator for 15 minutes to allow Matrigel to solidify ( Figure 3 View D).

[0075] 4. Remove the steel balls on both sides ( Figure 3 E) and culture medium was added for cultivation.

[0076] Example 2: 3D directional growth of organoid axons

[0077] After a period of culturing of the retinal organoid sample obtained by the method of Example 1, it can be seen that the axons of the retinal organoid in the first chamber are in the middle channel filled with Matrigel (such as Figure 4 View A) (other growth factors and other substances can be added) and reach the right end of the tube on the 20th day, with a length of 5.8 mm (such as Figure 4 (as shown in view B).

[0078] Example 3: Implantation of a Second Organoid

[0079] When the axons of the organoids in the first chamber grow to a certain length, the second organoid is implanted. A steel ball ( Figure 5 The purpose of this is to prevent Matrigel from flowing out of the chamber.

[0080] Use the Pasteur tube to suck up the second organoid (here, the thalamus organoid is used as an example) and implant it into the second chamber ( Figure 5 After aspirating the medium ( Figure 5 C), the chamber is filled with Matrigel ( Figure 5 Place the culture device in the incubator for 15 minutes to allow the Matrigel to solidify. Remove the steel ball from the right channel of the second chamber ( Figure 5 View E).

[0081] Example 4: Baffle Installation and Projection Formation

[0082] Place the PC baffle in the middle of the two chambers, and seal the contact surface between the baffle and Matrigel with 3% agarose solution ( Figure 6 A, the baffle is shown by the white arrow, and 3% agarose is sealed on both sides of the baffle, as shown by the orange arrow). The purpose of the baffle is to isolate different types of culture medium liquid.

[0083] Add 1 ml of retinal organoid culture medium to the left side of the baffle and 1 ml of thalamic organoid culture medium to the right side of the baffle for subsequent culture ( Figure 6 View B). After a while, axons from the retinal organoids (expressing EGFP) grew across the tubes into the thalamic organoids ( Figure 6 View C), dense retinal organoid-derived axons are visible in the thalamic organoid ( Figure 7 Figure D). Immunostaining was used to detect the expression of presynaptic protein Synaptophysin (SYP) and postsynaptic protein postsynaptic density protein 95 (PSD95) in the thalamus. Figure 7 View E and Figure 7 View F shows the expression of presynaptic protein SYP in thalamic organoids and the expression of postsynaptic protein PSD95 in thalamic organoids. Most of the SYP and PSD95 signals co-localize, and in some areas, SYP, PSD95 and EGFP signals triple co-localize ( Figure 7 (View G, white arrows indicate the signal triplet overlap region).

[0084] The results showed that axons derived from retinal organoids grew into thalamic organoids and formed synapses to complete the projection.

[0085] Example 5: Constructing a human optic nerve regeneration model using the culture device

[0086] As mentioned above, the 3D directional growth of neuronal axons is exposed in the tube, so it is very convenient to cut the axons and directly observe the axon regeneration. The following demonstration uses retinal organoids as an example to build a human optic nerve regeneration model.

[0087] The retinal organoids were planted in the first chamber and filled with Matrigel in the middle channel. After a period of time, the channel was filled with axons growing to the right, similar to the human optic nerve. The retinal organoid axons grew in the channel ( Figure 8 View A). The axon is severed using a special blade ( Figure 8 View B, white arrows indicate the site of severance), and new axons can be seen a few days later ( Figure 8 View C, newly formed axons, white arrows indicate the cut site, blue arrows indicate newly formed axons).

[0088] Experiments have shown that this device can be used to conveniently build various neuron axon regeneration models.

[0089] It should be noted that in the application documents of this patent, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including one" do not exclude the existence of other identical elements in the process, method, article or device including the elements. In the application documents of this patent, if it is mentioned that an action is performed according to an element, it means that the action is performed at least according to the element, which includes two situations: performing the action only according to the element, and performing the action according to the element and other elements. Expressions such as multiple, multiple, and multiple include 2, 2 times, 2 kinds, and more than 2, more than 2 times, and more than 2 kinds.

[0090] This specification includes combinations of the various embodiments described herein. Individual references to "one embodiment" or a particular embodiment, etc., do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless indicated as mutually exclusive or clear to those skilled in the art. It should be noted that the word "or" is used in this specification in a non-exclusive sense unless the context clearly indicates or requires otherwise.

