Method for producing three-dimensional neural cell aggregate and cell aggregate produced thereby

Abstract

The present invention relates to a method for producing a three-dimensional neuronal cell cluster, and a three-dimensional neuronal cell cluster produced thereby, wherein stem cells are differentiated into neuroepithelial cells, separated into individual cells, and then cultured in a three-dimensional re-aggregation mode by limiting the initial number of cells to a specific count of cells. According to the production method of the present invention, central necrosis is suppressed even during cultivation, and neural cell characteristics are expressed, whereby the method can be highly useful in various applications such as clinical and research fields and enables the production of micro-sized neural cell aggregates suitable for transplantation and injectable applications.

Description

Method for manufacturing three-dimensional neural cell aggregates and cell aggregates manufactured thereby

[0001] The present invention relates to a method for producing a three-dimensional neural cell aggregate manufactured in an injectable micro-size, in which central necrosis is suppressed, and to a cell aggregate manufactured thereby.

[0002] Organoids are a novel stem cell differentiation technique that utilizes the differentiation, self-renewal, and self-organization capacities of stem cells to recreate the cell composition and structure of internal organs through three-dimensional culture. By simulating an environment similar to internal organs, organoids can be utilized for research on simulating various diseases and screening therapeutic drugs. Furthermore, through the production of patient-specific organoids, they are being evaluated as a future technology that can produce personalized treatments for various diseases at low cost and in a short period of time.

[0003] However, core necrosis has been reported to frequently occur in 3D-cultured neural cell organoids. This is primarily due to the insufficient supply of oxygen, nutrients, and growth factors as the organoids grow in size. Deficiency of these essential elements induces cell necrosis in the organoid core, compromising the reliability of experimental results and preventing the organoids from replicating the normal physiological environment.

[0004] Therefore, developing technologies to suppress or prevent central necrosis is a critical challenge in organoid research. Various attempts are being made to achieve this goal, including limiting organoid size to prevent necrosis and utilizing microfluidics technology to effectively supply oxygen and nutrients. Furthermore, with the advancement of cell culture technologies utilizing scaffolds or hydrogels, methods are being proposed to minimize necrosis by improving the physical environment while maintaining cell-to-cell interactions.

[0005] The technical problem to be achieved by the present invention is to provide a method for producing a three-dimensional neural cell aggregate, characterized by differentiating stem cells into neuroepithelial cells, separating them into individual cells, and then limiting the initial cell number to a specific cell number and performing three-dimensional reaggregation culture.

[0006] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0007] In order to solve the above problem, the present inventors have developed a method comprising: 1) treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells;

[0008] 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and

[0009] 3) Step of culturing neuroepithelial cells in three dimensions;

[0010] A method for manufacturing a three-dimensional neural cell aggregate, characterized by including:

[0011] The neuroepithelial cells differentiated in step 2 above may be separated into single cells by treating them with accutase and then arranged in units of 6 to 75.

[0012] According to one aspect, the stem cell may be any one of a human embryonic stem cell (hESC), an induced pluripotent stem cell, and an adult stem cell.

[0013] According to one side, the neurodifferentiation promoting drug of step 1) may be at least one selected from the group consisting of SB431542, CHIR99021, Dorsomorphin, A-83-01, RepSox, BIO (6-bromoindirubin-3'-oxime) and LiCl (Lithium Chloride).

[0014] According to one aspect, the microwell plate may be a multiwell plate having a concave portion.

[0015] According to one aspect, the above manufacturing method may enable long-term continuous culture in the same microwell plate after step 2).

[0016] According to another embodiment of the present invention, a three-dimensional neural cell aggregate manufactured by the above manufacturing method is provided.

[0017] According to one side, the diameter of individual neurons of the three-dimensional neural cell aggregate may be 150 to 300 um, which corresponds to a size suitable for injection or transplantation.

[0018] According to one side, the three-dimensional neural cell aggregate may have a suppressed necrotic core.

[0019] According to one side, the three-dimensional neural cell aggregates may not express c-Casp3.

[0020] According to another embodiment of the present invention,

[0021] 1) A step of treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells;

[0022] 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and

[0023] 3) Step of culturing neuroepithelial cells in three dimensions;

[0024] A cell aggregate manufactured by a manufacturing method including preparing a cell aggregate,

[0025] A method for screening a neurological disease drug is provided, further comprising a step of contacting a candidate substance with the above cell aggregate.

[0026] According to one side, the above neurological disease may be one or more of Parkinson's disease, Alzheimer's disease, stroke, cerebral palsy, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, autism, and brain development disorder syndrome.

[0027] According to the manufacturing method of the present invention, when stem cells are differentiated into neuroepithelial cells, then separated into individual cells and cultured at a specific initial cell count range to form a three-dimensional cell aggregate, central necrosis is suppressed and neural cell characteristics are expressed even during long-term culture. Furthermore, by manufacturing as described above, injectable, micro-sized neural cell aggregates suitable for transplantation can be obtained.

