Component for culture medium

Abstract

Provided is a culture medium for culturing cells containing specific components. Provided is a culture medium comprising at least one component selected from a group consisting of (1) halcinonides, (2) ALK4 / 7 inhibitor or TGFβ receptor type 1 kinase inhibitor, (3) BRD7 / 9 dual degradation factor or BRD7 / 9 binding molecule (degradation factor), (4) LATS1 / 2 inhibitor, PKA inhibitor, or AKT1 inhibitor, (5) CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor, or PKC inhibitor or adenosine kinase inhibitor, (6) E-selectin inhibitor, VCAM1 inhibitor, or ICAM1 inhibitor, and (7) LATS1 inhibitor + LATS2 inhibitor.

Description

Culture medium components

[0001] This disclosure relates to a culture medium for cells containing specific components (particularly various stem cells such as mesenchymal stem cells (MSCs)). This disclosure particularly concerns the composition and use of a culture medium suitable for the proliferation and maintenance of various stem cells, such as mesenchymal stem cells.

[0002] Cell culture typically requires a culture medium containing specific components. These components are essential for promoting cell proliferation, maintenance, and the expression of specific functions. However, conventional culture media may lack certain components necessary for optimizing cell proliferation. This disclosure provides an improved culture medium to address these issues.

[0003] In recent years, research into the development of pharmaceuticals and regenerative medicine utilizing living cells or tissues has been progressing and attracting attention. In particular, research into organ regeneration technologies using pluripotent stem cells such as ES cells and iPS cells is accelerating. On the other hand, cell therapy using somatic stem cells such as bone marrow stem cells utilizes the inherent function of somatic stem cells to repair tissue damaged by disease, and is attracting attention as a more feasible form of regenerative medicine, with research progressing accordingly.

[0004] Various stem cells, such as mesenchymal stem cells (hereinafter also referred to as MSCs), are pluripotent somatic stem cells that can usually be isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood, and have the ability to differentiate into many different types of cells.

[0005] As a result of various investigations, the Disclosing Party has found that various stem cells, particularly mesenchymal stem cells, can be obtained by treating cells, especially various types of stem cells, with at least one specific component, and has completed this disclosure.

[0006] In other words, this disclosure relates to at least the following:

[0007] <Form 1> [Item 1-1] (1) The following hallucinonides

[0008]

[0009] (Here, R １ and R ２is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), or R １ and R ２ together form a ring, R ３ is halo or hydrogen, R ４ is halo (preferably chloro) or optionally substituted hydroxy), or a pharmaceutically acceptable salt or solvate thereof, a medium. [Item 1-2] (1') R １ and R ２ are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), the medium according to the above item. [Item 1-3] (1") R １ and R ２ together form a ring, the medium according to any one of the above items. [Item 1-4] (1A)

[0010] or or a pharmaceutically acceptable salt or solvate thereof, the medium according to any one of the above items.

[0011] [Item 1-5] (1B) A culture medium according to any one of the above items, comprising halcinonide, clobetasol, fluocinonide, or desoside (preferably halcinonide or clobetasol) or a pharmaceutically acceptable salt or solvate thereof. [Item 1-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 1-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 1-6B] A serum-free culture medium according to any one of the above items. [Item 1-6C] A culture medium according to any one of the above items, free from heterologous raw materials. [Item 1-7] A method for producing cells, comprising the step of culturing cells using a culture medium according to any one of the above items. [Item 1-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 1-8A] The cell count range mentioned above is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A moderate amount is sufficient, and more preferably, 500-1000 / cm ２ ~10,000 / cm ２ For example, 5000 cells / cm² ２The method according to any one of the above items, although often seeded before or after. [Item 1-8B] The method according to any one of the above items, wherein the range of the cell number is a specific value such as 1500 cells / well during screening, and / or is carried out under conditions such as 4500 cells / well which are estimated to be confluent. [Item 1-8C] The method according to any one of the above items, wherein the specific container is a container of a suitable material (including with or without coating). [Item 1-8D] The method according to any one of the above items, wherein the specific temperature is a suitable temperature (25-37°C, or 30°C, etc.). [Item 1-9] A cell culture apparatus comprising a culture medium according to any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 1-10] A method for preserving cells, comprising the step of preserving cells using the culture medium according to any one of the above items.

[0012] [Item 1-11] A composition for preserving cells, comprising the component described in any one of the above items. [Item 1-12] A cell-containing composition comprising the component described in any one of the above items and cells. [Item 1-13] Cells cultured using the component described in any one of the above items. [Item 1-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced. [Item 1-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 1-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. [Item 1-13D] Cells or cell populations according to any one of the above items, wherein the expression of CD44 usually does not decrease, but the others often decrease, for example, it is a desirable and advantageous feature that the expression of CD73 and CD90, or CD44, CD90 and CD105, does not decrease. [Item 1-13E] Cells or cell populations according to any one of the above items, wherein the expression of CD90 is maintained at a high level. [Item 1-13E] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 1-13F] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 8 or more. [Item 1-13G] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 1-13H] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 8 or more.[Item 1-13I] The cells or cell population described in any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 1-13J] The cells or cell population described in any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage. [Item 1-13AA] The cells described in any one of the above items, wherein the properties of the cells are improved (for example, enhanced cartilage differentiation ability). [Item 1-13AB] A cell population containing about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or more of the cells described in any one of the above items.

[0013] [Item 1-14] A pharmaceutical product comprising the cells described in any one of the above items. [Item 1-15] A method for differentiating cells, comprising the step of culturing the cells obtained using the medium described in any one of the above items under conditions that induce differentiation. [Item 1-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation ability. [Item 1-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 1-15C] The method according to any one of the above items, wherein the cells are not likely to become cartilage and / or have high cartilage differentiation ability. [Item 1-16] Differentiated cells obtained by differentiating stem cells by the method according to any one of the above items. [Item 1-17] A method for treating or preventing a subject, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmaceutical product described in any one of the above items to a subject that requires it. [Item 1-18] A method for quality control of stem cells, comprising determining whether the cell has a coefficient C that correlates with the culture medium described in any one of the above items. [Item 1-18A] The method according to any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to cell markers. [Item 1-18B] The method according to any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 1-18C] The method according to any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that are expressed over a long period of time. [Item 1-18D] The method according to any one of the above items, wherein the marker, in the case of culture medium, includes information relating to the identification of the component itself. [Item 1-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a culture medium supply section for providing the culture medium or its raw materials as described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting culture conditions as necessary.

[0014] [Item 1-20] A culture medium suitable for transporting cells containing the culture medium components described in any one of the above items. [Item 1-21] A method for managing cell culture for cells suitable for culturing using the culture medium described in any one of the above items, comprising: 1) providing information about the culture medium with respect to cells provided by the user; 2) assigning identifiable labels to the cells and the culture medium; and 3) managing whether the cells and the culture medium are compatible by referring to the labels.

[0015] [Item 1-22] Use of hallucinonides as described in Item 1-1, or pharmaceutically acceptable salts or solvates thereof, for culturing cells (preferably mesenchymal stem cells). [Item 1-23] Use of cells (preferably mesenchymal stem cells) as described in Item 1-22, comprising the features of any one of Items 1-2 to 1-6C. [Item 1-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, at least one expression level selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 1-1. [Item 1-25] The method according to Item 1-24, comprising the features of any one of Items 1-2 to 1-6C. [Item 1-26] Cells or cell populations as described in any one of Items 1-13 to 1-13AB, or differentiated cells or cell populations as described in Item 1-16, for use in therapeutic purposes. [Item 1-27] Cells or cell populations described in any one of items 1-13 to 1-13AB, or differentiated cells or cell populations described in item 1-16, for use in regenerative medicine or the treatment of cartilage diseases. [Item 1-28] Use of cells or cell populations described in any one of items 1-13 to 1-13AB, or differentiated cells or cell populations described in item 1-16, in the manufacture of a drug for regenerative medicine or the treatment of cartilage diseases. [Item 1-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 1-13 to 1-13AB, or differentiated cells or cell populations described in item 1-16, to the subject.

[0016] <Form 2> [Item 2-1] (2) A culture medium containing ALK4 / 7 inhibitor or TGFβ receptor type 1 kinase inhibitor. [Item 2-2] (2') The culture medium described in the above item, containing TGFβ receptor type 1 kinase inhibitor. [Item 2-3] A culture medium according to any one of the above items, comprising (2") an ALK4 / 7 inhibitor. [Item 2-4] A culture medium according to any one of the above items, comprising (2A) a TGFβ receptor type 1 kinase inhibitor, which is also an ALK4 / 7 inhibitor. [Item 2-5] The culture medium according to any one of the above items, wherein (2) comprises (2B) AZ12601011 or a pharmaceutically acceptable salt or solvate thereof. [Item 2-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 2-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 2-6B] A serum-free culture medium according to any one of the above items. [Item 2-6C] A culture medium according to any one of the above items, which does not contain biological raw materials.

[0017] [Item 2-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 2-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 2-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A density of approximately 500 cells / cm² is acceptable, and more preferably 500 cells / cm². ２ ~10000 cells / cm ２ , or 1000 cells / cm ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２The method according to any one of the above items, although seeding is often performed before and after, but is not limited to these. [Item 2-8B] The method according to any one of the above items, wherein the range of the cell number is adopted at the time of screening, such as 1500 cells / well, and / or is carried out under conditions such as 4500 cells / well which are estimated to become confluent. [Item 2-8C] The method according to any one of the above items, wherein the specific container is a container of a suitable material (including with or without coating). [Item 2-8D] The method according to any one of the above items, wherein the specific temperature is a suitable temperature (25-37°C, or 30°C, etc.). [Item 2-9] A cell culture apparatus comprising a culture medium according to any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary.

[0018] [Item 2-10] A method for preserving cells, comprising the step of preserving the cells using a culture medium described in any one of the above items. [Item 2-11] A composition for preserving cells, comprising the component described in any one of the above items. [Item 2-12] A cell-containing composition comprising the component described in any one of the above items and cells. [Item 2-13] Cells cultured using the component described in any one of the above items. [Item 2-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced. [Item 2-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 2-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. [Item 2-13D] Cells or cell populations according to any one of the above items, wherein the expression of CD44 usually does not decrease, but the others often decrease, for example, it is a desirable and advantageous feature that the expression of CD73 and CD90, or CD44, CD90 and CD105, does not decrease. [Item 2-13E] Cells or cell populations according to any one of the above items, wherein the expression of CD90 is maintained at a high level. [Item 2-13E] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 2-13F] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 8 or more. [Item 2-13G] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 2-13H] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 8 or more.[Item 2-13I] The cells or cell population described in any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 2-13J] The cells or cell population described in any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage.

[0019] [Item 2-13AA] Cells according to any one of the above items, wherein the properties of the cells are improved (for example, the ability to differentiate into cartilage is enhanced). [Item 2-13AB] A cell population containing about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or more of the cells according to any one of the above items. [Item 2-14] A pharmaceutical product containing the cells according to any one of the above items. [Item 2-15] A method for differentiating cells, comprising the step of culturing cells obtained using the medium according to any one of the above items under conditions that induce differentiation. [Item 2-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation ability. [Item 2-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 2-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high cartilage differentiation ability.

[0020] [Item 2-16] Differentiated cells obtained by differentiating stem cells by the method described in any one of the above items. [Item 2-17] A method for treating or preventing a subject in need, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmacopoeia described in any one of the above items to the subject in need. [Item 2-18] A method for quality control of stem cells, comprising determining whether they have a coefficient C correlated with the culture medium described in any one of the above items. [Item 2-18A] The method described in any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 2-18B] The method described in any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 2-18C] The method described in any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that exhibit long-term expression. [Item 2-18D] The method described in any one of the above items, wherein the marker, in the case of culture medium, includes information relating to the identification of the component itself. [Item 2-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a medium supply section for providing the medium or raw materials described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting the culture conditions as necessary. [Item 2-20] A medium suitable for transporting cells containing the medium components described in any one of the above items. [Item 2-21] A method for managing cell culture for cells suitable for culturing using the medium described in any one of the above items, comprising: 1) providing information about the medium for cells provided by the user; 2) assigning identifiable labels to the cells and the medium; and 3) managing whether the cells and the medium are compatible by referring to the labels.

[0021] [Item 2-22] Use of an ALK4 / 7 inhibitor or a TGFβ receptor type 1 kinase inhibitor as described in (2) of Item 2-1 for culturing cells (preferably mesenchymal stem cells). [Item 2-23] Use of a cell (preferably mesenchymal stem cells) as described in Item 2-22, comprising the features of any one of Items 2-2 to 2-6C. [Item 2-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 2-1. [Item 2-25] The method according to Item 2-24, comprising the features of any one of Items 2-2 to 2-6C. [Item 2-26] Cells or cell populations described in any one of items 2-13 to 2-13AB, or differentiated cells or cell populations described in item 2-16, for use in treatment. [Item 2-27] Cells or cell populations described in any one of items 2-13 to 2-13AB, or differentiated cells or cell populations described in item 2-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 2-28] Use of cells or cell populations described in any one of items 2-13 to 2-13AB, or differentiated cells or cell populations described in item 2-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 2-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 2-13 to 2-13AB, or differentiated cells or cell populations described in item 2-16, to the subject.

[0022] <Form 3> [Item 3-1] (3) A culture medium containing a BRD7 / 9 dual degradation factor or a BRD7 / 9 binding molecule (degradation factor). [Item 3-2] (3') The culture medium described in the above item, containing a BRD7 / 9 binding molecule (degradation factor). [Item 3-3] A culture medium according to any one of the above items, comprising (3") a BRD7 / 9 double degradation factor. [Item 3-4] A culture medium according to any one of the above items, comprising (3A) a BRD7 / 9 binding molecule (degradation factor) which is also a BRD7 / 9 double degradation factor. [Item 3-5] The culture medium according to any one of the above items, wherein (3) comprises (3B) VZ185 or a pharmaceutically acceptable salt or solvate thereof. [Item 3-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 3-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 3-6B] A serum-free culture medium according to any one of the above items. [Item 3-6C] A culture medium according to any one of the above items, which does not contain biological raw materials.

[0023] [Item 3-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 3-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 3-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A certain level is sufficient, and more preferably, 500-1000 cells / cm². ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２The method according to any one of the above items, although seeding is often performed before and after, but is not limited to these. [Item 3-8B] The method according to any one of the above items, wherein the range of the cell number is adopted at the time of screening, such as 1500 cells / well, and / or carried out under conditions such as 4500 cells / well which are estimated to become confluent. [Item 3-8C] The method according to any one of the above items, wherein the specific container is a container of a suitable material (including with or without coating). [Item 3-8D] The method according to any one of the above items, wherein the specific temperature is a suitable temperature (such as 25-37°C or 30°C). [Item 3-9] A cell culture apparatus comprising a culture medium according to any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 3-10] A method for preserving cells, comprising the step of preserving cells using the culture medium according to any one of the above items.

[0024] [Item 3-11] A composition for preserving cells, comprising the component described in any one of the above items. [Item 3-12] A cell-containing composition comprising the component described in any one of the above items and cells. [Item 3-13] Cells cultured using the component described in any one of the above items. [Item 3-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced. [Item 3-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 3-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. [Item 3-13D] Cells or cell populations according to any one of the above items, wherein the expression of CD44 usually does not decrease, but the others often decrease, for example, it is a desirable and advantageous feature that the expression of CD73 and CD90, or CD44, CD90 and CD105, does not decrease. [Item 3-13E] Cells or cell populations according to any one of the above items, wherein the expression of CD90 is maintained at a high level. [Item 3-13E] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 3-13F] Cells or cell populations according to any one of the above items, wherein the expression of at least one of the markers (preferably at least one of CD73, CD90 and CD105) does not decrease significantly when the number of passages is 8 or more. [Item 3-13G] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 5 or more, preferably 6 or more, and more preferably 7 or more. [Item 3-13H] Cells or cell populations according to any one of the above items, which maintain differentiation ability at passage number 8 or more.[Item 3-13I] The cells or cell population described in any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 3-13J] The cells or cell population described in any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage.

[0025] [Item 3-13AA] Cells according to any one of the above items, wherein the properties of the cells are improved (for example, their cartilage differentiation potential is enhanced). [Item 3-13AB] A cell population containing approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, or more of the cells according to any one of the above items. [Item 3-14] A pharmaceutical product comprising the cells according to any one of the above items. [Item 3-15] A method for differentiating the cells, comprising the step of culturing the cells under conditions that induce differentiation using the medium described in any one of the above items. [Item 3-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming their differentiation potential. [Item 3-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 3-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high cartilage differentiation potential. [Item 3-16] Differentiated cells obtained by differentiating stem cells by the method described in any one of the above items. [Item 3-17] A method for treating or preventing a subject in need, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmacopoeia described in any one of the above items to the subject in need. [Item 3-18] A method for quality control of stem cells, comprising determining whether they have a coefficient C correlated with the culture medium described in any one of the above items. [Item 3-18A] The method described in any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 3-18B] The method described in any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 3-18C] The method described in any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that exhibit long-term expression. [Item 3-18D] The method described in any one of the above items, wherein the marker, in the case of culture medium, includes information relating to the identification of the component itself.[Item 3-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a medium supply section for providing the medium or raw materials described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting the culture conditions as necessary. [Item 3-20] A medium suitable for transporting cells containing the medium components described in any one of the above items. [Item 3-21] A method for managing cell culture for cells suitable for culturing using the medium described in any one of the above items, comprising: 1) providing information about the medium for cells provided by the user; 2) assigning identifiable labels to the cells and the medium; and 3) managing whether the cells and the medium are compatible by referring to the labels.

[0026] [Item 3-22] Use of (3) BRD7 / 9 dual degradation factor or BRD7 / 9 binding molecule (degradation factor) as described in Item 3-1 for culturing cells (preferably mesenchymal stem cells). [Item 3-23] Use of Item 3-22 for culturing cells (preferably mesenchymal stem cells), comprising the features of any one of Items 3-2 to 3-6C. [Item 3-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 3-1. [Item 3-25] The method according to Item 3-24, comprising the features of any one of Items 3-2 to 3-6C. [Item 3-26] Cells or cell populations described in any one of items 3-13 to 3-13AB, or differentiated cells or cell populations described in item 3-16, for use in treatment. [Item 3-27] Cells or cell populations described in any one of items 3-13 to 3-13AB, or differentiated cells or cell populations described in item 3-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 3-28] Use of cells or cell populations described in any one of items 3-13 to 3-13AB, or differentiated cells or cell populations described in item 3-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 3-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 3-13 to 3-13AB, or differentiated cells or cell populations described in item 3-16, to the subject.

[0027] <Form 4> [Item 4-1] (4) A culture medium containing LATS1 / 2 inhibitor, PKA inhibitor, or AKT1 inhibitor. [Item 4-2] (4') The culture medium described in the above item, containing AKT1 inhibitor. [Item 4-3] A culture medium according to any one of the above items, comprising (4") LATS1 / 2 inhibitor or PKA inhibitor. [Item 4-4] A culture medium according to any one of the above items, comprising (4A) AKT1 inhibitor, which is also LATS1 / 2 inhibitor or PKA inhibitor. [Item 4-5] A culture medium according to any one of the above items, wherein (4) comprises (4B) A-674563 or a pharmaceutically acceptable salt or solvate thereof. [Item 4-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 4-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 4-6B] A serum-free culture medium according to any one of the above items. [Item 4-6C] A culture medium according to any one of the above items, which does not contain heterologous raw materials.