[0091] All documents mentioned in this application are considered to be included in the disclosure of this application as a whole, so that they can be used as the basis for modification when necessary. In addition, it should be understood that after reading the above disclosure of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A culture device for preparing separate combined organoids, characterized in that: The culture device comprises: at least one combined organoid culture unit and its corresponding culture chamber; Wherein, the combined organoid culture unit comprises at least a first cell culture chamber, a second cell culture chamber, and one or more directional channels for connecting the cell culture chambers in the combined organoid culture unit; Wherein, the first cell culture chamber is configured to culture a first cell, and the first cell is a human or animal cell; The second cell culture chamber is configured to culture second cells, wherein the second cells are human or animal cells; The directional channel is filled with a directional growth induction culture medium, thereby allowing the first cell and / or the second cell to produce directional axon induced growth in the directional growth induction culture medium; The first cell culture chamber is located in the first culture chamber, and the second cell culture chamber is located in the second culture chamber; the first culture chamber is configured to contain a first culture medium suitable for the growth of first cells, and the second culture chamber is configured to contain a second culture medium suitable for the growth of second cells; and the first culture medium and the second culture medium are configured not to mix with each other.

2. The culture device according to claim 1, characterized in that The first cell and the second cell are the same or different.

3. The culture device according to claim 1 or 2, characterized in that: The first cell and the second cell are different.

4. The culture device according to claim 1, characterized in that: The first cell is selected from any one of the following: Neurons, brain organoids, retinal organoids, spinal cord organoids; or Glial cells, muscle organoids, heart, pancreas, small intestine and other visceral organoids, tumor organoids.

5. The culture device according to claim 1, characterized in that: The second cell is selected from the group consisting of neurons, brain organoids, retinal organoids, and spinal cord organoids; or Glial cells, muscle organoids, cardiac organoids, pancreatic organoids, intestinal organoids, tumor organoids.

6. The culture device according to claim 1, characterized in that: The directional induced growth culture medium is an axon directional induced growth culture medium.

7. The culture device according to claim 1 or 6, characterized in that: The directional induction growth culture medium is Matrigel.

8. The culture device according to claim 1, characterized in that: The cell origin is any one of the following: mammals, rodents, non-human primates, and humans.

9. The culture device according to claim 1, characterized in that: After culturing, a first cell culture formed by the first cells and a second cell culture formed by the second cells form a combined state in the directional channel, thereby forming a separated combined organoid; In another preferred embodiment, the first cells form a first cell culture, and / or the second cells form a second cell culture; wherein, The first cell culture and / or the second cell culture is an organoid.

10. A method for culturing separated combined organoids, characterized in that: The method is configured to construct a combined organoid in a separate method; the culture method comprises: - Using a culture device, by occupying the channels of the culture device and applying Matrigel, the directional growth of axons in three-dimensional space is achieved.

11. The culture method according to claim 10, characterized in that: The method specifically comprises the following steps: S1: Place isolated homologous or heterologous organoids in different chambers on both sides; S2: The organoids in different chambers on both sides are connected by axons to form combined organoids.

12. The culture method according to claim 11, characterized in that The method also The following steps are involved: S3: When the organoids in the two side chambers need to be grown in different culture media, a baffle is set in the middle of the two side chambers to separate the two side chambers, so that the heterogeneous organoids can grow in their respective most suitable culture media.

13. The culture method according to claim 10, characterized in that: The culture device comprises a plurality of chambers, and the chambers are connected by pipelines.

14. The culture method according to claim 10, characterized in that: The culture device includes a plurality of culture areas, and the culture areas are communicatively connected to each other.

15. A method for culturing separated combined organoids, characterized in that: The culture method cultures neural cells in a plurality of culture regions, and the neural cells in the culture regions perform neural projection between the plurality of culture regions.

16. The culture method according to claim 15, characterized in that: The plurality of culture areas are configured with corresponding culture media according to the nerve cells in the culture areas, and the culture media are not mixed with each other.

17. The culture method according to claim 15, characterized in that: A directional channel is arranged between the culture areas, and a directional induction growth culture medium is arranged in the directional channel. The nerve cells produce directional axon induced growth through the directional induction growth culture medium, thereby forming nerve projection.

18. The culture method according to claim 17, characterized in that: The directed induction growth culture medium is Matrigel.

19. A separated composite organoid, characterized in that: The separated combined organ includes a plurality of nerve aggregation regions, and projections are formed between the plurality of nerve aggregation regions through the directional growth of axons.

20. The split combined organ according to claim 19, characterized in that: The separated combined organoids are human retinal organoids and the human retinal organoids form projections through three-dimensional oriented growth of long-range axons and thalamic organoids.

21. An axon regeneration model, characterized in that: The method is constructed using the culture device according to any one of claims 1 to 9.

22. An axon myelination model, characterized in that: The method is constructed using the culture device according to any one of claims 1 to 9.

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