[0028] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0029] Figure 1 shows a method for producing microorganoids.

[0030] Figure 2 shows the measurements of the size of neural organoids.

[0031] Figure 3 compares the differentiation markers of organoids with an initial cell count of 75 and organoids with an initial cell count of 5,000 according to the present invention.

[0032] Figure 4A shows organoids transplanted into the spinal cord of a nude rat, and Figure 4B shows the transplanted organoids differentiating into mature neural cells.

[0033] Figure 5 shows the connectivity of host blood vessels after micro-organoid transplantation.

[0034] The present inventors confirmed that when neuroepithelial cells derived from stem cells are separated into single cells and then cultured in three dimensions in a microwell plate by limiting a specific initial cell number range, central necrosis is suppressed and neuronal characteristics are expressed even during long-term culture, and that relatively small-sized neuronal cell aggregates that are injectable and suitable for transplantation are formed, thereby completing the invention of the present manufacturing method.

[0035] In order to solve the above problem, the present inventors have developed a method comprising: 1) treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells;

[0036] 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and

[0037] 3) Step of culturing neuroepithelial cells in three dimensions;

[0038] A method for manufacturing a three-dimensional neural cell aggregate, characterized by including:

[0039] The neuroepithelial cells differentiated in step 2) above can be separated into single cells by treating them with accutase and then arranged in groups of 6 to 75 cells. Culturing in this manner allows for precise control of the initial cell number in organoid culture and enables three-dimensional reaggregation culture.

[0040] The term "stem cell" as used herein refers to a cell that has the ability to self-replicate and differentiate into two or more cells, and can be classified into totipotent stem cells, pluripotent stem cells, multipotent stem cells, and induced pluripotent stem cells. Preferably, it may be an induced pluripotent stem cell.

[0041] In the present invention, the three-dimensional cell aggregate may be an organoid, for example, a neural cell organoid.

[0042] The term "organoid" as used herein refers to a cell aggregate created by re-aggregating or recombining cells isolated from stem cells or organ-derived cells using a 3D culture method, and may include organoids or cell clusters formed from suspension cell cultures. The organoids may also be referred to as miniature organoids, organ analogs, or pseudo-organs. Specifically, the organoids comprise one or more cell types among the various cell types that constitute an organ or tissue, and can reproduce the structure and function of the tissue or organ.

[0043] According to one side, the neurodifferentiation promoting drug of step 1) may be at least one selected from the group consisting of SB431542, CHIR99021, Dorsomorphin, A-83-01, RepSox, BIO (6-bromoindirubin-3'-oxime) and LiCl (Lithium Chloride).

[0044] According to one aspect, the microwell plate may be a multiwell plate having a concave portion.

[0045] According to one aspect, the above manufacturing method may enable long-term continuous culture in the same microwell plate after step 2).

[0046] In the present invention, after forming a three-dimensional cell aggregate, additional shaking or non-shaking culture may be performed. Long-term culture may be possible through the additional culture.

[0047] According to another embodiment of the present invention, a three-dimensional neural cell aggregate manufactured by the above manufacturing method is provided.

[0048] According to one side, the diameter of individual neurons of the three-dimensional neural cell aggregate may be 150 to 300 um.

[0049] According to one side, the three-dimensional neural cell aggregates may have suppressed necrotic cores. This can be confirmed by the absence of expression of c-Casp3, a related marker, as confirmed in the examples below.

[0050] According to another embodiment of the present invention,

[0051] 1) A step of treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells;

[0052] 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and

[0053] 3) Step of culturing neuroepithelial cells in three dimensions;

[0054] A cell aggregate manufactured by a manufacturing method including preparing a cell aggregate,

[0055] A method for screening a neurological disease drug is provided, further comprising a step of contacting a candidate substance with the above cell aggregate.

[0056] According to one side, the above neurological disease may be one or more of Parkinson's disease, Alzheimer's disease, stroke, cerebral palsy, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, autism, and brain development disorder syndrome.

[0057]

[0058] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0059] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0060] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0061] Example 1. Production of neural organoids

[0062] To generate neural organoids, human pluripotent stem cells (hPSCs) were treated daily with 10 mM SB431542 and 3 mM CHIR99021 for 3 days to induce neuroepithelial cells. The induced neuroepithelial cells were dissociated into single cells by treating them with Accutase. A small number (8–75) of these cells were then seeded in 96-well low-attachment culture dishes containing growth factor-supplemented media, where they were allowed to re-aggregate into three-dimensional structures.