[0028] [Item 4-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 4-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 4-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A certain level is sufficient, and more preferably, 500-1000 cells / cm². ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２The method according to any one of the above items, although often seeded before or after. [Item 4-8B] The method according to any one of the above items, wherein the range of the cell number is adopted at the time of screening, such as 1500 cells / well, and / or carried out under conditions such as 4500 cells / well, which are estimated to be confluent. [Item 4-8C] The method according to any one of the above items, wherein the specific container is a container of appropriate material (including with or without coating).

[0029] [Item 4-8D] The method according to any one of the above items, wherein the specific temperature is an appropriate temperature (25-37°C, or 30°C, etc.). [Item 4-9] A cell culture apparatus comprising a culture medium according to any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 4-10] A method for preserving cells, comprising the step of preserving cells using the culture medium according to any one of the above items. [Item 4-11] A composition for preserving cells, comprising the component according to any one of the above items. [Item 4-12] A cell-containing composition comprising the component according to any one of the above items and cells. [Item 4-13] Cells cultured using the component according to any one of the above items. [Item 4-13A] The cells according to any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced.

[0030] [Item 4-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 4-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. Typically, CD44 does not decrease, but the others often decrease, and it is a favorable characteristic that CD73 and CD90, or CD44, CD90, and CD105, do not decrease, as described in any one of the above items. [Item 4-13D] A cell or cell population described in any one of the above items in which the expression of CD90 is maintained at a high level. [Item 4-13E] Cells or cell populations according to any one of the above items, wherein the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more, the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced. [Item 4-13F] Cells or cell populations according to any one of the above items, wherein the number of passages is 8 or more, and the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced. [Item 4-13G] Cells or cell populations according to any one of the above items, wherein the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more, the differentiation ability is maintained. [Item 4-13H] Cells or cell populations according to any one of the above items, wherein the number of passages is 8 or more, and the differentiation ability is maintained. [Item 4-13I] The cells or cell population described in any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 4-13J] The cells or cell population described in any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage.

[0031] [Item 4-13AA] Cells according to any one of the above items, wherein the properties of the cells are improved (for example, enhanced cartilage differentiation ability). [Item 4-13AB] A cell population containing approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, or more of the cells according to any one of the above items. [Item 4-14] A pharmaceutical product comprising the cells according to any one of the above items. [Item 4-15] A method for differentiating the cells, comprising the step of culturing the cells under conditions that induce differentiation using the medium described in any one of the above items. [Item 4-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation ability. [Item 4-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 4-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high cartilage differentiation ability.

[0032] [Item 4-16] Differentiated cells obtained by differentiating stem cells by the method described in any one of the above items. [Item 4-17] A method for treating or preventing a subject in need, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmacopoeia described in any one of the above items to the subject in need. [Item 4-18] A method for quality control of stem cells, comprising determining whether they have a coefficient C correlated with the culture medium described in any one of the above items. [Item 4-18A] The method described in any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 4-18B] The method described in any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 4-18C] The method described in any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that exhibit long-term expression. [Item 4-18D] The method described in any one of the above items, wherein the marker, in the case of culture medium, includes information relating to the identification of the component itself. [Item 4-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a medium supply section for providing the medium or raw materials described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting the culture conditions as necessary. [Item 4-20] A medium suitable for transporting cells containing the medium components described in any one of the above items. [Item 4-21] A method for managing cell culture for cells suitable for culturing using the medium described in any one of the above items, comprising: 1) providing information about the medium for cells provided by the user; 2) assigning identifiable labels to the cells and the medium; and 3) managing whether the cells and the medium are compatible by referring to the labels.

[0033] [Item 4-22] Use of (4) LATS1 / 2 inhibitor, PKA inhibitor or AKT1 inhibitor as described in Item 4-1 for culturing cells (preferably mesenchymal stem cells). [Item 4-23] Use of cells (preferably mesenchymal stem cells) as described in Item 4-22, comprising the features of any one of Items 4-2 to 4-6C. [Item 4-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 4-1. [Item 4-25] The method according to Item 4-24, comprising the features of any one of Items 4-2 to 4-6C. [Item 4-26] Cells or cell populations described in any one of items 4-13 to 4-13AB, or differentiated cells or cell populations described in item 4-16, for use in treatment. [Item 4-27] Cells or cell populations described in any one of items 4-13 to 4-13AB, or differentiated cells or cell populations described in item 4-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 4-28] Use of cells or cell populations described in any one of items 4-13 to 4-13AB, or differentiated cells or cell populations described in item 4-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 4-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 4-13 to 4-13AB, or differentiated cells or cell populations described in item 4-16, to the subject.

[0034] <Form 5> [Item 5-1] (5) A culture medium comprising a CK1 / 2 inhibitor, an insulin receptor Tyr kinase inhibitor, a PKA inhibitor or a PKC inhibitor or an adenosine kinase inhibitor. [Item 5-2] (5') A culture medium according to any one of the above items, comprising an adenosine kinase inhibitor. [Item 5-3] A culture medium according to any one of the above items, comprising (5") a CK1 / 2 inhibitor, an insulin receptor Tyr kinase inhibitor, a PKA inhibitor, or a PKC inhibitor. [Item 5-4] A culture medium according to any one of the above items, comprising (5A) an adenosine kinase inhibitor which is also a CK1 / 2 inhibitor, an insulin receptor Tyr kinase inhibitor, a PKA inhibitor, or a PKC inhibitor. [Item 5-5] The culture medium according to any one of the above items, wherein (5) comprises (5B) 5-iodotubercidin or a pharmaceutically acceptable salt or solvate thereof. [Item 5-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 5-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 5-6B] A serum-free culture medium according to any one of the above items. [Item 5-6C] A culture medium that does not contain raw materials of a different origin, as described in any one of the above items.

[0035] [Item 5-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 5-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 5-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A moderate amount is sufficient, and more preferably, 500-1000 / cm ２ ~10,000 / cm ２ For example, 2000 cells / cm² ２The method according to any one of the above items, although seeding is often performed before or after. [Item 5-8B] The method according to any one of the above items, wherein the range of the cell number is adopted at the time of screening, such as 1500 cells / well, and / or is carried out under conditions such as 4500 cells / well, which are estimated to be confluent. [Item 5-8C] The method according to any one of the above items, wherein the specific container is a container of appropriate material (including with or without coating). [Item 5-8D] The method according to any one of the above items, wherein the specific temperature is an appropriate temperature (25-37°C, or 30°C, etc.).

[0036] [Item 5-9] A cell culture apparatus comprising a culture medium described in any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 5-10] A method for preserving cells, comprising the step of preserving cells using the culture medium described in any one of the above items. [Item 5-11] A composition for preserving cells, comprising the component described in any one of the above items. [Item 5-12] A cell-containing composition comprising the component described in any one of the above items and cells. [Item 5-13] Cells cultured using the component described in any one of the above items. [Item 5-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced. [Item 5-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 5-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. Typically, CD44 does not decrease, but the others often decrease, for example, it is a preferred and advantageous feature that CD73 and CD90, or CD44, CD90, and CD105, do not decrease, as described in any one of the above items. [Item 5-13D] The expression of CD90 is maintained at a high level, as described in any one of the above items. [Item 5-13E] The expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) does not significantly decrease when the passage number is 5 or more, preferably 6 or more, and more preferably 7 or more, as described in any one of the above items. [Item 5-13F] Cells or cell populations according to any one of the above items, wherein the passage number is 8 or more and the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced.[Item 5-13G] Cells or cell populations according to any one of the above items, wherein the differentiation ability is maintained for 5 or more passages, preferably 6 or more, and more preferably 7 or more. [Item 5-13H] Cells or cell populations according to any one of the above items, wherein the differentiation ability is maintained for 8 or more passages. [Item 5-13I] Cells or cell populations according to any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, than before passage. [Item 5-13J] Cells or cell populations according to any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, than before passage.

[0037] [Item 5-13AA] Cells according to any one of the above items, wherein the properties of the cells are improved (for example, enhanced cartilage differentiation ability). [Item 5-13AB] A cell population containing about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or more of the cells according to any one of the above items. [Item 5-14] A pharmaceutical product comprising the cells according to any one of the above items. [Item 5-15] A method for differentiating the cells, comprising the step of culturing the cells under conditions that induce differentiation using the medium according to any one of the above items. [Item 5-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation ability. [Item 5-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 5-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high cartilage differentiation ability. [Item 5-16] Differentiated cells obtained by differentiating stem cells using the method described in any one of the above items.

[0038] [Item 5-17] A method for treating or preventing a subject in need, comprising the step of administering an effective amount of the cells or the pharmaceutical described in any one of the above items to the subject in need. [Item 5-18] A method for quality control of stem cells, comprising determining whether they have a coefficient C correlated with the culture medium described in any one of the above items. [Item 5-18A] The method according to any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 5-18B] The method according to any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 5-18C] The method according to any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that exhibit long-term expression. [Item 5-18D] The method according to any one of the above items, wherein the marker, in the case of culture medium, includes information relating to the identification of the component itself. [Item 5-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a medium supply section for providing the medium or raw materials described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting the culture conditions as necessary. [Item 5-20] A medium suitable for transporting cells containing the medium components described in any one of the above items. [Item 5-21] A method for managing cell culture for cells suitable for culturing using the medium described in any one of the above items, comprising: 1) providing information about the medium for cells provided by the user; 2) assigning identifiable labels to the cells and the medium; and 3) managing whether the cells and the medium are compatible by referring to the labels.

[0039] [Item 5-22] Use of (5) CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor or PKC inhibitor or adenosine kinase inhibitor as described in Item 5-1 for culturing cells (preferably mesenchymal stem cells). [Item 5-23] Use of Item 5-22 for culturing cells (preferably mesenchymal stem cells), comprising the features of any one of Items 5-2 to 5-6C. [Item 5-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 5-1. [Item 5-25] The method according to Item 5-24, comprising the features of any one of Items 5-2 to 5-6C. [Item 5-26] Cells or cell populations described in any one of items 5-13 to 5-13AB, or differentiated cells or cell populations described in item 5-16, for use in treatment. [Item 5-27] Cells or cell populations described in any one of items 5-13 to 5-13AB, or differentiated cells or cell populations described in item 5-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 5-28] Use of cells or cell populations described in any one of items 5-13 to 5-13AB, or differentiated cells or cell populations described in item 5-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 5-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 5-13 to 5-13AB, or differentiated cells or cell populations described in item 5-16, to the subject.

[0040] <Form 6> [Item 6-1] (6) A culture medium comprising an E-selectin inhibitor, a VCAM1 inhibitor, or an ICAM1 inhibitor. [Item 6-2] (6') A culture medium according to any one of the above items, comprising an ICAM1 inhibitor. [Item 6-3] A culture medium according to any one of the above items, comprising (6") an E-selectin inhibitor or a VCAM1 inhibitor. [Item 6-4] A culture medium according to any one of the above items, comprising (6A) an ICAM1 inhibitor which is also an E-selectin inhibitor or a VCAM1 inhibitor. [Item 6-5] The culture medium according to any one of the above items, wherein (6) comprises (6B) ICAM-1-IN-1 or a pharmaceutically acceptable salt or solvate thereof. [Item 6-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 6-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 6-6B] A serum-free culture medium according to any one of the above items. [Item 6-6C] A culture medium according to any one of the above items, which does not contain heterologous raw materials. [Item 6-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 6-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 6-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A certain level is sufficient, and more preferably, 500-1000 cells / cm². ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２ The method described in any one of the above items, although sowing is often done before or after these methods.

[0041] [Item 6-8B] The method according to any one of the above items, wherein the range of the cell number is a specific value such as 1500 cells / well during screening, and / or is carried out under conditions such as 4500 cells / well which are estimated to be confluent. [Item 6-8C] The method according to any one of the above items, wherein the specific container is a container of a suitable material (including with or without coating). [Item 6-8D] The method according to any one of the above items, wherein the specific temperature is a suitable temperature (such as 25-37°C or 30°C). [Item 6-9] A cell culture apparatus comprising a culture medium or means for providing the culture medium according to any one of the above items, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 6-10] A method for preserving cells, comprising the step of preserving cells using the culture medium according to any one of the above items. [Item 6-11] A composition for preserving cells comprising the components according to any one of the above items. [Item 6-12] A cell-containing composition comprising the components according to any one of the above items and cells. [Item 6-13] Cells cultured using any one of the above items. [Item 6-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced.

[0042] [Item 6-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 6-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. Typically, CD44 does not decrease, but the others often decrease, and it is a favorable characteristic that CD73 and CD90, or CD44, CD90, and CD105, do not decrease, as described in any one of the above items. [Item 6-13D] A cell or cell population described in any one of the above items in which the expression of CD90 is maintained at a high level. [Item 6-13E] Cells or cell populations according to any one of the above items, wherein the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more, in which case the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced. [Item 6-13F] Cells or cell populations according to any one of the above items, wherein the number of passages is 8 or more, and the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced. [Item 6-13G] Cells or cell populations according to any one of the above items, wherein the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more, in which case differentiation ability is maintained. [Item 6-13H] Cells or cell populations according to any one of the above items, wherein the number of passages is 8 or more, in which case differentiation ability is maintained. [Item 6-13I] The cells or cell population according to any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 6-13J] The cells or cell population according to any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage. [Item 6-13AA] The cells according to any one of the above items, wherein the properties of the cells are improved (for example, enhanced cartilage differentiation ability).[Item 6-13AB] A cell population containing approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, or more of the cells described in any one of the above items.

[0043] [Item 6-14] A pharmaceutical product comprising the cells described in any one of the above items. [Item 6-15] A method for differentiating the cells, comprising the step of culturing the cells under conditions that induce differentiation using the medium described in any one of the above items. [Item 6-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation ability. [Item 6-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 6-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high cartilage differentiation ability. [Item 6-16] Differentiated cells obtained by differentiating stem cells by the method according to any one of the above items. [Item 6-17] A method for treating or preventing a subject, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmaceutical product described in any one of the above items to a subject in need of it. [Item 6-18] A method for quality control of stem cells, comprising determining whether the cell has a coefficient C that correlates with the culture medium described in any one of the above items. [Item 6-18A] The method according to any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 6-18B] The method according to any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 6-18C] The method according to any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that are expressed over a long period of time. [Item 6-18D] The method according to any one of the above items, wherein the marker, in the case of a culture medium, includes information relating to the identification of the component itself. [Item 6-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a culture medium supply section for providing the culture medium or its raw materials as described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting culture conditions as necessary. [Item 6-20] A culture medium suitable for transporting cells containing any one of the culture medium components described in any of the above items.[Item 6-21] A method for managing cell culture for cells suitable for cultivation using the culture medium described in any one of the above items, comprising: 1) providing information about the culture medium with respect to cells provided by the user; 2) assigning identifiable labels to the cells and the culture medium; and 3) managing whether the cells and the culture medium are compatible by referring to the labels.

[0044] [Item 6-22] Use of (6) E-selectin inhibitor, VCAM1 inhibitor or ICAM1 inhibitor as described in Item 6-1 for culturing cells (preferably mesenchymal stem cells). [Item 6-23] Use of cells (preferably mesenchymal stem cells) as described in Item 6-22, comprising the features of any one of Items 6-2 to 6-6C. [Item 6-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 6-1. [Item 6-25] The method according to Item 6-24, comprising the features of any one of Items 6-2 to 6-6C. [Item 6-26] Cells or cell populations described in any one of items 6-13 to 6-13AB, or differentiated cells or cell populations described in item 6-16, for use in treatment. [Item 6-27] Cells or cell populations described in any one of items 6-13 to 6-13AB, or differentiated cells or cell populations described in item 6-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 6-28] Use of cells or cell populations described in any one of items 6-13 to 6-13AB, or differentiated cells or cell populations described in item 6-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 6-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 6-13 to 6-13AB, or differentiated cells or cell populations described in item 6-16, to the subject.

[0045] <Form 7> [Item 7-1] (7) A culture medium containing LATS1 inhibitor + LATS2 inhibitor (inhibiting either one is sufficient). [Item 7-2] (7') A culture medium according to any one of the above items, containing LATS1 inhibitor. [Item 7-3] A culture medium according to any one of the above items, comprising (7") a LATS1 / 2 inhibitor (that inhibits both). [Item 7-4] A culture medium according to any one of the above items, comprising (7A) a LATS1 / 2 inhibitor. [Item 7-5] A culture medium according to any one of the above items, wherein the (7) LATS1 / 2 inhibitor comprises (7B) LATS-IN-1 or a pharmaceutically acceptable salt or solvate thereof. [Item 7-6] A culture medium according to any one of the above items, for culturing various stem cells such as mesenchymal stem cells. [Item 7-6A] A culture medium according to any one of the above items, for culturing mesenchymal stem cells. [Item 7-6B] A serum-free culture medium according to any one of the above items. [Item 7-6C] A culture medium according to any one of the above items, which does not contain heterologous raw materials.

[0046] [Item 7-7] A method for producing cells, comprising the step of culturing cells using a culture medium described in any one of the above items. [Item 7-8] The method according to any one of the above items, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. [Item 7-8A] The range of cell numbers is such that MSCs are not seeded at low densities, and is preferably 500 cells / cm². ２ ~20000 cells / cm ２ A certain level is sufficient, and more preferably, 500-1000 cells / cm². ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２The method according to any one of the above items, although often seeded before or after. [Item 7-8B] The method according to any one of the above items, wherein the range of the cell number is adopted at the time of screening, such as 1500 cells / well, and / or carried out under conditions such as 4500 cells / well, which are estimated to be confluent. [Item 7-8C] The method according to any one of the above items, wherein the specific container is a container of appropriate material (including with or without coating). [Item 7-8D] The method according to any one of the above items, wherein the specific temperature is an appropriate temperature (25-37°C, or 30°C, etc.).

[0047] [Item 7-9] A cell culture apparatus comprising a culture medium described in any one of the above items or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. [Item 7-10] A method for preserving cells, comprising the step of preserving cells using the culture medium described in any one of the above items. [Item 7-11] A composition for preserving cells, comprising the component described in any one of the above items. [Item 7-12] A cell-containing composition comprising the component described in any one of the above items and cells. [Item 7-13] Cells cultured using the component described in any one of the above items. [Item 7-13A] The cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced. [Item 7-13B] A cell population comprising the cells described in any one of the above items, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. [Item 7-13C] The marker is at least one of CD44, CD73, CD90, and CD105, and at least one of these does not decrease. Typically, CD44 does not decrease, but the others often decrease, for example, it is a preferred and advantageous feature that CD73 and CD90, or CD44, CD90, and CD105, do not decrease, as described in any one of the above items. [Item 7-13D] The expression of CD90 is maintained at a high level, as described in any one of the above items. [Item 7-13E] The expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) does not significantly decrease when the number of passages is 5 or more, preferably 6 or more, and more preferably 7 or more, as described in any one of the above items. [Item 7-13F] Cells or cell populations according to any one of the above items, wherein the passage number is 8 or more and the expression of at least one of the markers (preferably at least one of CD73, CD90, and CD105) is not significantly reduced.[Item 7-13G] Cells or cell populations according to any one of the above items, wherein the differentiation ability is maintained for 5 or more passages, preferably 6 or more, and more preferably 7 or more. [Item 7-13H] Cells or cell populations according to any one of the above items, wherein the differentiation ability is maintained for 8 or more passages. [Item 7-13I] Cells or cell populations according to any one of the above items, wherein the expression of CD90, CD44 and / or CD73 is maintained at 70% or more, usually 80% or more, preferably 90% or more, and more preferably 95% or more, before passage. [Item 7-13J] Cells or cell populations according to any one of the above items, wherein the expression of CD105 is maintained at 30% or more, usually 40% or more, preferably 50% or more, and more preferably 70% or more, before passage.

[0048] [Item 7-13AA] Cells as described in Item 7-13, wherein the properties of the cells are improved (for example, enhanced chondrogenic potential). [Item 7-13AB] A cell population comprising approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, or more of the cells described in any one of the above items. [Item 7-14] A pharmaceutical product comprising the cells described in any one of the above items. [Item 7-15] A method for differentiating cells, comprising the step of culturing the cells under conditions that induce differentiation using the medium described in any one of the above items. [Item 7-15A] The method according to any one of the above items, wherein the culture comprises culturing the cells after proliferation using a different differentiation medium while confirming the differentiation potential. [Item 7-15B] The method according to any one of the above items, wherein the conditions that induce differentiation also include differentiation conditions that are commonly used for MSCs. [Item 7-15C] The method according to any one of the above items, wherein the cells are less likely to become cartilage and / or have high chondrogenic potential. [Item 7-16] Differentiated cells obtained by differentiating stem cells by the method described in any one of the above items. [Item 7-17] A method for treating or preventing a subject, comprising the step of administering an effective amount of the cells described in any one of the above items or the pharmaceutical agent described in any one of the above items to a subject in need.

[0049] [Item 7-18] A method for quality control of stem cells, comprising determining whether it has a coefficient C that correlates with the culture medium described in any one of the above items. [Item 7-18A] The method according to any one of the above items, wherein the coefficient C, in the case of cells, includes a coefficient relating to a cell marker. [Item 7-18B] The method according to any one of the above items, wherein the cell marker includes a general marker, such as a cell marker associated with proliferation. [Item 7-18C] The method according to any one of the above items, wherein the cell marker includes markers described herein, such as those that enhance differentiation ability or those that are expressed over a long period. [Item 7-18D] The method according to any one of the above items, wherein the marker, in the case of a culture medium, includes information relating to the identification of the component itself. [Item 7-19] A system for culturing cells, comprising: 1) a cell culture section for containing cells intended for culture; 2) a culture medium supply section for providing the culture medium or its raw materials as described in any one of the above items; and 3) a cell culture condition adjustment section for adjusting culture conditions as necessary. [Item 7-20] A culture medium suitable for transporting cells containing the culture medium components described in any one of the above items. [Item 7-21] A method for managing cell culture for cells suitable for cultivation using the culture medium described in any one of the above items, comprising: 1) providing information about the culture medium with respect to cells provided by the user; 2) assigning identifiable labels to the cells and the culture medium; and 3) managing whether the cells and the culture medium are compatible by referring to the labels.

[0050] [Item 7-22] Use of (7) LATS1 inhibitor + LATS2 inhibitor (inhibiting either one) as described in Item 7-1 for culturing cells (preferably mesenchymal stem cells). [Item 7-23] Use of Item 7-22 for culturing cells (preferably mesenchymal stem cells), comprising the features of any one of Items 7-2 to 7-6C. [Item 7-24] A method for maintaining or improving the quality of cells (preferably mesenchymal stem cells) (preferably, the expression level of at least one selected from the group consisting of CD44, CD73, CD90 and CD105, and / or differentiation potential after long-term passage), comprising the step of contacting the cells in a culture medium containing the components described in Item 7-1. [Item 7-25] The method according to Item 7-24, comprising the features of any one of Items 7-2 to 7-6C. [Item 7-26] Cells or cell populations described in any one of items 7-13 to 7-13AB, or differentiated cells or cell populations described in item 7-16, for use in treatment. [Item 7-27] Cells or cell populations described in any one of items 7-13 to 7-13AB, or differentiated cells or cell populations described in item 7-16, for use in regenerative medicine or in the treatment of cartilage diseases. [Item 7-28] Use of cells or cell populations described in any one of items 7-13 to 7-13AB, or differentiated cells or cell populations described in item 7-16, in the manufacture of a drug for regenerative medicine or in the treatment of cartilage diseases. [Item 7-29] A method for performing regenerative medicine or treating a cartilage disease in a subject in need thereof, comprising the step of administering an effective amount of cells or cell populations described in any one of items 7-13 to 7-13AB, or differentiated cells or cell populations described in item 7-16, to the subject.

[0051] In this disclosure, the one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary.

[0052] This disclosure provides a technology for effectively proliferating and maintaining various stem cells, such as mesenchymal stem cells.

[0053] Figure 1 shows stained images of DAPI, CD73, and CD44 with and without hallucinonide (1 μM). Figure 2 shows stained images of DAPI, CD73, and CD44 with and without AZ12601011 (1 μM). Figure 3 shows stained images of DAPI, CD73, and CD44 with and without VZ185 (250 nM). Figure 4 shows stained images of DAPI, CD73, and CD44 with and without A-674563 (250 nM). Figure 5 shows stained images of DAPI, CD73, and CD44 with and without 5-IOdotubercidin (500 nM). Figure 6 shows stained images of DAPI, CD73, and CD44 with and without ICAM-1-IN-1 (250 nM). Figure 7 shows stained images of DAPI, CD73, and CD44 with and without LATS-IN-1 addition (2.5 μM). Figure 8 shows the results of Example 1, where the ratio of growth rate (%) to concentration (nM) was calculated. Figure 9 shows the results of Example 2, where the ratio of the results in Figure 8 to the amount (yen / mM) for each concentration was calculated in order to calculate the growth efficiency for each amount for each component. Figure 10 shows the first container of one embodiment. Figure 11 shows four of the first containers of one embodiment stacked on top of each other. Figure 12 shows the second container of one embodiment. Figure 13 shows two of the second containers of one embodiment stacked on top of each other.

[0054] Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0055] (Definitions) First, we will define the terms and general technologies used in this disclosure.

[0056] (Definition of the highest-level concept) In this disclosure, "halcinonides" means:

[0057] Here, R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), or R1 and R2 together form a ring, or refer to a pharmaceutically acceptable salt or solvate thereof. Representative examples include halcinonides and clobetasol. While we do not wish to be bound by theory, halcinonides are primarily thought to bind to specific receptors within cells and affect cellular signaling pathways, promoting cell proliferation by suppressing the expression of genes that control cell growth, although this is not the only possible mechanism. Alternatively, halcinonides may bind to nuclear receptors, suppressing the production of cytokines and chemokines, thereby minimizing tissue damage by suppressing excessive inhibitory responses. Halcinonides, including fluocinonide, desoside, and clobetasol, are thought to have the ability to cross the cell membrane and act on nuclear receptors within cells to regulate gene transcription.

[0058] In this specification, "ALK4" refers to a protein belonging to the family of receptor serine / threonine kinases, which plays a particularly important role in the activin signaling pathway. It has been assigned GeneID 91, and its synonyms are ACVR1B, SKR2, ACTRIB, and ACVRLK4. ALK4 itself is well known in the field, as seen in Dronkers E et al., "Activin A and ALK4 Identified as Novel Regulators of Epithelmilal to Mesenchymal Transition (EMT) in Human Epicardial Cells," Front Cell Dev Biol. 2021 Dec 16;9:765007. [PMID: 34977017], Zhang YC et al. , “Up-regulated expression of miR-576 inhibits ALK4 expression, regulates JAK / STAT signaling pathway and promotes "Proliferation and migration of prosstatic cancer cells" Biomed Pharmacother. 2018 Feb; 98:440-445. Epub 2017 Dec 27. This topic is described in numerous publications, including [PMID: 37462480], Tsuchida K, “Signal translation pathway through active receivers as a therapeutic target of muscle skeletal diseases and cancer” Endocr J. 2008 Mar;55(1):11-21. Epub 2007 Sep 14. [PMID: 17878607]. In this disclosure, ALK4 and other gene-related terms may be represented in lowercase or uppercase + lowercase, but these all refer to the same gene and, depending on the context, are understood to refer to the concept of a protein, nucleic acid, or gene, or any combination thereof.

[0059] In this specification, "ALK5" refers to the major receptor of the TGF-β signaling pathway, which regulates many physiological processes such as cell proliferation, differentiation, apoptosis (programmed cell death), and development. It has been assigned GeneID 7046, and its synonyms are AAT5, TGFβR1, SKR4, LDS1A, LDS2A, and TGFR-1. ALK5 itself is well-known in this field; see Mansour MA et al., “Advances in the discovery of activin receptor-like kinase 5 (ALK5) inhibitors,” Bioorg Chem. 2024 Jun;147:107332. Epub 2024 Apr 3. This has been documented in numerous publications, including [PMID: 38581966], Ling LE et al., “Tgf-beta type I receptor (Alk5) kinase inhibitors in oncology.” Curr Pharma Biotechnol. 2011 Dec;12(12):2190-202. [PMID: 21619541].

[0060] In this specification, "ALK7" refers to a protein belonging to the family of receptor serine / threonine kinases, which is primarily involved in signal transduction by activin and other TGF-β (Transforming Growth Factor Beta) family ligands. It is assigned GeneID 130399, and its synonym is ACVR1C.ALK7 is well known in this field, as seen in Ibanez CF. et al., “Regulation of metabolic homeostasis by the TGF-β superfamily receptor ALK7” FEBS J. 2022 Oct;289(19):5776-5797. doi: 10.1111 / febs.16090. Epub 2021 Jul 11. [PMID: 34173336], and Cheng WL et al. , “ALK7 Acts as a Positive Regulator of Macrophage Activation through Down-Regulation of PPARγ Expression”J Atheroscler Thromb. 2021 Apr 1;28(4):375-384. doi: 10.5551 / jat. 54445. Epub 2020 Jul 9. [PMID: 32641645], Zhao Z, Lu et al. , “ALK7 Inhibition Protections Osteoblast Cells Against High Glucoseinduced ROS Production via Nrf2 / HO-1 Signaling Pathway” Curr Mol Med. 2022;22(4):354-364. [PMID: 34126915], Izumi T. This topic is discussed in numerous publications, such as "The GDF3-ALK7 signaling axis in adipose tissue: a possible therapeutic target for obesity and associated diabetes?" Endocr J. 2023 Aug 28;70(8):761-770. Epub 2023 Apr 19. [PMID: 37081691].

[0061] In this specification, "ALK4 inhibitor" refers to a substance or entity that inhibits the activity of ALK4. Specific examples include, but are not limited to, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, EW-7197, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, IN-1130, and SM16.

[0062] In a particular embodiment, Cao J et al., “SB431542 partially inhibitors high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells” Cell Commun Signal. 2024 Jan 5;22(1):17. doi: 10.1186 / s12964-023-01372-1. [PMID: 38183022], Suzuki H et al. This includes inhibitors disclosed in “Direct TGF-s signaling via alk4 / 5 / 7 pathway is involved in gut binding in sea urchin embryos” Dev Dyn. 2022 Jan;251(1):226-234. doi: 10.1002 / dvdy.442. Epub 2021 Nov 27. [PMID: 34816532].

[0063] In this specification, "ALK5 inhibitor" refers to a substance or entity that inhibits the activity of ALK5. It is also referred to as a TGFβR1 inhibitor. Specific examples include, but are not limited to, SB-525334, SB-505124, SB-431542, SB-202474, LY2157299, LY2109761, LY-364947, IN-1130, GW-788388, D4476, SD-208, EW-7197, A83-01, RepSox, and SM16.

[0064] In certain embodiments, Tojo M, et al., The ALK-5 inhibitor A-83-01 inhibitors Smad signaling and epithelial-to-mesenchymal transformation by transforming growth factor-beta. Cancer Sci. 2005 Nov;96(11):791-800. [PMID: 16271073], Engebretsen KV, et al. , “Attenuated development of cardiac fibrosis in left ventricular pressure overload by SM16, an orally active inhibitor of ALK5”. J Mol Cell Cardiol. 2014 Nov;76:148-57. [PMID: 25169971], Fjodorova M et al. , “A role for TGFβ signaling in medium spiny neuron differentiation of human pluripotent stem cells.” Neuronal Signal. 2020 May 6;4(2) [PMID: 32714602], Enza Lonardo et al. This includes the inhibitor disclosed in “Nodal / Activin signaling drives self-renewal and tumorigeneity of pancreatic cancer stem cells and provides a target for combined drug therapy” Cell Stem Cell. 2011 Nov 4;9(5):433-46. doi: 10.1016 / j. stem. 2011.10.001. [PMID: 22056140].

[0065] In this specification, "ALK7 inhibitor" refers to a substance or entity that inhibits the activity of ALK7. Specific examples include, but are not limited to, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, and IN-1130. In certain embodiments, Sunil K. Halder et al. This includes the inhibitor disclosed in “A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potential inhibitor agent for human cancers,” Neoplasia 2005 May;7(5):509-21. [PMID: 15967103].

[0066] In this specification, "ALK4 / 5 inhibitor" refers to a substance or entity that inhibits the activity of ALK4 or ALK5. Examples include, but are not limited to, SM16, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, EW-7197, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, SB-202474, LY2109761, IN-1130, D4476, and RepSox.

[0067] In a particular embodiment, Fujiki K, et al., “Blockade of ALK4 / 5 signaling suppresses cadmium- and erastin-induced cell death in renal proximal tubular epithelial cells via distinct signaling mechanisms” Cell Death Differ. 2019 Nov;26(11):2371-2385. Epub 2019 Feb 25. [PMID: 30804470], Thielen NGM et al. , “Separating friend from foe: Inhibition of TGF-β-induced detrimental SMAD1 / 5 / 9 phosphorylation while maintaining protective SMAD2 / 3 signaling in OA chondrocytes” Osteoarthritis Cartilage. 2023 Nov;31(11):1481-1490. Epub 2023 Aug 29. [PMID: 37652257], Sun Zet al. This includes the inhibitor disclosed in “The TGF-β pathway mediates doxorubicin effects on cardiac endotherial cells” J Mol Cell Cardiol. 2016 Jan;90:129-38. Epub 2015 Dec 11. [PMID: 26686989].

[0068] In this specification, "ALK4 / 7 inhibitor" refers to a substance or entity that inhibits the activity of ALK4 or ALK7. Specific examples include, but are not limited to, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, EW-7197, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, and IN-1130.

[0069] In a particular embodiment, Enza Lonardo et al., “Nodal / Activin signaling drives self-renewal and tumorigeneity of pancreatic cancer stem cells and provides a target for combined drug therapy” Cell Stem Cell. 2011 Nov 4;9(5):433-46. [PMID: 22056140], Spender LC, et al. , “Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor βSuperfamily Type 1 Receptors.”Mol Pharmacol. 2019 Feb; 95(2):222-234. [PMID: 30459156], Petersen M et al. This includes the inhibitor disclosed in “Oral administration of GW788388, an inhibitor of TGF-beta type I and II receptor kinases, decreases renal fibrosis.” Kidney Int, 2008, 73(6), 705-715. [PMID: 18075500].

[0070] In this specification, "ALK5 / 7 inhibitor" refers to a substance or entity that inhibits the activity of ALK5 or ALK7. Specific examples include, but are not limited to, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, SB-202474, LY2109761, IN-1130, D4476, EW-7197, and RepSox.

[0071] In certain embodiments, Tang Feng, et al., Pyrazoles. Patent WO2021129621 International Publication or Son JY et al., “EW-7197, a novel ALK-5 kinase inhibitor, potentially inhibitors breast to lung metastasis” Mol Cancer Ther. 2014 Jul;13(7):1704-16. doi: 10.1158 / 1535-7163. MCT-13-0903. Epub 2014 May 9. [PMID: 24817629], Lee GT et al. , “Effect of IN-1130, a small molecule inhibitor of transforming growth factor-beta type I receptor / activin receptor-like kinase-5, on prostate cancer cells” J Urol. 2008 Dec; 180(6):2660-7. doi: 10.1016 / j. juro. 2008.08.008. Epub 2008 Oct 31. This includes the inhibitors disclosed in [PMID: 18951571].

[0072] In this specification, "ALK4 / 5 / 7 inhibitor" refers to a substance or entity that inhibits the activity of ALK4, ALK5, or ALK7. Specific examples include, but are not limited to, SB-431542, LY-364947, A-83-01, SB-505124, LY2157299, SD-208, SB525334, GW788388, Dorsomorphin, LDN-193189, DMH1, AZ12601011, AZ12799734, SB-202474, LY2109761, IN-1130, D4476, EW-7197, and RepSox.

[0073] In a particular embodiment, Inman GJ et al., “SB-431542 is a potential and specific inhibitor of transforming growth factor-beta superfamily type I-activating receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7” Mol Pharmacol. 2002 Jul;62(1):65-74. [PMID: 12065756], Li Y et al. , “Activin A Increases Human Trophoblast Invasion by Inducing SNAIL-Mediated MMP2 Up-Regulation Through ALK4” J Clin Endocrinol Metab. 2015 Nov;100(11):E1415-27. doi: 10.1210 / jc. 2015-2134. Epub 2015 Aug 25. [PMID: 26305619], Bollum LK et al. This includes the inhibitor disclosed in “BMP-7 causes apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5” PLOS One. 2017 May 10;12(5):e0177188. [PMID: 28489883].

[0074] Whether a cell is an ALK4 / 7 inhibitor is determined by evaluating the decrease in specific phosphorylation markers of ALK4 / 7 activity in cell culture experiments. It can also be confirmed by measuring the IC50 value of the inhibitor using a kinase activity assay (Inman et al., “SB-431542 is a potential and specific inhibitor of transforming growth factor-beta superfamily type I-activating receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7,” Molecular Pharmacology, 2002). Generally, the following methods are used: 1. 1. Western blotting (measuring the level of phosphorylated Smad2 / 3 to evaluate the inhibitory effect of ALK4 / 7); 2. ELISA (measuring the level of phosphorylated Smad2 / 3 in proteins extracted from cells or tissues using a specific antibody); 3. Reporter assay (measuring the expression of a reporter gene that reflects the activation of the TGFβ signaling pathway (e.g., luciferase bound to the PAI-1 promoter)); 4. Cell proliferation and differentiation assays (performing cell proliferation assays (e.g., MTT assay) or cell differentiation assays to evaluate the inhibitory effect of ALK4 / 7); 5. Real-time PCR (measuring mRNA levels of genes related to the ALK4 / 7 signaling pathway to evaluate the effect of the inhibitor). By combining these methods, the effects of ALK4 / 7 inhibitors can be evaluated in detail, and their impact on cell signaling and function can be analyzed.The same method can be used to confirm whether each of the inhibitors ALK4, ALK5, and ALK7 is present (Fujiki K. “Involvement of Notch1 and ALK4 / 5 Signaling Pathways in Renal Tubular Cell Death: Their Application to Clarification of Cadmium Toxicity” Nihon Eiseigaku Zashi. 2020;75(0). doi: 10.1265 / jjh.20007. [PMID: 33342936]).