[0063] The protocol for producing neural organoids, which involves inducing neuroepithelial cells by treating SB / CHIR drugs for 3 days in 2D, then isolating them into single cells and re-aggregating a small amount of neuroepithelial cells in 3D for culture, is shown in Fig. 1. The size of the neural organoids was measured and shown in Fig. 2. A typical neural organoid produced by injecting 5,000 neuroepithelial cells reaches a size where necrosis occurs in 13 days, but a micro-organoid produced by injecting 75 neuroepithelial cells did not reach a size where necrosis occurs even after culture for a month, indicating that micro-organoids can be cultured as organoids that do not form necrosis.

[0064]

[0065] Example 2. Confirmation of marker expression

[0066] Microorganoids were cultured for one month and compared with existing organoids through immunostaining, and the results are shown in Figure 3. The organoids created by seeding 75 cells did not show c-Casp3 (apoptotic marker) inside the organoids, indicating that necrosis (necrotic core) was not formed, and SOX2 (neuronal cell marker) and NF-M (neuronal fiber marker) confirmed that they were well differentiated like larger organoids.

[0067]

[0068] Example 3. Transplantation experiment

[0069] After transplanting microorganoids into the normal spinal cord and damaged spinal cord of nude rats with suppressed immune function, the presence of organoids was confirmed through HuNu (human nuclear marker), as shown in Fig. 4A. Through this result, it was confirmed that the organoids were stably established, and through the expression of SOX2 (neuronal cell marker), it was confirmed that some of the transplanted organoids remained in the neural stem cell stage.

[0070] To confirm the differentiation of transplanted organoid-derived neural stem cells into mature neural cells, immunohistochemical staining was performed with the neural cell marker MAP-2 antibody, as shown in Figure 4B, and MAP-2-positive organoid cells were identified. In particular, an increase in the proportion of MAP-2-positive cells was confirmed in the damaged spinal cord, confirming that organoids can smoothly differentiate into neural cells within the damaged spinal cord.

[0071] Connectivity with the host is crucial for the growth and differentiation of transplanted microorganoids. The presence of host blood vessels within the organoids was confirmed through Reca1 (a vascular marker) immunostaining, as shown in Figure 5. This suggests that sufficient oxygen and nutrients are supplied to the organoids, enabling the microorganoids to grow stably within the host.

[0072]

[0073] Example 4. Drug screening experiment

[0074] To determine whether micro-organoids can be applied to drug screening, micro-organoids were exposed to hypoxic and hypoglycemic (HXHG) conditions, and then a total of four drugs were screened using Propidium iodide / Acridine Orange staining. The results are shown in Fig. 6. It was confirmed that two drugs, ISRIB and Rapa (Rapamycin), were effective. This confirmed that the micro-organoids of the present invention can also be utilized in a drug screening system.

[0075]

[0076] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0077] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

Claims

1. 1) A step of treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells; 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and 3) Step of culturing neuroepithelial cells in three dimensions; A method for producing a three-dimensional neural cell aggregate, characterized by including:

2. A method for producing a three-dimensional neural cell aggregate according to claim 1, wherein the stem cell is any one of a human embryonic stem cell (hESC), an induced pluripotent stem cell, and an adult stem cell.

3. A method for producing a three-dimensional neural cell aggregate in the first paragraph, wherein the neural differentiation promoting drug of step 1) is at least one selected from the group consisting of SB431542, CHIR99021, Dorsomorphin, A-83-01, RepSox, BIO (6-bromoindirubin-3'-oxime), and LiCl (Lithium Chloride).

4. A method for producing a three-dimensional neural cell aggregate, wherein the microwell plate in claim 1 is a multi-well plate having a concave portion.

5. In the first paragraph, the manufacturing method is a method for manufacturing a three-dimensional neural cell aggregate, which can be continuously cultured for a long period of time in the same microwell plate after step 2).

6. A three-dimensional neural cell aggregate manufactured by the manufacturing method of clauses 1 to 5.

7. A cell aggregate in accordance with claim 6, wherein the diameter of individual nerve cells of the three-dimensional nerve cell aggregate is 150 to 300 um.

8. In the 6th paragraph, the three-dimensional neural cell aggregate is a cell aggregate in which the necrotic core is suppressed.

9. A cell aggregate according to claim 6, wherein the three-dimensional neural cell aggregate does not express c-Casp3. 10.1) A step of treating stem cells with a drug that promotes neural differentiation to differentiate them into neuroepithelial cells; 2) A step of arranging differentiated neuroepithelial cells in units of 6 to 75 on a microwell plate; and 3) Step of culturing neuroepithelial cells in three dimensions; A cell aggregate manufactured by a manufacturing method including preparing a cell aggregate, A method for screening a neurological disease drug, further comprising the step of contacting a candidate substance with the above cell aggregate.

11. A screening method according to claim 10, wherein the neurological disease is at least one of Parkinson's disease, Alzheimer's disease, stroke, apoplexy, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, autism, and brain development disorder syndrome.

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