[0075] Whether a cell is a TGFβ receptor type 1 kinase (ALK5) inhibitor is determined by evaluating the decrease in a specific phosphorylation marker of ALK5 (Gene ID: 7046) activity in a cell culture experiment. Furthermore, this can also be confirmed by measuring the IC50 value of the inhibitor using a kinase activity assay (Inman et al., “SB-431542 is a potential and specific inhibitor of transforming growth factor-beta superfamily type I-activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7,” Molecular Pharmacology, 2002. [PubMed ID: 12065756]; Laping et al., “Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I Receptor kinase activity: SB-431542, “Molecular Pharmacology, 2002. [PMID: 12065755], Taniguchi Y, et al., Enhanced antitumor efficacy of folate-linked liposomal doxorubicin TGF-β type I receptor inhibitor. Cancer Sci. 2010 Oct;101(10):2207-13. [PMID: 20608940]), Krishnaiah M et al. , “Synthesis and biological evaluation of 5-(fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles as inhibitors of "transforming growth factor-β type I receptor kinase" Bioorg Med Chem Lett. See also 2015 Nov 15;25(22):5228–31. Epub 2015 Sep 26. [PMID: 26483198].

[0076] Generally, the following methods are used: 1. Western blotting (measuring the level of phosphorylated Smad2 / 3 to evaluate the inhibitory effect on TGFβR1); 2. ELISA (measuring the level of phosphorylated Smad2 / 3 in proteins extracted from cells or tissues using specific antibodies); 3. Reporter assay (measuring the expression of a reporter gene (e.g., luciferase bound to the PAI-1 promoter) that reflects the activation of the TGFβ signaling pathway); 4. Cell proliferation and differentiation assays (performing cell proliferation assays (e.g., MTT assay) or cell differentiation assays to evaluate the inhibitory effect on TGFβR1); 5. Real-time PCR (measuring mRNA levels of genes related to the TGFβ signaling pathway (e.g., PAI-1, Snail, Slug) to evaluate the effect of inhibitors). By combining these methods, the effects of TGFβ receptor type 1 kinase inhibitors can be evaluated in detail, and their impact on cellular signaling and function can be analyzed.

[0077] In this specification, "BRD7" refers to a protein possessing a bromodomain, which is involved in chromatin modification and transcriptional regulation. It has been assigned GeneID 29117, and its synonyms are BP75, NAG4, CELTIX, and SMARCI1. BRD7 is well known in the field, as seen in Park SW et al., "Emerging Roles of BRD7 in Pathogenesis." Int J Mol Sci. 2020 Sep 27;21(19):7127. doi: 10.3390 / ijms21197127 [PMID: 32992509], and Yu X et al. This is described in publications such as, “BRD7: a novel tumor suppressor gene in different cancers.” Am J Transl Res. 2016 Feb 15;8(2):742-8. eCollection 2016. [PMID: 27158366].

[0078] In this specification, "BRD9" refers to a protein possessing a bromodomain, which is involved in chromatin modification and transcriptional regulation. It has been assigned GeneID 65980, and its synonyms are PRO9856, SMARCI2, and LAVS3040. It is a paralog of BRD7. BRD9 is well known in the field and has been described in publications such as Zhu X et al., "Targeting BRD9 for Cancer Treatment: A New Strategy." Onco Targets Ther. 2020 Dec 24;13:13191-13200. eCollection 2020. [PMID: 33380808].

[0079] In this specification, "BRD7 / 9 dual degradation factor" refers to a substance or entity that targets and degrades both BRD7 and BRD9 proteins. Specific examples include, but are not limited to, VZ185, dBRD9, GNE-987, BI-894999, and MS2126. In certain embodiments, Zoppi V, et al. This includes factors disclosed in “Iterative Design and Optimization of Initially Inactive Protein Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7”. J Med Chem. 2019 Jan 24;62 [PMID: 30540463].

[0080] In this specification, “BRD7 / 9 binding (degradation) molecule” refers to a substance or entity that binds to the BRD7 and BRD9 proteins and inhibits or degrades their function. Specific examples include, but are not limited to, dBRD9, GNE-987, BI-7273, BI-9564, LP99, I-BRD9, BIC1, BIC2, BI-894999, and GNE-375. In a particular embodiment, see Clark PG et al., “Development of chemical probes for the bromodmains of BRD7 and BRD9,” Drug Discov Today Technol. 2016 Mar;19:73-80. Epub 2016 Aug 6. [PMID: 27769361], Karim RM et al. , “Structural Basis of Inhibitor Selection in the BRD7 / 9 Subfamily of Bromodomains” J Med Chem. 2020 Mar 26;63(6):3227-3237. doi: 10.1021 / acs. jmedchem. 9b01980. Epub 2020 Mar 6. [PMID: 32091206], Clark PG et al. , “LP99: Discovery and Synthesis of the First Selective BRD7 / 9 Bromodomain Inhibitor” Angew Chem Weinheim Bergstr Ger. 2015 May 18;127(21):6315-6319. doi: 10.1002 / ange.201501394. Epub 2015 Apr 13. [PMID: 27346896], including the BRD7 / 9 inhibitor disclosed in WO2014 / 144721.

[0081] Whether a substance is a BRD7 / 9 dual degradation factor is determined by evaluating the reduction in BRD7 (Gene ID: 29117) and BRD9 (Gene ID: 65980) protein levels in cell culture experiments. The efficiency of degradation can be confirmed using Western blotting or proteasome activity assays. (See Clark PG et al., “Development of chemical probes for the bromodogens of BRD7 and BRD9” Drug Discov Today Technol. 2016 Mar;19:73-80. Epub 2016 Aug 6. [PMID: 27769361], Winter et al.) This can be confirmed by referring to publicly available literature such as, "Drug development. Phythalimid conjugation as a strategy for in vivo target protein degradation," Science 21 May 2015 Vol 348, Issue 6241 pp. 1376-1381. [PMID: 25999370].

[0082] Whether a molecule binds to BRD7 / BRD9 is determined by evaluating its binding affinity to the BRD7 and BRD9 binding sites in cell culture experiments. Binding affinity can be confirmed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) (Gilan et al., "Selective targeting of BET bromodomains," Nature Chemical Biology, 2013. [PubMed ID: 23817068]; Filippakopoulos et al., "Selective inhibition of BET bromodomains," Nature, 2010. [PubMed ID: 20871596]; Park SW et al., "Emerging Roles of BRD7 in Pathophysiology. ” Int J Mol Sci. 2020 Sep 27; 21 (19): 7127. doi: 10.3390 / ijms21197127. [PMID: 32992509], Yu X et al., “BRD7: a novel Tumor suppressor gene in different cancers. ” Am J Transl Res. 2016 Feb 15;8(2):742-8. eCollection 2016. [PMID: 27158366]).Generally, the following methods are used: 1. Western blotting (Western blotting is used to measure the degradation status of BRD7 and BRD9 proteins and evaluate the effect of binding molecules); 2. Immunoprecipitation assay (Immunoprecipitation is used to evaluate the presence and degradation status of these proteins using antibodies specific to BRD7 and BRD9); 3. Real-time PCR (The mRNA levels of BRD7 and BRD9 are measured to evaluate the effect of binding molecules on gene expression); 4. Proteasome activity assay (Proteasome activity is measured to confirm that the degradation of BRD7 and BRD9 is due to the ubiquitin-proteasome system); 5. Cell proliferation and apoptosis assay (MTT assay or flow cytometry is used to evaluate the effects of BRD7 and BRD9 degradation on cell proliferation and apoptosis).

[0083] In this specification, "LATS1" refers to a major kinase of the Hippo signaling pathway, which plays a crucial role in tumor suppression through the regulation of cell proliferation and apoptosis. It has been assigned GeneID 9113, and its synonyms are wts and WARTS. LATS1 is well known in this field, as seen in: Furth N et al., “The LATS1 and LATS2 tumor suppressors: beyond the Hippo pathway” Cell Death Differ. 2017 Sep;24(9):1488-1501. Epub 2017 Jun 23. [PMID: 28644436], Ma Set et al. This is described in publications such as “The Hippo Pathway: Biology and Pathophysiology” Annu Rev Biochem. 2019 Jun 20;88:577–604. Epub 2019 Dec 19. [PMID: 30566373].

[0084] In this specification, "LATS2" refers to a major kinase in the Hippo signaling pathway, playing a crucial role in tumor suppression through the regulation of cell proliferation and apoptosis. Compared to LATS1, LATS2 is particularly strongly involved in the G1 / S checkpoint. It has been assigned GeneID 26524, and its synonyms are KPM and LATS2. LATS2 is well-known in this field; see Furth N et al., "The LATS1 and LATS2 tumor suppressors: beyond the Hippo pathway," Cell Death Differ. 2017 Sep;24(9):1488-1501. Epub 2017 Jun 23. This is described in publications such as [PMID: 28644436], Ma Setal., “The Hippo Pathway: Biology and Pathophysiology” Annu Rev Biochem. 2019 Jun 20;88:577-604. Epub 2019 Dec 19. [PMID: 30566373].

[0085] In this specification, "LATS1 / 2 inhibitor" refers to a substance or entity that inhibits the activity of LATS1 or LATS2. Specific examples include, but are not limited to, LATS-IN-1 (TRULI), MGH-CP1, VT02956, NIBR-LTSi, KMH-233, BCH, GPNA (hydrochloride), GA-017, and TDI-011536.In certain embodiments, the method described by Nathaniel Kastan, et al. , “Small-molecule inhibition of LATS kinases promotes Yap-dependent proliferation in postmitotic mammalian tissues”, bioRxiv 2020.02.11.944157. , [PMID: 34035288], Li Q, Sun Y, Jarugumilli GK, et al. “LATS1 / 2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription”, Cell Stem Cell. 2020;26(5):675-692. e8. doi:10.1016 / j. stem. 2020.03.00, [PMID: 32259481], Aihara A, et al. , “Small molecule LATS kinase inhibitors block the Hippo signaling pathway and promote cell growth under 3D culture conditions”, J Biol Chem. 2022 Apr;298(4):101779. , [PMID: 35231442], Huttunen KM, et al. This includes the inhibitor disclosed in “A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentials Antiproliferative Drug Efficacy in Cancer Cells”, J Med Chem. 2016;59(12):5740-5751. [PMID: 27253989].

[0086] Whether a cell is a LATS1 / 2 inhibitor is determined by evaluating the decrease in Yap phosphorylation markers, which are indicators of LATS1 (Gene ID: 9113) and LATS2 (Gene ID: 26524) activity, in a cell culture experiment. Furthermore, this can also be confirmed by measuring the IC50 value of the inhibitor using a kinase activity assay (Kastan N. et al., “Small-molecule inhibition of LATS kinases may promote Yap-dependent preference in postmitotic mammalian tissues”, Nat Commun. 2021, [PMID: 34035288], Furth N. et al., “The LATS1 and LATS2 tumor suppressors: beyond the Hippo pathway”, Cell Death Differ.). 2017 Sep;24(9):1488-1501. Epub 2017 Jun 23. [PMID: 28644436], Ma S et al. , “The Hippo Pathway: Biology and Pathophysiology” Annu Rev Biochem. 2019 Jun 20;88:577-604. Epub 2019 Dec 19. [PMID: 30566373], Ma S, et al. “Transcriptional expression of estrogen receptor alpha by YAP “Reveals the Hippo pathway as therapeutic target for ER+ breast cancer”, Nat Commun. 2022 Feb 25;13(1):1061.).

[0087] In this specification, "PKA" refers to cAMP-dependent protein kinases, enzymes that play a crucial role in intracellular signal transduction. PKAs are genes and proteins that control many physiological processes, including cell proliferation, differentiation, metabolism, and gene expression. See also Yuening Liu et al., “Physiological and pathological roles of protein kinase A in the heart”, Cardiovasc Res. 2022 Jan 29;118(2):386-398, [PMID: 33483740]. It has been assigned GeneID 5573, and its synonyms are CAR, CNC, CNC1, PKR1, TSE1, ADOHR, PPNAD1, PRKAR1, ACRDYS1, and Prkar1alpha.

[0088] In this specification, "PKA inhibitor" refers to a substance or entity that inhibits the activity of cAMP-dependent protein kinase (PKA). Specific examples include, but are not limited to, H-89, KT5720, Rp-cAMP, PKI (14-22), amide, SQ22536, Myr-PKI, 6-Benzoyl-cAMP, cAMPS-Rp, PKAi, cAMPS-Sp, H7, H8, H9, HA1077, HA1004, and chererythrine. Z X Lu et al. See also “Selective inhibition of cyclo AMP-dependent protein kinase by isoquinoline derivative”, Biol Chem Hoppe Seyler. 1996 Jun;377(6):373-8, [PMID: 8839983]. In certain embodiments, the PKA inhibitor disclosed in WO2014 / 170342 is included.

[0089] Whether a substance is a PKA inhibitor can be determined by measuring intracellular cAMP levels or by evaluating the phosphorylation status of the PKA substrate. See also Yuening Liu et al., “Physiological and pathological roles of protein kinase A in the heart”, Cardiovasc Res. 2022 Jan 29;118(2):386-398, [PMID: 33483740]. Generally, the following methods are used: 1. Western blotting (Western blotting is used to measure the level of phosphorylated PKA substrate and evaluate the inhibitor effect); 2. ELISA (Specific antibodies are used to measure the level of phosphorylated PKA substrate in proteins extracted from cells or tissues); 3. 1. Real-time PCR (to measure mRNA levels of genes related to PKA signaling and evaluate the effects of inhibitors); 2. Cell proliferation and apoptosis assays (to evaluate the effects of inhibitors, cell proliferation assays (e.g., MTT assay) and apoptosis assays (e.g., flow cytometry) are performed); 3. Protein kinase assays (to directly measure the activity of specific kinases, assays using radiophosphate or fluorescently labeled substrates are performed).

[0090] In this specification, "AKT1" refers to a protein kinase that plays a crucial role in cellular signaling pathways and is a member of the AKT (or protein kinase B) family. See also Juan Angel Fresno Vara et al., "PI3K / Akt signaling pathway and cancer", Cancer Treat Rev. 2004 Apr;30(2):193-204, [PMID: 15023437]. It has been assigned GeneID 207, and its synonyms are AKT, PKB, RAC, PRKBA, PKB-ALPHA, and RAC-ALPHA.

[0091] In this specification, "AKT1 inhibitor" refers to a substance or entity that inhibits the activity of AKT1 and is interchangeable with "protein kinase B inhibitor". Specific examples include, but are not limited to, A-674563, MK-2206, AZD5363 (Capivacertib), GSK690693, GSK2141795 (uprocertib), GSK2110183 (afurecertib), AZD5363, GDC-0068 (ipatacertib), Perifosine, Triciribine (API-2), A-443654, AT7867, CCT128930, RX-0201, BAY 1125976, and LY294002. Kumar JS et al. See also “Ultrasound assisted one-pot synthesis of rosuvastantin-based novel azaidole derivatives via coupling-cyclization strategy under Pd / Cu-catalysis: Their evaluation as potential cytotoxic agents”, Bioorg Chem. 2022;124:105857, [PMID: 35594765]. In certain embodiments, this includes PKA inhibitors as disclosed in WO2023 / 114948 and WO2024 / 064026.

[0092] Whether or not a substance is an AKT1 inhibitor can be determined by evaluating the phosphorylation state of AKT1 or by observing changes in downstream signaling molecules. See also Juan Angel Fresno Vara et al. “PI3K / Akt signaling pathway and cancer”, Cancer Treat Rev. 2004 Apr;30(2):193-204, [PMID: 15023437].

[0093] In this specification, "CK1" refers to a protein kinase belonging to the serine / threonine kinase family, an enzyme that primarily phosphorylates serine or threonine residues. It is involved in many physiological processes and plays an important role in cellular signaling, cell cycle regulation, transcriptional regulation, and maintenance of cellular polarity. See also Uwe Knippschild et al., "The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis," Front Oncol. 2014 May 19:4:96, [PMID: 24904820]. It has been assigned GeneID 1452, and its synonyms are CSNK1A1, CK1a, CKIa, HLCDGP1, PRO2975, and HEL-S-77p.

[0094] In this specification, "CK2" refers to a protein kinase belonging to the serine / threonine kinase family, an enzyme that primarily phosphorylates serine, threonine, and tyrosine residues. It is involved in various intracellular physiological processes and plays a crucial role in regulating cell growth, proliferation, differentiation, and apoptosis. See also Mathias Montenarh and Claudia Gotz, “Protein kinase CK2 and ion channels (Review)”. Biomed Rep. 2020 Dec;13(6):55, [PMID: 33082952]. It has been assigned GeneID 1457, and its synonyms are CSNK2A1, CKII, Cka1, Cka2, CK2A1, and OCNDS.

[0095] In this specification, "CK1 inhibitor" refers to a substance or entity that inhibits the activity of CK1. Specific examples of CK1 inhibitors include, but are not limited to, D4476, IC261, CX-4945 (Silmitaserb), TBB (4,5,6,7-Tetrabromo-benzotriazole), DMAT (2-Dimethylamino-4,5,6,7-Tetrabromo-1H-benzimidazole), PF-670462, PF-4800567, PF-5006739, SR-3029, BIO (6-Bromoindirubin-3'-oxyme), LH846, TA01, TA02, TAK715, CKI-7, LY2090314, and others. See also Laco F, et al., “Cardiomyocyte difference of pluripotent stem cells with SB203580 analogues correlations with Wnt pathway CK1 inhibition independent of p38 MAPK signaling”, J Mol Cell Cardiol. 2015 Mar;80:56–70, [PMID: 25528965]. In certain embodiments, CK1 inhibitors disclosed in WO2018 / 112342 and WO2015 / 119579A are included, the contents of which are incorporated herein by reference in their entirety. See also [PMID: 22908959].

[0096] In this specification, "CK2 inhibitor" refers to a substance or entity that inhibits the activity of CK2. Specific examples of CK2 inhibitors include, for example, D4476, IC261, CX-4945 (Silmitaserb), TBB (4,5,6,7-Tetrabromo-benzotriazole), DMAT (2-Dimethylamino-4,5,6,7-Tetrabromo-1H-benzimidazole), PF-670462, SR-3029, BIO (6-Bromoindirubin-3'-oxyme), CKI-7, LY2090314, CX-5011, CX-5279, TBI, TBCA, CIBG-300, Electric Acid, K64 / PBIN, Examples include, but are not limited to, K66 / TMCB, IQA, Fisetin, Hematein, FLC21, TID46, Quinolone 9, Quinolone 7, TTP22, and FNH79. Wang Y, et al., “Discovery of 5-(3-Chromophenylamino)benzo[c][2,6]naphthyridine Derivatives as Highly Selective CK2 Inhibitors with Potent Cancer Cell Stemness Inhibition”, J Med Chem. See also 2021 Apr 22;64(8):5082-5098, [PMID: 33834781]. In certain embodiments, the CK1 inhibitors disclosed in WO2016 / 22538 are included, the contents of which are incorporated herein by reference in their entirety. See also Giorgio Cozza et al., “Protein kinase CK2 inhibitors: a patent review”, Expert Opin Ther Pat. 2012 Sep;22(9):1081-97, [PMID: 22908959].

[0097] In this specification, "CK1 / 2 inhibitor" means a substance or entity that inhibits the activity of CK1 or CK2.

[0098] Whether a molecule is a CK1 / 2 inhibitor can be determined by evaluating the phosphorylation state of the target kinase and the changes in downstream signaling molecules. (Uwe Knippschild et al., “The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis”, Front Oncol.) See also 2014 May 19:4:96, [PMID: 24904820] and Mathias Montenarh and Claudia Gotz, “Protein kinase CK2 and ion channels (Review)”, Biomed Rep. 2020 Dec;13(6):55. [PMID: 33082952]. Generally, the phosphorylation level of kinase substrates is measured using Western blotting. In addition, MTT assays and flow cytometry are used to evaluate cell viability and apoptosis. Furthermore, it is possible to visualize the phosphorylation state within cells using imaging techniques with fluorescently labeled probes.

[0099] In this specification, “insulin receptor” refers to a protein present on the cell surface that is involved in insulin signaling. Insulin is a hormone secreted by beta cells of the pancreas and is involved in important physiological processes such as blood glucose regulation and metabolic regulation. See also J Lee and P F Pilch, “The insulin receptor: structure, function, and signaling”, Am J Physiol. 1994 Feb;266(2 Pt 1):C319-34, [PMID: 8141246]. It has been assigned GeneID 3643, and synonyms are INSR, HHF5, and CD220.

[0100] The effect of inhibiting insulin receptors can be measured by evaluating the autophosphorylation status of insulin receptors or by measuring the phosphorylation status of downstream signaling molecules (e.g., AKT, ERK). See also J Lee and P F Pilch, “The insulin receptor: structure, function, and signaling”, Am J Physiol. 1994 Feb;266(2 Pt 1):C319-34, [PMID: 8141246]. Generally, Western blotting and assays using ELISA are used. Assays that measure cellular glucose uptake are also utilized.

[0101] In this specification, "Tyr kinase" refers to a protein kinase that phosphorylates tyrosine residues, catalyzing the phosphorylation of tyrosine residues and regulating cellular signaling pathways. It plays a crucial role in regulating physiological processes such as cell growth, proliferation, differentiation, cell migration, and apoptosis. See also S. R. Hubbard and J. H. Till, “Protein tyrosine kinase structure and function”, Annu Rev. Biochem. 2000:69:373-98, [PMID: 10966463]. Representative examples include, but are not limited to, the following: SRC (Gene ID: 6714, synonyms: ASV, SRC1, THC6, c-SRC, p60-Src), ABL1 (Gene ID: 25, synonyms: ABL, JTK7, p150, c-ABL, v-abl, CHDSKM, c-ABL1, BCR-ABL, bcr / abl), EGFR (Gene ID: 1956, synonyms: ERBB, ERRP, HER1, mENA, ERBB1, PIG61, NISBD2), JAK2 (Gene ID: 3717, synonym: JTK10), MET (Gene ID: 4233, Synonyms: DA11, HGFR, AUTOS9, RCCP2, c-Met, DFNB97), RET (Gene ID: 5979, Synonyms: PTC, MTC1, HSCR1, MEN2A, MEN2B, CDHF12, CDHR16, RET-ELE1), PDGFRB (Gene ID: 5159, Synonyms: IMF1, KOGS, IBGC4, JTK12, PDGFR, PENTT, CD140B, PDGFR1, PDGFR-1), KIT (Gene ID: 3815, Synonyms: PBT, SCFR, C-Kit, CD117, MASTC), FLT3 (Gene ID: 2322, synonyms: FLK2, STK1, CD135, FLK-2), ALK (Gene ID: 238, synonyms: ALK1, CD246, NBLST3).

[0102] In this specification, "Tyr kinase inhibitor," also known as "tyrosine kinase inhibitor," refers to a substance or entity that inhibits the activity of tyrosine kinase (Protein Tyrosine Kinase), thereby regulating cell proliferation, differentiation, metabolism, and signal transduction. "Insulin receptor Tyr kinase inhibitor," also known as "insulin receptor tyrosine kinase inhibitor," refers to a substance or entity that inhibits the activity of tyrosine kinase (Protein Tyrosine Kinase) endogenous in the insulin receptor (Gene ID: 3643). Specifically, insulin receptor Tyr kinase inhibitors suppress abnormal cell proliferation and tumor formation by inhibiting the phosphorylation activity of receptor tyrosine kinase in the insulin receptor. Insulin receptor tyr kinase inhibitors are widely used as therapeutic agents for cancer and other growth disorders. Specific examples of insulin receptor tyr kinase inhibitors include Paul MK et al., “Tyrosine kinase - Role and signature in Cancer”, Int J Med Sci. 2004;1(2):101-115. [PMID: 15912202] and Moon K et al. See also, “The design, synthesis, and biological evaluation of potential receptor tyrosine kinase inhibitors,” Bioorganic & Medical Chemistry Letters 22 (2012) 4979-4985, [PMID: 22765894]. Alternatively, insulin receptor (Tyr kinase) inhibitors regulate cellular glucose uptake, metabolism, and growth through the insulin signaling pathway. Specifically, they reduce insulin-dependent intracellular signaling by inhibiting the tyrosine kinase activity of the insulin receptor. These inhibitors are important tools in diabetes and cancer research.Specific examples of insulin receptor Tyr kinase inhibitors include, but are not limited to, BMS-536924, OSI-906 (Linsitinib), AG-1024, HNMPA-(AM)3, S961, GSK1904529A, AEW541, NVP-AEW541, PQIP (pyrrolo[2,3-d]pyrimidine derivative), Tyrphostin AG 538, and BMS-754807. In certain embodiments, inhibitors disclosed in WO2021 / 237022 are included.

[0103] The effect of a Tyr kinase inhibitor can be measured by evaluating the phosphorylation state of the target tyrosine kinase and the changes in downstream signaling molecules (e.g., STAT, MAPK). (S. R. Hubbard and J. H. Till, “Protein tyrosine kinase structure and function”, Annu Rev. Biochem.) See also 2000:69:373-98, [PMID: 10966463] and Levitzki A, “Tyrosine Kinase Inhibitors: Views of Selection, Sensitivity, and Clinical Performance” Annu Rev Pharmacol Toxicol. 2013;53:161-185 [PMID: 23043437]. Generally, Western blotting and ELISA assays are used. Biological effects are also evaluated through cell proliferation assays and apoptosis assays.

[0104] In this specification, "PKC" refers to enzymes belonging to the serine / threonine kinase family, which are involved in many intracellular physiological processes. PKCs are important enzymes for regulating cellular signaling pathways, particularly cell proliferation, differentiation, apoptosis, intercellular interactions, and cell cycle regulation. See also Takahito Kawano et al., "Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials", Pharmaceutics. 2021 Oct 20;13(11):1748, [PMID: 34834162]. The following are some representative examples, but are not limited to these: PRKCA (Gene ID: 5578, synonyms: AAG6, PKCA, PRKACA, PKCI+ / -, PKCalpha, PKC-alpha), PRKCB (Gene ID: 5579, synonyms: PKCB, PRKCB1, PRKCB2, PKCI(2), PKCbeta, PKC-beta), PRKCG (Gene ID: 5582, synonyms: PKCC, PKCG, SCA14, PKCI(3), PKCgamma, PKC-gamma), PRKCD (Gene ID: 5580, Synonyms: MAY1, PKCD, ALPS3, CVID9, nPKC-delta), PRKCE (Gene ID: 5581, Synonyms: PKCE, nPKC-epsilon), PRKCH (Gene ID: 5583, synonyms: PKCL, PKC-L, PRKCL, uORF2, nPKC-eta), PRKCQ (Gene ID: 5588, synonyms: PRKCT, nPKC-theta), PRKCZ (Gene ID: 5590, synonyms: PKC2, PKC-ZETA).

[0105] In this specification, "PKC inhibitor," also known as "protein kinase C inhibitor," is a substance or entity that inhibits the activity of PKC (Protein Kinase C) and regulates cell proliferation, differentiation, metabolism, and signal transduction. Specifically, PKC inhibitors regulate intracellular signal transduction pathways by inhibiting the phosphorylation activity of PKC, thereby controlling abnormal cell proliferation and other pathological conditions. PKC inhibitors play an important role in therapeutic research for cancer, cardiovascular disease, neurodegenerative diseases, and other conditions. Specific examples of PKC inhibitors include, but are not limited to, Bisindolylmaleimide I (GF 109203X), Ro-31-8220, Staurosporine, Chererythrine, Go 6983, Enzastaurin (LY317615), AEB071 (Sotrastaurin), Ruboxistaurin (LY333531), H7, Calphostin C, GO6976, UCN-01, CGP-53353 solid, K-252B solution, K252C, and ZIP. (Theodore Letal.) See also “Intranional delivery of protein kinase C pseudosubstrate leads to growth cone collapse”, J Neurosci. 1995 Nov;15(11):7158-67, [PMID: 7472470]. Certain embodiments include PKC inhibitors disclosed in WO2019 / 055589 and WO2015 / 179847, the contents of which are incorporated herein by reference in their entirety.

[0106] The effect of PKC inhibitors can be measured by evaluating the phosphorylation state of the PKC substrate or by measuring changes in downstream signaling molecules (e.g., MAPK, NF-κB). (See Takahito Kawano et al., “Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials”, Pharmaceutics. 2021 Oct 20;13(11):1748, [PMID: 34834162] and Martiny-Baron G et al.) See also “Selective inhibition of protein kinase C isozymes by the indolocarbazol Go 6976” J Biol Chem. 1993;268(13):9194-7, [PMID: 8486620]. Generally, Western blotting or assays using ELISA are used.

[0107] In this specification, “adenosine kinase” refers to an enzyme that catalyzes the transfer of gamma phosphate from ATP to adenosine, and functions as a regulator of both extracellular adenosine and intracellular adenine nucleotide concentrations. It plays a particularly important role in the nervous and immune systems. See also Alhanouf Alhusani et al., “Adenosine Kinase Deficiency: Report and Review”, Neuropediatrics. 2019 Feb;50(1):46-50, [PMID: 30477030]. It has been assigned GeneID 132, and its synonyms are AK and ADK.

[0108] In this specification, "adenosine kinase inhibitor" refers to a substance or entity that inhibits the activity of adenosine kinase (AK, Gene ID: 132) and regulates adenosine metabolism. Specifically, adenosine kinase inhibitors increase intracellular and extracellular adenosine levels by inhibiting the activity of adenosine kinase, resulting in neuroprotective, anti-inflammatory, and antitumor effects. These inhibitors are important tools in research into the treatment of neurological diseases and cancer. Specific examples of adenosine kinase inhibitors include, but are not limited to, ABT-702, A-134974, GP515, 5-Iodotubercidin, AMI-5, ITU (Iodotubercidin), ABT-702 Mesylate, A-286982, Tubercidin, ABT-546, GP683, and 2'-deoxycoformycin. (Jarvis MF et al.) , “ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2,3-d]pyrimidine), a novel orally effective Adenosine kinase inhibitor with analgesic and anti-inflammatory properties: I. In vitro characterization and acute Antinociceptive effects in the mouse”, J Pharmacol Exp Ther. See also 2000 Dec;295(3):1156-64, [PMID: 11082453]. In certain embodiments, the ADK inhibitor disclosed in WO01 / 44265 is included.

[0109] The effect of an adenosine kinase inhibitor can be measured by measuring adenosine levels or evaluating changes in adenosine-dependent signaling pathways. (See Alhanouf Alhusani et al., “Adenosine Kinase Deficiency: Report and Review”, Neuropediatrics. 2019 Feb;50(1):46-50, [PMID: 30477030] and Boison D, “Adenosine kinase: extraction for therapeutic gain”, Pharmacol Rev.) See also 2013;65(3):906-43 [PMID: 23592612]. Generally, adenosine concentration is measured using HPLC (high-performance liquid chromatography). Cell-based assays and behavioral analyses using animal models are also used to evaluate efficacy.

[0110] In this specification, "E-selectin" refers to a type of cell adhesion molecule, a protein primarily expressed on the surface of vascular endothelial cells. It plays a crucial role in regulating the attachment and migration of leukocytes to vascular endothelial cells during inflammatory responses. See also Jinjin Zhang et al., "E-selectin in vascular pathogenesis," Front Immunol. 2024 Jul 19:15:1401399, [PMID: 39100681]. It has been assigned GeneID 6401, and its synonyms are SELE, ELAM, ESEL, CD62E, ELAM1, LECAM2, and selectin-e.

[0111] In this specification, "E-selectin kinase inhibitor" refers to a substance or entity that inhibits the activity of E-selectin (Gene ID: 6401). E-selectin inhibitors modulate the function of molecules involved in cell adhesion and inflammatory responses. Specifically, E-selectin inhibitors suppress inflammatory responses by inhibiting ligand binding to E-selectin, thereby reducing the migration of leukocytes to inflammatory sites. These inhibitors are promising tools in the treatment of inflammatory diseases and cancer. Specific examples of E-selectin inhibitors include, but are not limited to, GMI-1070 (Rivipansel), Bimosiamose, Uproleselan (GMI-1271), TBC-1269 (Selbexin), Fucoidan, Heparin, P-Selectin Glycoprotein Ligand-1 (PSGL-1) blocks, Small molecular inhibitors (e.g., Glycomimetic inhibitors), Sialyl Lewis X analogs, and Inclacumab. (Dampier CD et al.) See also “A Randomized Clinical Trial of the Efficacy and Safety of Rivipansel for Sickle Cell Vaso-occlusive Crisis (VOC)”, Blood. 2022 Aug 18:blood. 2022015797. [PMID: 35981565]. In certain embodiments, the invention includes an E-selectin kinase inhibitor disclosed in WO2008 / 011094.

[0112] The effect of E-selectin inhibitors can be measured using cell adhesion assays or competitive inhibition tests for E-selectin ligand binding. (Jinjin Zhang et al., “E-selectin in vascular pathogenesis”, Front Immunol. 2024 Jul 19:15:1401399. [PMID: 39100681] and Ley K, et al., “Getting to the site of inflation: the leukocyte adhesion cascade updated”, Nat Rev Immunol.) See also 2007;7(9):678-89 [PMID: 17717539]. Generally, flow cytometry or ELISA can be used to measure leukocyte adhesion and migration after inhibitor treatment. Analysis of inflammatory responses using animal models is also used to evaluate the effects.

[0113] In this specification, "VCAM-1" refers to a type of cell adhesion molecule that is primarily expressed in vascular endothelial cells. VCAM-1 plays a crucial role in inflammatory and immune responses, particularly regulating the process by which leukocytes (e.g., monocytes and lymphocytes) adhere to vascular endothelium and migrate to the site of inflammation. See also Deok-Hoon Kong et al., "Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer", Int J Mol Sci. 2018 Apr 2;19(4):1057, [PMID: 29614819]. It has been assigned GeneID 7412, and its synonyms are CD106 and INCAM-100.

[0114] In this specification, "VCAM-1 inhibitor," also known as "Vascular Cell Adhesion Molecule-1 inhibitor," refers to a substance or entity that inhibits the activity of VCAM-1 (Gene ID: 7412). VCAM-1 inhibitors modulate the function of molecules involved in cell adhesion and inflammatory responses. Specifically, VCAM-1 inhibitors suppress inflammatory responses by inhibiting ligand binding to VCAM-1, thereby reducing the migration of leukocytes to the site of inflammation. These inhibitors are promising tools in the treatment research of inflammatory diseases, cardiovascular diseases, and cancer. Specific examples of VCAM-1 inhibitors include, but are not limited to, Firategrast (SB-683699), Anti-VCAM-1 inhibitors, Adherex ADH-1, TBC-1269 (Selbexin), AL-1576, BIO5192, 7H9, Small molecular inhibitors (e.g., glycomimetics), Heparin derivatives, PSMA-1, AGI-1067, and BAY 11-7082. (Nizamutdinova IT et al.) , “Hesperidin, hesperidin methyl chlorone and phellopterin from Poncirus trifoliata (Rutaceae) differentially regulate the expression of adhesion molecules in tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells”, Int Immunopharmacol. See also 2008 May;8(5):670-8, [PMID: 18387509] and Hutchinson M. “Natalizumab: A new treatment for relating remitting multiple sclerosis”, The Clin Risk Manag. 2007 Jun;3(2):259-68, [PMID: 18360634].In certain embodiments, the VCAM-1 inhibitor disclosed in WO2020 / 236890 is included.

[0115] The effectiveness of VCAM-1 inhibitors can be measured using cell adhesion assays or competitive inhibition studies of VCAM-1 ligand binding. See also Deok-Hoon Kong et al., “Emerging Roles of Vascular Cell Adhesion Molecule-1 (VCAM-1) in Immunological Disorders and Cancer”, Int J Mol Sci. 2018 Apr 2;19(4):1057, [PMID: 29614819]. Generally, flow cytometry or ELISA is used to measure leukocyte adhesion and migration after inhibitor treatment. Analysis of inflammatory responses and atherosclerosis using animal models is also used to evaluate the effectiveness.

[0116] In this specification, “ICAM-1” refers to a type of cell adhesion molecule, a protein that plays a crucial role in immune and inflammatory responses. ICAM-1 is primarily expressed in endothelial cells, epithelial cells, macrophages, and some immune cells. See also Triet M Bui et al., “ICAM-1: A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis”, J Leukoc Biol. 2020 Sep;108(3):787-799, [PMID: 32182390]. It has been assigned GeneID 3383, and its synonyms are BB2, CD54, and P3.58.

[0117] In this specification, “ICAM-1 inhibitor” is also referred to as “Intercellular Adhesion Molecule-1 inhibitor,” and modulates the function of molecules involved in cell adhesion and inflammatory responses. Specifically, ICAM-1 inhibitors suppress inflammatory responses by inhibiting ligand binding to ICAM-1, thereby reducing the migration of leukocytes to the site of inflammation. These inhibitors are promising tools in the therapeutic research of inflammatory diseases, cardiovascular diseases, and cancer. Specific examples of ICAM-1 inhibitors include, but are not limited to, Enlimomab (R6.5), Alicaforsen, BMS-539, BIRB 796, Anti-ICAM-1 inhibitors, Small molecular inhibitors (e.g., LFA-1 inhibitors), ICAM-1 inhibitor oligonucleotides, Fostamatib, Heparin derivatives, Ginsenoside Rg3, myricetin, ezrin (EZR), CD18, NFκB activation inhibitor, RAPTIVA, and A-205804.Also, Zhu GD et al. , “Selective inhibition of ICAM-1 and E-selectin expression in human endothelial cells. 2. Arylmodifications of 4-(aryloxy)thieno[2,3-c]pyridines with fine-tuning at C-2 carbamides”, J Med Chem. 2001 Oct 11;44(21):3469-87, [PMID: 11585452] and Stewart AO et al. See also “Discovery of inhibitors of cell adhesion molecular expression in human endotherial cells. 1. Selective inhibition of ICAM-1 and E-selectin expression”, J Med Chem. 2001 Mar 15;44(6):988-1002, [PMID: 11300880]. In certain embodiments, the ICAM-1 inhibitor disclosed in WO2017 / 147540 is included.

[0118] The effectiveness of ICAM-1 inhibitors can be measured using cell adhesion assays or competitive inhibition studies of ICAM-1 ligand binding. (riet M Bui et al., “ICAM-1: A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis” J Leukoc Biol. 2020 Sep;108(3):787-799, [PMID: 32182390]) Generally, flow cytometry or ELISA is used to measure leukocyte adhesion and migration after inhibitor treatment. Analysis of inflammatory responses and atherosclerosis using animal models is also used to evaluate the effectiveness.

[0119] In this specification, the following methods are used to distinguish between LATS1 inhibitors and LATS2 inhibitors. LATS1 and LATS2 are protein kinases involved in the Hippo signaling pathway, each possessing specific functions and roles. The following methods are effective for distinguishing between LATS1 inhibitors and LATS2 inhibitors: 1. Gene-specific expression analysis: For example, since the expression of LATS1 and LATS2 is controlled by different genes, they can be distinguished by analyzing their respective gene-specific expression. For this purpose, real-time PCR (RT-PCR) can be used to measure the mRNA levels of LATS1 and LATS2 using specific primers. Alternatively, RNA sequencing (RNA-Seq) can be used to identify the expression of LATS1 and LATS2 from the overall gene expression profile; 2. Protein level detection using specific antibodies can be employed. In this case, specific antibodies against LATS1 and LATS2 can be used to evaluate protein-level expression and activity. Western blotting: The presence of proteins can be detected using antibodies specific to LATS1 and LATS2, respectively. Immunoprecipitation (IP): LATS1 or LATS2 can be precipitated using a specific antibody and then detected by Western blotting. 3. Functional analysis using specific inhibitors can be used, as this allows for the differentiation of LATS1 and LATS2 functions, suggesting that some inhibitors are selective for specific kinases. 3. Differentiation can also be achieved through downstream signal analysis, where the functional differences between LATS1 and LATS2 can be evaluated by analyzing the changes in downstream signaling molecules (e.g., YAP, TAZ) associated with the activation of LATS1 and LATS2. LATS1-specific signals and functions include the regulation of cell division (LATS1 is considered to play a particularly important role in the process of cell division).For example, LATS1 has been shown to regulate centrosome division during the M phase of cell division, and to regulate apoptosis (LATS1 also plays a role in promoting apoptosis, and has been reported to activate the apoptotic pathway in response to certain stress responses and DNA damage).

[0120] LATS2 may be involved in specific signaling and functional regulation of the G1 / S phase of the cell cycle (LATS2 is particularly emphasized for its role in regulating the progression of the G1 / S phase of the cell cycle, and LATS2 deficiency has been reported to cause specific abnormalities in cell cycle regulation), and interaction with the p53 pathway (LATS2 has been reported to interact with the tumor suppressor p53, and is thought to play a role in regulating cell proliferation through this pathway. LATS2 contributes to apoptosis and arrest of cells by assisting in the stabilization and activation of p53). Regarding common downstream signaling, both kinases (LATS1 and LATS2) regulate gene expression by phosphorylating the major downstream signaling molecules YAP (Yes-associated protein) and TAZ (Transscriptual co-activator with PDZ-binding motif), inhibiting their translocation to the nucleus. These processes are involved in cell proliferation, apoptosis, and stem cell maintenance. While LATS1 and LATS2 share many common functions and downstream signaling molecules, they are suggested to have specific physiological roles and signaling pathways. In particular, LATS1 is thought to play an important role in cell division and apoptosis, while LATS2 plays a crucial role in the regulation of the G1 / S phase of the cell cycle and the p53 pathway. These distinctions can be made using methods such as fluorescence imaging (observing the intracellular localization of downstream signaling molecules and confirming the effects of LATS1 or LATS2 inhibition) and reporter assays (using a reporter system that measures the transcriptional activity of YAP / TAZ to evaluate changes in LATS1 or LATS2 activity). By combining these methods, LATS1 and LATS2 inhibitors can be effectively distinguished, and their specific roles and functions can be analyzed.

[0121] In one embodiment, the “halcinonides” of this disclosure are defined by the following formula (1): It can be expressed as follows: Here, R １ and R ２ Each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), or R １ and R ２ They come together to form a ring, R ３ is a halo or hydrogen, and R ４ These substituents can be a halo or an optionally substituted hydroxyl group, and these substituents can be in various combinations as follows:

[0122] In this specification, "alkyl" means a linear or branched saturated hydrocarbon group, for example, "C １－６ "Alkyl" refers to alkyl groups with 1 to 6 carbon atoms. The same applies to other numbers. In this specification, it is understood that even when subscripts attached to carbon atoms are not subscripts, they have the same meaning. That is, C １－６ C1-6 and C1-6 have the same meaning. In this specification, the term "group" is understood to have the same meaning whether it is included or omitted. That is, "alkyl group" and "alkyl" are understood to have the same meaning.

[0123] In this specification, alkyl is defined as "C １－８ It can be "alkyl" and "C １－８ "Alkyl" refers to a linear or branched saturated hydrocarbon group with 1 to 8 carbon atoms. １－８ Preferably, "C" is used as "alkyl". １－６ Examples include "alkyl", and more preferably "C １－３ "Alkyl" is one example. １－３ Specific examples of "alkyl" include, for example, methyl, ethyl, propyl, 1-methylethyl, etc. １－６ A specific example of "alkyl" is, for example, the aforementioned "C １－３In addition to specific examples of "alkyl," other examples include butyl, 2-methylpropyl, 1-methylpropyl, 1,1-dimethylethyl, pentyl, 3-methylbutyl, 2-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, etc. １－８ A specific example of "alkyl" is, for example, the aforementioned "C １－６ In addition to specific examples of "alkyl," heptyl and octyl are also mentioned.

[0124] In this specification, "alkoxy" means an oxy group substituted with "alkyl". １－６ "Alkoxy" refers to the aforementioned "C １－６ This refers to an oxy group substituted with "alkyl". １－６ Preferably, "C" is used as "alkoxy". １－４ "Alkoxy" is one example, and more preferably "C １－３ "Alkoxy" is one example. １－３ Specific examples of "alkoxy" include, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, etc. １－４ A specific example of "alkoxy" is, for example, the aforementioned "C １－３ In addition to specific examples of "alkoxy," other examples include butoxy, 2-methylpropoxy, 1-methylpropoxy, and 1,1-dimethylethoxy. １－６ A specific example of "alkoxy" is, for example, the aforementioned "C １－４ In addition to specific examples of "alkoxy," other examples include pentyloxy, 3-methylbutoxy, 2-methylbutoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, 1,1-dimethylpropoxy, hexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 1-methylpentyloxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, and 1,2-dimethylbutoxy.

[0125] In this specification, "hydroxyl" or "hydroxy" means the -OH group.

[0126] In this specification, the term "hydroxyalkyl" means at least one -OH group added to the parent molecule by an alkylene group as defined herein.

[0127] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents. Substituents include, but are not limited to, halogens, =O (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0128] term This refers to a single bond. or double bond or triple bond It represents.

[0129] term This refers to a single bond. or double bond It represents.

[0130] In this specification, "R １ and R ２ In this specification, "together form a ring" means R １ and R ２ Toga, R １ and R ２ This refers to the formation of a fused ring with a part of a ring that has a specific ring. Examples of such rings include dioxolanes.

[0131] (Halcinonides) Examples of hallucinonides include, for example,

[0132] (1A) or

[0133]

[0134]

[0135] Examples include pharmaceutically acceptable salts or solvates thereof.

[0136] (TGFβ receptor type 1 kinase inhibitor, which is also an ALK4 / 7 inhibitor) Description of ALK4 / 7 inhibitor and TGFβ receptor type 1 kinase inhibitor ALK4 / 7 inhibitor and TGFβ receptor type 1 kinase inhibitor are substances or entities that inhibit the activity of ALK4 (Activin Receptor-Like Kinase 4, Gene ID: 92), ALK7 (Activin Receptor-Like Kinase 7, Gene ID: 9825), and TGFβ receptor type 1 (TGFBR1, Gene ID: 7046), and regulate cell proliferation, differentiation, and signal transduction. These kinases play a central role in the TGFβ (Transforming Growth Factor-beta) signaling pathway, particularly in development, physiological homeostasis, and pathological processes. Generally, the following methods are used: 1. Western blotting (measuring the level of phosphorylated Smad2 / 3 to evaluate the inhibitory effects on ALK4 / 7 and TGFβR1); 2. ELISA (using specific antibodies to measure the level of phosphorylated Smad2 / 3 in proteins extracted from cells or tissues); 3. Reporter assays (measuring the expression of reporter genes (e.g., luciferase bound to the PAI-1 promoter) that reflect activation of the TGFβ signaling pathway); 4. Cell proliferation and differentiation assays (performing cell proliferation assays (e.g., MTT assays) and cell differentiation assays to evaluate the inhibitory effects on ALK4 / 7 and TGFβR1); 5. Real-time PCR (measuring mRNA levels of genes related to the TGFβ signaling pathway (e.g., PAI-1, Snail, Slug) to evaluate the effects of inhibitors). By combining these methods, the effects of ALK4 / 7 and TGFβ receptor type 1 kinase inhibitors can be evaluated in detail, and their impact on cellular signaling and function can be analyzed.

[0137] Specific examples of ALK4 / 7 and TGFβ receptor type 1 kinase inhibitors include, for example, SB-431542, LY364947, A-83-01, GW788388, RepSox, Galunicertib (LY2157299), SD-208, SB-505124, Vactosertib (TEW-7197), and EW-7197. The effects of ALK4 / 7 and TGFβ receptor type 1 kinase inhibitors can be measured by evaluating the phosphorylation state of the target kinase and the changes in downstream signaling molecules (e.g., Smad2 / 3) (Inman GJ, et al. “SB-431542 is a potential and specific inhibitor of transforming growth factor-beta superfamily type I-activating receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7” Mol Pharmacol. 2002;62(1):65-74).

[0138] (BRD7 / 9 binding molecules (degradation factors) that are also BRD7 / 9 dual degradation factors) BRD7 / 9 binding molecules (degradation factors) that are also BRD7 / 9 dual degradation factors refer to substances or entities designed to specifically target and degrade the activity of BRD7 (Bromodomain Containering 7, Gene ID: 29117) and BRD9 (Bromodomain Containering 9, Gene ID: 26038). These degradation factors are designed using PROTAC (Proteolytic Targeting Chimera) technology and inhibit the function of BRD7 and BRD9 by degrading them via the ubiquitin-proteasome system. This makes it possible to treat conditions involving the activation of BRD7 and BRD9. Examples of BRD7 inhibitors include BRD7-IN-2, BRD7-IN-1, LP99, TP-472, BI-7273, BI-9564, BRD7-IN-1 free base, BI7273, BRD7-IN-3, (2S,3R)-LP99, Bromodomain IN-2, and VZ185. Examples of BRD9 inhibitors include PROTAC BRD9-binding moiety 1 hydrochloride, LP99, GNE-375, TP-472, BI-7273, BI-9564, PROTAC BRD9-binding moiety 1, (2S,3R)-LP99, dBRD9 dihydrochloride, BRD7-IN-3, TP-238, TP-238 hydrochloride, GNE-886, BRD7-IN-2, Bromodomain IN-2, FHD-609, GSK8573, QA-68, QA-68-ZU81, and VZ185. These are BRD7 / 9 dual degradation factors. Specific examples of BRD7 / 9 binding molecules (degradation factors) include dBET1, MZ1, ARV-771, AT1, BSJ-03-123, A1874, BI-894999, JQ1-PROTAC, BETd-246, ZXH-3-26, and VZ185.

[0139] The effect of BRD7 / 9 binding molecules (degradation factors), which are also BRD7 / 9 dual degradation factors, can be measured by evaluating the degradation status of BRD7 and BRD9 and the fluctuations of downstream signaling molecules (Lu J, et al. “Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4” Chemistry & Biology. 2015;22(6):755-763). Generally, the following methods are used: 1. Western blotting (using Western blotting to measure the degradation status of BRD7 and BRD9 proteins and evaluate the effect of binding molecules); 2. Immunoprecipitation assay (using antibodies specific to BRD7 and BRD9 to evaluate the presence and degradation status of these proteins by immunoprecipitation); 3. 1. Real-time PCR (measuring mRNA levels of BRD7 and BRD9 to evaluate the effect of binding molecules on gene expression); 2. Proteasome activity assay (measuring proteasome activity to confirm that BRD7 and BRD9 degradation is mediated by the ubiquitin-proteasome system); 3. Cell proliferation and apoptosis assay (using MTT assays or flow cytometry to evaluate the effects of BRD7 and BRD9 degradation on cell proliferation and apoptosis). By combining these methods, the effects of BRD7 / 9 binding molecules (degradation factors) can be evaluated in detail, and their impact on intracellular signaling and function can be analyzed.

[0140] (AKT1 inhibitor, which is also a LATS1 / 2 inhibitor or PKA inhibitor) AKT1 inhibitor, which is also a LATS1 / 2 inhibitor and a PKA inhibitor, is a multifunctional substance or entity that inhibits the activity of AKT1 (Gene ID: 207), LATS1 (Gene ID: 9113), LATS2 (Gene ID: 26524), and PKA (Protein Kinase A). These inhibitors are used to regulate cell proliferation, apoptosis, and metabolic signaling pathways. In particular, inhibiting the phosphorylation of AKT1 leads to tumor suppression and promotion of apoptosis, and regulating cell proliferation by regulating the Hippo signaling pathway through the inhibition of LATS1 / 2. Examples of LATS1 inhibitors include TRULI (LATS-IN-1), VT02956, MGH-CP1, GA-017, KMH-233, BCH (2-Amino-2-norbornanecarboxyl acid; LAT1-IN-1), GPNA hydrochloride, VT02956 hydrochloride, (R)-KMH-233, and A-674563. Examples of LATS2 inhibitors include TRULI (LATS-IN-1), VT02956, GA-017, VT02956 hydrochloride, and A-674563. Examples of LATS1 / 2 inhibitors include MGH-CP1, TRULI (LATS-IN-1), VT02956, GA-017, GPNA hydrochloride, KMH-233, BCH (2-amino-2-norbornanecarboxyl acid), LATS1 Human Pre-designed siRNA Set A, VT02956 hydrochloride, (R)-KMH-233, QBS10072S, and A-674563. PKA inhibitors include AKT1-IN-1, BAY1125976, Vevorisertib trihydrochloride, AKT1-IN-3, AKT1-IN-5, AKT1-IN-6, AKT1-IN-7, AKT1-IN-4, AKT1 / Akt2-IN-2, AKT1&PKA-IN-2,AKT1 / Akt2-IN-1, 3CAI, A-674563 hydrochloride, A-674563, AKT1&PKA-IN-1, AKT1-IN-2, AKT-IN-2, Tubulin / AKT1-IN-1, AKT-IN-5, Capivasertib, Uprosertib, Uprosertib hydrochloride, Miransertib, Miransertib hydrochloride, CaMKII-IN-1, MS15, A-443654, AKT-IN-8, MS15 TFA, Ipatasertib dihydrochloride, DB07107, Afuresertib, Afuresertib hydrochloride, AKT-IN-6, AKT inhibitor VIII, Hu7691, AT7867, AT7867 dihydrochloride, AKT-IN-13, Oridonin, Maximum Cited Publications, MK-2206 dihydrochloride, Hu7691 free base, 24-Methylenecycloartanyl ferulate, AT13148, ALM301, M2698, Pifusertib hydrochloride, Pifusertib, Oridonin (Standard), GSK-690693, INY-03-041, INY-03-041 trihydrochloride, MK-2206, Ipatasertib, NTQ1062, SK1-I, SK1-I hydrochloride, Vevorisertib, MK-2206 free Examples include base, ZLY06, AKT-IN-3, (32-Carbonyl)-RMC-5552, Gamendazole, 9-Decyn-1-ol, SC79, APN / AKT-IN-1, SQLE-IN-1, BX-912, Azurin p28 peptide, SMART1, Azurin p28 peptide TFA, PTP1B-IN-24, Z / E-Guggulsterone, A-674563, 5-iodotubercidin or its pharmaceutically acceptable salts or solvates. Examples of PKA inhibitors include H-89, KT5720, Rp-cAMP, PKI (14-22)amide.Examples include SQ22536, Myr-PKI, 6-Benzoyl-cAMP, cAMPS-Rp, PKAi, cAMPS-Sp, H7, H8, H9, HA1077, HA1004, chererythrine, and A-674563. Specific examples of AKT1 inhibitors that are also LATS1 / 2 inhibitors or PKA inhibitors include MK-2206, XMU-MP-1, Verteporfin, AZD5363 (Capivasertive), GSK690693, Perifosine, Triciribine (API-2), A-443654, Ipatasertive (GDC-0068), AT7867, and A-674563.

[0141] The effect of the AKT1 inhibitor, which is also a LATS1 / 2 inhibitor or a PKA inhibitor, can be measured by evaluating the phosphorylation state of the target kinase and the changes in downstream signaling molecules (Meng Z, et al. "Mechanisms of Hippo pathway regulation." Genes Dev. 2016;30(1):1-17) (Alessi DR, et al. "Mechanism of activation of protein kinase B by insulin and IGF-1" EMBO J. 1996;15(23):6541-51). Generally, the following method is used: 1. 1. Western blotting (Western blotting is used to measure the levels of phosphorylated AKT1, LATS1 / 2, and PKA substrates to evaluate the inhibitor's effect); 2. ELISA (Specific antibodies are used to measure the levels of phosphorylated AKT1, LATS1 / 2, and PKA substrates in proteins extracted from cells or tissues); 3. Real-time PCR (MRNA levels of genes related to AKT1, LATS1 / 2, and PKA signaling are measured to evaluate the inhibitor's effect); 4. Cell proliferation and apoptosis assays (Cell proliferation assays (e.g., MTT assay) and apoptosis assays (e.g., flow cytometry) are performed to evaluate the inhibitor's effect); 5. Protein kinase assays (Assemblages using radiophosphate or fluorescently labeled substrates are performed to directly measure the activity of specific kinases). By combining these methods, the effects of LATS1 / 2 inhibitors and AKT1 inhibitors can be evaluated in detail, and their impact on intracellular signaling and function can be analyzed.

[0142] (Adenosine kinase inhibitor that is also a CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor, or PKC inhibitor) This multifunctional adenosine kinase inhibitor, which is also a CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor, and PKC inhibitor, is adenosine kinase inhibitor that is also a CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor, and PKC inhibitor. A) A substance or entity that inhibits the activity of PKC (Protein Kinase C) and adenosine kinase (Gene ID: 132). These kinases play important roles in cell proliferation, differentiation, metabolism, and signal transduction, and their inhibition is useful in therapeutic research for cancer, diabetes, neurological disorders, inflammatory diseases, and other conditions. Specific examples of this inhibitor include AG-1024 (insulin receptor Tyr kinase inhibitor), H-89 (PKA inhibitor), Staurosporine (PKC inhibitor), 5-Iodotubercidin (adenosine kinase inhibitor), D4476 (CK1 / 2 inhibitor), IC261 (CK1 inhibitor), CX-4945 (Silmitasertib) (CK2 inhibitor), LY294002 (insulin receptor Tyr kinase inhibitor), Bisindolylmaleimide I (GF 109203X) (PKC inhibitor), and Rp-cAMP (PKA inhibitor), any of which may possess all of these functions.

[0143] The effect of this multifunctional inhibitor can be measured by evaluating the phosphorylation state of the target kinase and the changes in downstream signaling molecules (Boison D, “Adenosine kinase: exploitation for therapeutic gain” Pharmacol Rev. 2013;65(3):906-43) (Martiny-Baron G, et al. “Selective inhibition of protein kinase C isozymes by the indolocarbazol Go 6976” J Biol Chem. 1993;268(13):9194-7). Generally, the following methods are used: 1. 1. Western blotting (to measure the phosphorylation status of each kinase and evaluate the effect of the inhibitor. Specific antibodies are used to detect the phosphorylation levels of PKA, PKC, CK1, CK2, insulin receptor, and Tyr kinase); 2. ELISA (ELISA with specific antibodies is used to quantitatively measure the phosphorylation status of each kinase); 3. Real-time PCR (to measure the mRNA levels of genes associated with each kinase signaling and evaluate the effect of the inhibitor); 4. Cell proliferation and apoptosis assays (MTT assays and flow cytometry are used to evaluate the effects of inhibition of each kinase on cell proliferation and apoptosis); 5. Protein kinase assays (assays using radiophosphate or fluorescently labeled substrates are performed to directly measure the activity of each kinase). By combining these methods, the effects of this multifunctional inhibitor can be evaluated in detail, and its impact on intracellular signaling and function can be analyzed.

[0144] (ICAM1 inhibitor, which is also an E-selectin inhibitor or VCAM1 inhibitor) ICAM-1 inhibitor, which is also an E-selectin inhibitor or VCAM-1 inhibitor, is a multifunctional substance or entity that inhibits the activity of ICAM-1 (Intercellular Adhesion Mole-1, Gene ID: 3383), E-selectin (Gene ID: 6401), and VCAM-1 (Vascular Cell Adhesion Mole-1, Gene ID: 7412). These molecules play important roles in cell adhesion and inflammatory responses, and their inhibition is useful in therapeutic research for inflammatory diseases, cardiovascular diseases, and cancer. Specific examples of these inhibitors include Enlimomab (R6.5) (ICAM-1 inhibitor), Alicaforsen (ICAM-1 inhibitor), BMS-539 (ICAM-1 inhibitor), BIRB 796 (ICAM-1 inhibitor), Anti-ICAM-1 inhibitors (ICAM-1 inhibitor), GMI-1070 (Rivipansel) (E-selectin inhibitor), Bimosiamose (E-selectin inhibitor), Uproleselan (GMI-1271) (E-selectin inhibitor), Firategrast (SB-683699) (VCAM-1 inhibitor), and Adherex. This could be ADH-1 (VCAM-1 inhibitor), among other things.

[0145] The effect of this multifunctional inhibitor can be measured by evaluating ligand binding inhibition of ICAM-1, E-selectin, and VCAM-1, as well as fluctuations in downstream signaling molecules (Rothlein R, et al. “A human intercellular addiction molecule (ICAM-1) inhibit from LFA-1” J Immunol. 1986;137(4):1270-4) (Gearing AJ, Newman W. “Circulating addiction molecules in disease” Immunol Today. 1993;14(10):506-12). Generally, the following methods are used: 1. 1. Western blotting (evaluating the inhibitor's effect by measuring the expression levels of ICAM-1, E-selectin, and VCAM-1 using specific antibodies); 2. ELISA (quantitatively measuring the levels of ICAM-1, E-selectin, and VCAM-1 in proteins extracted from cells or tissues using specific antibodies); 3. Flow cytometry (evaluating the inhibition of ICAM-1, E-selectin, and VCAM-1 activity by measuring the adhesion and migration of leukocytes after inhibitor treatment); 4. Cell adhesion assay (measuring the effect of the inhibitor by adding cells to plates coated with ICAM-1, E-selectin, and VCAM-1); 5. Analysis of inflammatory responses in animal models (analyzing inflammatory responses and atherosclerosis induced by inhibitor administration using animal models). By combining these methods, the effects of this multifunctional inhibitor can be evaluated in detail, and its impact on signal transduction and function of cell adhesion molecules can be analyzed.

[0146] (LATS1 / 2 inhibitors (those that inhibit both) LATS1 / 2 inhibitors (those that inhibit both) are LATS1 (Large Tumor Suppressor Kinase 1, Gene ID: 9113) and LATS2 (Large Tumor Suppressor Kinase 2, Gene ID: LATS1 / 2 inhibitors are substances or entities that simultaneously inhibit the activity of LATS1 / 2 protein kinases, regulating cell proliferation, apoptosis, and tissue homeostasis through the Hippo signaling pathway. These kinases control cell proliferation and play an important role in tumor suppression. By inhibiting LATS1 / 2 activity, they promote cell proliferation and regulate the expression of specific genes. Specific examples of LATS1 / 2 inhibitors include XMU-MP-1, Verteporfin, CA3, D4476, Triciribine (API-2), GNE-7915, Flufenamic acid, Dasatinib, Tizoxandide, and Fasudil.

[0147] The effect of LATS1 / 2 inhibitors can be measured by evaluating the phosphorylation status of LATS1 and LATS2, or by measuring changes in downstream signaling molecules (e.g., YAP, TAZ) (Meng Z, et al. “Mechanisms of Hippo pathway regulation.” Genes Dev. 2016;30(1):1-17). Generally, the following methods are used: 1. Western blotting (Western blotting is used to measure the levels of phosphorylated LATS1 and LATS2 and evaluate the effect of the inhibitor); 2. ELISA (Specific antibodies are used to measure the levels of phosphorylated LATS1 and LATS2 in proteins extracted from cells or tissues); 3. 1. Real-time PCR (measuring mRNA levels of genes related to LATS1 and LATS2 signaling (e.g., CTGF, CYR61, AMOTL2) to evaluate the effects of inhibitors); 2. Cell proliferation and apoptosis assays (using MTT assays or flow cytometry to evaluate the effects of LATS1 / 2 inhibition on cell proliferation and apoptosis); 3. Fluorescence imaging (using fluorescently labeled probes to visualize the intracellular localization of downstream signaling molecules (e.g., YAP, TAZ) associated with LATS1 and LATS2 activation); 4. Reporter assays (measuring the expression of reporter genes (e.g., luciferase reporters containing TEAD binding sites) to measure the transcriptional activity of YAP / TAZ). By combining these methods, the effects of LATS1 / 2 inhibitors can be evaluated in detail, and their impact on intracellular signaling and function can be analyzed.

[0148] In this specification, "halcinonide or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0149] In this specification, “pharmaceutically acceptable salt” means an inorganic or organic acid addition salt of the compound of this disclosure that is relatively nontoxic. These salts can be prepared temporarily during the final isolation and purification of the compound, or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid, and then isolating the salt thus formed.

[0150] The pharmaceutically acceptable salts of the compounds disclosed herein are not particularly limited as long as they are pharmaceutically acceptable, but specifically include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; aliphatic monocarboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid; aromatic monocarboxylic acids such as benzoic acid and salicylic acid; aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; and aliphatic tricarboxylic acids such as citric acid. Examples include organic carboxylic acids; aliphatic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, and 2-hydroxyethanesulfonic acid; aromatic sulfonic acids such as benzenesulfonic acid and p-toluenesulfonic acid; acid addition salts with acidic amino acids such as aspartic acid and glutamic acid; salts with metals such as alkali metals or alkaline earth metals such as sodium, potassium, magnesium, and calcium; salts with organic bases such as methylamine, ethylamine, ethanolamine, pyridine, lysine, arginine, and ornithine; and ammonium salts.

[0151] These salts can be obtained by conventional methods, for example, by mixing an equivalent amount of the compounds of this disclosure with a solution containing a desired acid or base, and then filtering out the desired salt or by distilling off the solvent. Furthermore, the compounds of this disclosure or their salts can form solvates with solvents such as water, ethanol, and glycerol.

[0152] In this specification, "solvate" refers to a solvate of the compound disclosed herein or a pharmaceutically acceptable salt thereof, and includes, for example, solvates with organic solvents (e.g., alcohol (ethanol, etc.) dihydrates), hydrates, etc. When forming a hydrate, it may be coordinated with any number of water molecules. Examples of hydrates include monohydrates, dihydrates, etc.

[0153] In this specification, "clobestazol or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0154] In this specification, "AZ12601011 or its pharmaceutically acceptable salt or solvate" means or a pharmaceutically acceptable salt or solvate thereof.

[0155] In this specification, "VZ185 or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0156] In this specification, "A-674563 or its pharmaceutically acceptable salt or solvate" means or a pharmaceutically acceptable salt or solvate thereof.

[0157] In this specification, "5-iodotubercidin or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0158] In this specification, "ICAM-1-IN-1 or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0159] In this specification, "LATS-IN-1 or a pharmaceutically acceptable salt or solvate thereof" means or a pharmaceutically acceptable salt or solvate thereof.

[0160] (Method for culturing cells) A method for culturing cells or cell populations, such as mesenchymal stem cells and other types of stem cells, using the components of this disclosure can be carried out by any method known in the art. In a method for culturing cells or cell populations, such as mesenchymal stem cells and other types of stem cells, cells obtained from various tissues are cultured using the components of this disclosure appropriately. The method for culturing cells or cell populations, such as mesenchymal stem cells and other types of stem cells isolated from tissues, is not particularly limited as long as it is a method suitable for each type of cell, and conventional methods can be used. Typically, a temperature of 30°C to 37°C and 2% to 7% CO2 are used. ２ Under the environment, 5% to 21% O ２ The procedure is carried out in an environmental environment. Furthermore, the timing and method of subculturing various stem cells, such as mesenchymal stem cells, are not particularly limited as long as they are suitable for the various stem cells, and can be carried out in the same manner as before while observing the morphology of the various stem cells, such as mesenchymal stem cells.

[0161] The culture medium for various stem cells, such as mesenchymal stem cells, is not particularly limited as long as it is a medium suitable for culturing various stem cells, such as mesenchymal stem cells, and conventionally known media to those skilled in the art can be used together with the components of this disclosure. Examples of such media include culture media for various stem cells, such as mesenchymal stem cells, from companies such as Promo Cell, Life Line, and Lonza. The culture medium may contain biological raw materials (e.g., animal serum). Considering that the resulting cells may be used to treat diseases in animals (including humans), it is preferable to use a culture medium that contains as few biological raw materials as possible (e.g., serum-free medium).

[0162] After culturing the cells as described above, and passing them an appropriate number of times considering the state of the cells, the cells are usually detached by treatment with enzymes such as trypsin and collagenase one to five days later, preferably one to four days later, and more preferably one to three days later, and then recovered by centrifugation. Alternatively, the culture supernatant is removed.

[0163] In a preferred implementation liquid, the culturing step includes culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature.

[0164] Regarding the range of cell numbers, low-density seeding is rare for MSCs, and preferably, 500 cells / cm² is preferred. ２ ~20000 cells / cm ２ A certain level is sufficient, and more preferably, 500-1000 cells / cm². ２ ~10000 cells / cm ２ For example, 2000 cells / cm² ２ Seeding is often performed before or after seeding, but is not limited to these methods. During screening, a specific value such as 1500 cells / well may be adopted, and the process can also be carried out under conditions such as 4500 cells / well, which are estimated to result in confluence. In addition, appropriate materials (including whether or not they are coated) and appropriate temperatures (25-37°C, or 30°C, etc.) can be used.

[0165] The cells of this disclosure show no significant decrease in the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105, even after long-term passage.

[0166] It is preferable that none of the CD44, CD73, CD90, or CD105 levels decrease. For example, CD44 often does not decrease. Since the others often decrease, the absence of a decrease in at least one of these CD73, CD90, and CD105 can be considered an important characteristic. High CD90 levels can also be considered a characteristic. Maintaining the results after long-term culture means that it can be carried out up to the 8th passage or more, whereas conventionally the 5th passage was the maximum. "Not decreased" means that the levels do not decrease below the level at which they normally decrease. Expression levels usually decrease at the 5th passage, and it is common to use the culture up to the 6th passage, so being able to use it beyond the 6th passage is an unexpected effect. These can also be called long-term culture. Maintaining the above four markers (especially at least one of the latter three markers) even after the 6th passage can be considered a characteristic. Here, CD90, CD73, and CD44 can be independently evaluated as "high" if they are 80-90% or higher (95% or higher is also acceptable). This is because it only drops to about 70%. On the other hand, CD105 drops easily, so it can be considered "high" at 30% or 50% or higher.

[0167] (Maintenance of various cells) The maintenance of cells such as various stem cells including mesenchymal stem cells can be achieved by adding appropriate components to a solution containing various stem cells such as mesenchymal stem cells and incubating for a certain period of time. The solution containing various stem cells such as mesenchymal stem cells may be a medium as long as it contains the components of the present disclosure, or may be a processing solution that is not a medium.

[0168] The method for culturing various stem cells such as mesenchymal stem cells in a medium supplemented with various stem cells such as mesenchymal stem cells is not particularly limited as long as it is a method suitable for each cell, and a method similar to the conventional method is used. There is no limitation on the concentration of various stem cells such as mesenchymal stem cells in the medium, but preferably it is 0.0001 to 1.0% by weight, more preferably 0.0002 to 0.1% by weight, still more preferably 0.0003 to 0.05% by weight, and particularly preferably 0.0005 to 0.01% by weight. Usually, at a temperature of 30°C to 37°C, 2% to 10% CO ２ environment, and 5% to 21% O ２ It is carried out under the environment. Also, there is no limitation on the time for contacting various stem cells such as mesenchymal stem cells with various stem cells such as mesenchymal stem cells, and the timing and method of passage of various stem cells such as mesenchymal stem cells are not particularly limited as long as they are suitable for each of the various stem cells such as mesenchymal stem cells. It can be carried out in the same manner as in the conventional method while observing the morphology of various stem cells such as mesenchymal stem cells. The contact time with various stem cells such as mesenchymal stem cells is preferably 12 hours to 2 weeks, preferably 1 day to 10 days, and more preferably 2 days to 1 week. In addition, the culture may be carried out using a serum-free medium throughout the entire culture period of the cells.

[0169] When performing treatment with various stem cells such as mesenchymal stem cells in a processing solution that is not a medium, there is no limitation on the concentration of various stem cells such as mesenchymal stem cells in the processing solution, but preferably it is 0.0001 to 1.0% by weight, more preferably 0.0002 to 0.1% by weight, still more preferably 0.0003 to 0.05% by weight, and particularly preferably 0.0005 to 0.01% by weight. The contact time with various stem cells such as mesenchymal stem cells is preferably 12 hours to 2 weeks, preferably 1 day to 10 days.

[0170] (Cell Recovery) Cells or cell populations such as mesenchymal stem cells treated with the components of this disclosure are detached from the cells by treatment with enzymes such as trypsin and collagenase at an appropriate time considering the state of the cells, or the mesenchymal stem cells are recovered by centrifugation without treatment with enzymes such as trypsin and collagenase. The recovered mesenchymal stem cells can be used as therapeutic stem cells, either as is or after washing with phosphate buffer or the like. If storage is required, the cells should be stored in a storage container using a storage solution.

[0171] The various stem cells, such as mesenchymal stem cells, obtained by the preparation method of this disclosure have altered types and amounts of secreted proteins such as cytokines and intracellularly expressed proteins, and are stem cells with excellent therapeutic effects. The various stem cells, such as mesenchymal stem cells, obtained by the preparation method of this disclosure are cells in which at least one therapeutic function is enhanced compared to the various stem cells, such as mesenchymal stem cells, that have not been treated with the preparation method. Examples of such therapeutic functions include anti-inflammatory function, immunomodulatory function, antitumor function or tissue repair function, and barrier enhancement function through tight junction formation of epithelial cells.

[0172] One embodiment of this disclosure is a novel method for producing cells. This method includes, for example, a step of horizontally rotating a container containing cells and culture medium, wherein the bottom shape of the container is square. This method can achieve superior cell culture from the viewpoint of at least one of the following: improved cell proliferation rate, improved cell number after culture, improved cell dispersion, uniformity of cell shape, uniformity of cell size, or reduced shear stress. In the examples described later, it is demonstrated that the cell culture method using the square-shaped dish of the example is superior to the cell culture method using the round-shaped dish in terms of at least one of these effects. In particular, since iPS cells and MSCs (various stem cells such as mesenchymal stem cells) are sensitive to shear stress, it is preferable to rotate them at a low speed during culture. However, when rotating at a low speed in a container with a round bottom shape, there is a problem that the cells tend to gather too much in the center. On the other hand, it has been revealed that by using a container with a square bottom shape, it is possible to maintain a state in which the cells do not tend to gather in the center (i.e., are dispersed) even at a low rotation speed that reduces shear stress on the cells. Furthermore, it was found that when rotating at a low speed, it is preferable to keep the liquid level of the culture medium low, and even more so, setting the height of the container low is preferable from the viewpoint of large-scale cultivation.

[0173] In one embodiment of the present disclosure, a method for producing cells may include, for example, the steps of supplying a culture medium to a container, preparing a cell suspension containing cells and culture medium, supplying the cell suspension to a container, sealing the container, placing the container on a rotating device, rotating the container, rotating the cells, rotating and shaking the cells, suspension culturing the cells, or recovering the cells from the container. In another embodiment of the present disclosure, a method for producing cells may include, for example, the steps of replacing the culture medium in the container with an enzyme (e.g., proteolytic enzyme) solution, enzymatically treating the cells, dispersing the cells, converting cell aggregates into single cells, or rotating a container containing a cell suspension containing enzymatically treated or dispersed cells. The step of recovering the cells from the container may include, for example, the step of aspirating the culture medium or cells with a pipette. The rotating device may be, for example, a horizontal rotating device.

[0174] In one embodiment of this disclosure, a cell production method may include a step of horizontally rotating a container with a square base, thereby causing wave-like bouncing in the culture medium during cell culture. In this case, the wave-like bouncing is caused by the movement of the culture medium between two opposing sides, and the waves repeatedly travel back and forth between the two sides. On the other hand, if the container has a circular base, such wave-like bouncing does not occur. Such wave-like bouncing may be used as an indicator that the environment is conducive to cell dispersion during culture.

[0175] In one embodiment of this disclosure, the cells may be, for example, suspension cells, cell aggregates (spheroids), or single cells. The above production method is particularly useful in culturing spheroids that are prone to fusion because it makes it easier to maintain a dispersed state of cells. Furthermore, the above production method is particularly useful in culturing iPS cells and MSCs because it produces less shear stress. The cells may also be mammalian cells. Mammals include, for example, humans, monkeys, rodents (mice, hamsters, etc.). The cells include, for example, stem cells or somatic cells. Stem cells include, for example, cells that have the ability to self-renew and the ability to differentiate into other types of cells. Stem cells include pluripotent stem cells, multipotent stem cells, and unipotent stem cells. Pluripotent stem cells include, for example, iPS cells or ES cells. Pluripotent stem cells may express any undifferentiated marker to the same or greater extent than, for example, human induced pluripotent stem cell strain 253G1 (HPS0002). Pluripotent stem cells include, for example, various stem cells such as mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, and neural stem cells. Unipotent stem cells include, for example, muscle stem cells and pigment stem cells. Somatic cells include, for example, cells derived from skin, heart, liver, lungs, stomach, intestines, kidneys, uterus, brain, blood, or mesenchymal tissue. Cells also include, for example, T cells and CHO cells. Cells may be attached to a carrier or material (for example, a plastic material (for example, plastic beads)).

[0176] In one embodiment of the present disclosure, the culture medium may be a liquid medium as long as it contains the components of the present disclosure. Examples of culture media include stem cell media (e.g., Essential 8 Medium), mammalian cell media (e.g., GIBCO Advanced Medium (Thermo Fisher)), etc. Other examples of culture media include serum media (e.g., 10% FBS), equilibrium salt solutions (e.g., PBS), basal media (MEM), compound media (e.g., RPMI 1640), serum-free media, etc. The culture medium is not particularly limited as long as it can culture various stem cells such as mesenchymal stem cells or precursor cells derived therefrom, and examples include EGM-2 (Lonza), αMEM, Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium / Ham F-12 mixed medium (DMEM / F12), RPMI 1640, etc. These culture media may typically contain various additives applicable to conventional cell culture, such as serum, various vitamins, various antibiotics, various hormones, and various growth factors. Particularly preferred culture media include DMEM / F12, EGM-2, EGM-2MV (both from Lonza), etc.

[0177] The culture is performed using a culture vessel such as a flask, with 5% CO2. ２ It is preferable to carry out the culture at 37°C. The culture medium may be changed, for example, every two days. The oxygen concentration can be 1-21% during culture. Particularly preferable is to culture at an oxygen concentration of 2-6%. The culture period is not particularly limited, but for example, it can be 1 to 14 days. After culturing for 1 to 6 days, the cells may be subcultured and cultured again for, for example, 3 to 6 days. The number of subcultures and the number of cultures are not particularly limited, or they may be within the range of two values. Furthermore, from the above viewpoint, the ratio of the height of the liquid level of the culture medium in the container to the length of one side (e.g., length or width) of the bottom shape of the container is preferably 0.2 or less, and particularly preferably 0.06 or less. This ratio may be, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.1, 0.15, 0.2, 0.25, or 0.3, and may be less than or equal to any of these values, or within the range of any two of these values.

[0178] When the liquid level of the culture medium in a container is set low, the height of the container can also be set low accordingly. Furthermore, setting the height of the container low improves the stability when multiple containers are stacked. Also, by setting the height of the container low, more containers can be installed in the culture equipment (e.g., an incubator), enabling large-scale culture. From the viewpoint of stability when containers are stacked, it is preferable that the height of the container is less than or equal to the length of one side of the base shape. Furthermore, from the viewpoint of providing a culture medium for large-scale culture, improving the cell proliferation rate, improving the number of cells after culture, or improving cell dispersion, the ratio of the height to the length of one side of the base shape (with the length of one side of the base shape being 1) is preferably 0.07 to 0.35, and particularly preferably 0.08 to 0.3. This ratio may be, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15, 0.18, 0.2, 0.25, 0.26, 0.3, 0.35, 0.4, 0.5, 0.6, or 1, and may be less than or equal to any of these values, or within the range of any two of these values. Here, one side of the base shape includes either the longest side or the shortest side, but is preferably the shortest side. The height of the container may be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 80, or 100 cm, and may be within the range of any two of these values. The height of the container may also be expressed, for example, as the shortest distance between the bottom and top of the side. The lowest point may be where the side and bottom surfaces meet, and the highest point may be where the side and top surfaces meet. Alternatively, the height may be expressed as the shortest distance between the bottom and top surfaces. Alternatively, the height may be expressed as the length from the top to the bottom of the container when viewed from the side. If there is a cap on the top surface, the height may be expressed as the shortest distance between the bottom surface and the lowest edge of the cap.

[0179] In one embodiment of the present disclosure, the liquid volume of the culture medium in the container may be, for example, 4, 5, 8, 10, 20, 50, 60, 100, 200, 300, 400, 600, 800, 1000, 1500, or 2000 mL, or may be a liquid volume greater than any of those values, or within the range of any two of those values. In one embodiment of the present disclosure, the liquid volume of the culture medium in the container is, for example, per bottom area of 1 cm ２ It may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 1.5, 2, 4, 6, 8, or 10 mL, or may be a liquid volume less than any of those values, or within the range of any two of those values. In one embodiment of the present disclosure, the filling rate of the culture medium in the container may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or may be a liquid volume less than any of those values, or within the range of any two of those values. The filling rate can be expressed as the ratio of the volume of the culture medium to the volume of the container. In one embodiment of the present disclosure, the cell culture may be carried out, for example, for 1, 5, 10, 20, 24, 48, 72, 96, 120, or 150 hours, or may be carried out for a time less than any of those values, or within the range of any two of those values. The cell culture may be carried out, for example, in a cell culture incubator (e.g., at 37 °C, 5% CO ２ ). Alternatively, the culture conditions conventionally used for cell culture can be appropriately selected. In one embodiment of the present disclosure, the cell concentration in the culture medium is, for example, 5×10 ７ , 10×10 ７ , 30×10 ７ , 50×10 ７ , 80×10 ７ , 100×10 ７ , or 150×10 ７ cells / L, or may be a liquid volume greater than any of those values, or within the range of any two of those values. In one embodiment of the present disclosure, the culture time may be, for example, 0.25, 0.5, 1, 5, 12, 24, 48, 72, 96, 168, or 240 hours, or may be greater than any of those values, or within the range of any two of those values.

[0180] In one embodiment of this disclosure, the container may have an opening. The opening may be on the top, side, or bottom of the container. A biological sample (such as cells) or culture medium can be injected from a space outside the container into a space inside the container through the opening. The opening may have a cylindrical structure or a structure with a circular horizontal cross-section. The opening may be provided with a port or a cap. The port may have a structure (e.g., a cylindrical structure) that connects the space inside the container to the space outside the container. The cap may have a structure that blocks the space inside the container from the space outside the container by covering the port. The cap can seal the inside of the container by sealing the opening. The cap may have a cylindrical structure, for example, with one of its upper or lower ends closed. The cap may be, for example, a screw cap. The opening may be located, for example, at the four corners of a rectangular shape on the outside of the top or side. The opening may be located, for example, on the diagonals of a rectangular shape on the outside of the top or side. If the opening has a cylindrical structure or a circular horizontal cross-section, the central axis of the circle may be located, for example, on the diagonal of a square shape on the outside of the top or side surface. When the opening is installed on the top surface, four openings are particularly preferred. In this case, as shown in Figure 11, it becomes possible to stably stack multiple containers, or the center of gravity is less likely to be unevenly distributed when rotating or oscillating. When the opening is installed on the side surface, one or two openings are particularly preferred. In this case, as shown in Figure 13, it is possible to stack the containers as they are, and a large culture area can be secured. The number of openings may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8, or within the range of any two of these values. By installing multiple openings, it becomes possible to put in culture medium from one opening and remove it from the other opening. The height of the opening may be, for example, 1, 2, 2.5, 3, 3.5, 4, 5, or 10 cm, or within the range of any two of these values. The height of the opening may be measured with the side of the opening that is in contact with the surface as the lower end. If the opening is cylindrical, its diameter may be, for example, 1, 2, 2.5, 3, 3.5, 4, 5, or 10 cm, or within the range of any two of these values.The ratio of the diameter of the opening to the length of one side (e.g., length or width) of the base shape may be, for example, 0.05, 0.1, 0.11, 0.115, 0.12, 0.125, 0.13, 0.135, 0.14, 0.145, or 0.15, with the length of one side of the base shape being 1, and may be less than or equal to any of these values, or within the range of any two of these values. The shapes, lengths, or ratios of the base, sides, or top of the container described above or below may also be applied to the shapes, lengths, or ratios of the inside of the container (the side in contact with the space inside the container, or the side containing the culture medium inside the container). For example, the description that the base of the container is square includes the inside base of the container being square in shape.

[0181] In one embodiment of this disclosure, the area of ​​the bottom surface of the container is, for example, 1, 4, 9, 25, 50, 100, 500, 1000, 2500, 5000, or 10000 cm². ２The number may be 20, 30, 40, 50, 75, 100, 150, 200, 300, or 500 times the sum of the lengths of the two connections, and may be greater than or equal to any of these values, or within the range of any two of these values. In one embodiment of the present disclosure, the bottom and sides of the container may be positioned at approximately right angles to each other. The bottom and sides may be directly connected to form an angle, or connected via a connecting portion (e.g., a straight line or an arc). The connecting portion includes the portion between the straight section of the bottom and the straight section of the side when the container is viewed from the side, and which does not lie on the extension of the straight section of the bottom. Two connecting portions are visible when the container is viewed from the side. The length of the straight section of the bottom when the container is viewed from the side may be 20, 30, 40, 50, 75, 100, 150, 200, 300, or 500 times the sum of the lengths of the two connecting portions, and may be greater than or equal to any of these values, or within the range of any two of these values. The side may include any one side or four side sides. When the connection is arc-shaped, the radius of the curve R is preferably small from the viewpoint of improving cell dispersion, for example, preferably 0.5 cm or less, and particularly preferably 0.3 cm or less. The radius of the curve may be, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 cm, and may be less than or equal to any of these values, or within the range of any two of these values. The radius of the curve may also be the radius of the curved section where the bottom and side surfaces connect when the container is viewed from the side. The radius of the curve is, for example, the radius of the curve at the position with the smallest radius of the curve within the curved section. The top and side surfaces may be directly connected to form a corner, or they may be connected via a connection (for example, a straight line or an arc shape). One side surface may be directly connected to form a corner, or they may be connected via a connection (for example, a straight line or an arc shape).

[0182] In one embodiment of this disclosure, the constituent material of the container includes, for example, synthetic resin, natural resin, or glass. Synthetic resins include, for example, polystyrene resin, polypropylene resin, polyethylene resin, etc. By using a transparent material, the culture conditions can be observed from the outside. The material of the container can be any material used for conventional culture vessels. In one embodiment of this disclosure, the inner surface of the container may be made of a cell-non-adherent resin or coated with a cell adhesion inhibitor. In one embodiment of this disclosure, the container may be manufactured by mold molding technology, 3D printing, etc.

[0183] Figure 10 shows a first container as an example. This container has a square shape for its bottom surface 1 and top surface 2. The sides 3 are rectangular in shape. The top surface 2 has four openings 4. Because the openings 4 are located on the top surface 2, the culture medium is less likely to spill, and the operation of changing the culture medium and retrieving cells is excellent. Each opening 4 has a port 6 inside, and a screw cap 5 is installed to cover it. The openings 4 are located at the four corners of the top surface 2. Inside the container, surrounded by the bottom surface 1, top surface 2 and sides 3, there is an internal space capable of containing the culture medium. The length L1 of one side of the bottom surface 1 is particularly preferably 23 to 28 cm. The height L2 of the container is particularly preferably 2.5 to 3 cm. The height L3 of the openings 4 is particularly preferably 2.5 to 3 cm. The diameter L4 of the openings 4 is particularly preferably 3 cm.

[0184] One embodiment of the present disclosure is a culture vessel comprising a bottom surface, a top surface, and sides, wherein the bottom surface is square in shape and the top surface has an opening. The top surface is square in shape, and the opening may be located at the corners of the top surface (e.g., one to four corners). The opening may form a protrusion. Another embodiment is a culture vessel comprising a bottom surface, a top surface, and sides, wherein the bottom surface is square in shape and the sides have an opening on the top side. The top surface may be square in shape. The opening may be located at the corners on the top side of the sides (e.g., one to two corners). The opening may form a protrusion on the surface. Another embodiment is a suspension culture vessel comprising a culture medium container and a lid, wherein the bottom surface of the culture medium container is square in shape. The culture medium container may have a bottom surface and sides. The culture medium container may have four sides. The lid may be square in shape. Another embodiment is a multi-layer container having a configuration in which a plurality of culture vessels are stacked on top of each other. Multiple containers offer excellent operability and are suitable for large-scale cultures because operations such as culture medium injection and culture medium replacement can be performed on each container. The number of containers may be, for example, 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, or 50, and may be greater than or equal to any two of these values. Each culture container has the same central axis, but the containers as a whole may overlap each other at a 45-degree angle. Here, the central axis may be a line drawn from the center of the point where the diagonals of the square shape of the base intersect, in a direction perpendicular to the horizontal plane. The state of being offset by 45 degrees may mean the state formed when, starting from a state where multiple containers are stacked without angle offset, the even-numbered container from the bottom is rotated 45 degrees around the central axis, and the top surface of the odd-numbered container from the bottom is brought into contact with the bottom surface of the even-numbered container from the bottom. Another embodiment is a multiple container in which a second container is located on top of a first container. Another embodiment is a multi-layered container in which multiple culture vessels are stacked, and the top surface of the bottom culture vessel is in contact with the bottom surfaces of the other culture vessels. In one embodiment of this disclosure, the culture vessel is preferably a vessel used for horizontal rotation culture of cells (horizontal rotation cell culture vessel).The above embodiments of culture vessels and multi-layer containers can adopt any of the numerical values ​​and configurations listed in the above-described embodiments of containers.

[0185] As another example of a container, a second container is shown in Figure 12. This container has a square shape for its bottom surface 101 and top surface 102. The side surface 103 has a rectangular shape. There is one opening 104 on the side surface. The opening 104 has a port inside, and a screw cap 105 is installed to cover it. The opening 104 is on the side surface 103 and is located in one corner on the top surface 102 side. This makes decanting easier. Inside the container, surrounded by the bottom surface 101, top surface 102 and side surface 103, there is an internal space capable of accommodating culture medium. The second container is placed upright on its side when changing culture medium in a clean bench, making it easy to work with multiple containers simultaneously. The length L101 of one side of the bottom surface 101 is particularly preferably 23 to 40 cm. The height L102 of the container is particularly preferably 5 to 6 cm. The height L103 of the opening 104 is particularly preferably 2.5 to 3 cm. The diameter L104 of the opening 104 is preferably 3 cm. The distance L105 from the lower end of the opening 104 to the bottom surface 101 on the side surface 103 of the container is preferably 2.5 to 3 cm. Alternatively, the distance L105 may be determined by subtracting the length 104 of the diameter of the opening 104 from the height L102 of the container.

[0186] The second container can be used in stacks. As an example, Figure 20 shows four of the second containers stacked on top of each other. Since the opening 104 of the second container is located on the side 103, the containers can be stacked directly, allowing for a larger culture area. When stacking containers, the number of containers may be, for example, 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, or 50, and may be greater than or equal to any two of these values. By stacking many containers, large-scale cultures can be performed.

[0187] One embodiment of the present disclosure is a cell culture apparatus comprising a culture medium container and a rotating part. This apparatus may include a culture medium container and a rotating part for horizontally rotating the culture medium container, and the culture medium container may have a square-shaped base. The culture medium container may contain culture medium and cells. The culture medium container may have a bottom, sides, and top, and may have a closed system of space inside. The culture medium container may have an opening. The opening may have a structure (e.g., a cylindrical structure) that connects the space inside the culture medium container with the space outside the container. A biological sample (such as cells) or culture medium can be injected from the space outside the container into the space inside the container through the opening. The culture medium container may be a culture vessel. The size, shape, etc. of the culture medium container and opening can be any of the numerical values ​​and configurations listed in the above-described embodiment of the container (in this case, the culture medium container may be read as a container). The rotating part may be configured to rotate the culture medium container. The rotating part may have a base on which the culture medium container can be placed. The base may be positioned parallel to the horizontal plane. The base and the culture vessel may be in contact in a direction perpendicular to the horizontal plane. The base may rotate with the vessel on it. One or more culture medium containers may be placed on the base. The number of containers may be, for example, 2, 4, 6, 8, 10, 20, 30, 40, 50, or 100, and may be greater than or equal to any of these values, or within the range of any two of these values. The multiple culture medium containers on the base may be arranged without stacking, or they may be arranged in a stacked manner. The rotation speed, rotation diameter, etc. of the rotating part can be any of the values ​​and configurations listed in the above embodiments.

[0188] One embodiment of the present disclosure is a cell culture apparatus comprising a container and a rotating device. The container may have a square-shaped base. The rotating device may be a horizontal rotating device or a swivel device. The rotating device may include a base for supporting the container.

[0189] One embodiment of the present disclosure is a cell culture vessel that contains a suspension culture medium (e.g., liquid medium) and has a square-shaped base. Another embodiment is a cell culture vessel that has a square-shaped base and contains suspension cells, the suspension cells oscillating in accordance with the horizontal rotation and oscillation of the vessel. Another embodiment is a cell culture method that includes the step of horizontally rotating a vessel containing cells and a culture medium, and the base shape of the vessel is square. Another embodiment is the use of a vessel with a square-shaped base in cell culture (e.g., horizontal rotation culture). Another embodiment is a cell production method that includes the step of rotating a vessel containing cells and a culture medium, and the base shape of the vessel is square. Another embodiment is a cell production method that includes the step of horizontally rotating a vessel containing cells and a culture medium. Another embodiment is a cell suspension production method that includes the step of horizontally rotating a vessel containing cells and a culture medium, and the base shape of the vessel is square. Cell culture methods that involve horizontally rotating a rectangular container have the characteristic that cells do not gather in the center and do not adhere well. Therefore, methods such as creating cell sheets while culturing cells in a container may be excluded from this culture method.

[0190] One embodiment of the present disclosure is a container having a bottom, sides, and a top, comprising four sides (first to fourth sides), wherein the first and third sides are parallel to each other, and the second and fourth sides are parallel to each other. The first and second sides may be in contact, and the first and fourth sides may be in contact. The second and third sides may be in contact, and the third and fourth sides may be in contact. The four sides may be in contact with the bottom at their lower ends and with the top at their upper ends.

[0191] One embodiment of this disclosure is a method for dispersing cells, which includes the step of horizontally rotating a container containing cells and culture medium, wherein the bottom shape of the container is square. When cells are dispersed, nutrients are more easily distributed to the cells. In addition, shear stress is reduced, and cell viability is improved.

[0192] One embodiment of the present disclosure is a method for enzymatically treating cells, comprising the step of horizontally rotating a container containing cells and an enzyme solution, wherein the bottom shape of the container is square. This method can achieve excellent enzymatic treatment from the viewpoint of at least one of the following: improvement of the viability rate after treatment, improvement of the number of cells after treatment, or reduction of shear stress during treatment. The enzyme solution may be, for example, a solution containing a proteolytic enzyme. This solution may include, for example, a solution for dispersing cell aggregates or adherent cells into single cells. This solution may include, for example, TrypLE® Select (Gibco 12563-011), trypsin solution, acutase solution, or a collagenase solution. The cells contained with the enzyme solution may be, for example, cell aggregates or cells adhered to a plastic material. This method may include, for example, the steps of suspension culture of cells, contact of cells with the enzyme solution, preparation of a suspension containing cells and the enzyme solution, or replacement of the culture medium in the container with the enzyme solution. The enzyme content in the suspension may be an effective amount for cell dispersion. The above enzymatic treatment includes, for example, cell dispersion. The embodiments of the above enzymatic treatment method can adopt any of the numerical values, components (e.g., cells, etc.), and steps listed in the above embodiments of the cell production method. For example, the cells include stem cells (e.g., various stem cells such as iPS cells or mesenchymal stem cells).

[0193] One embodiment of the present disclosure provides a cell enzyme processing apparatus comprising a culture medium container and a rotating unit for horizontally rotating the culture medium container, wherein the culture medium container has a square-shaped base. Using this apparatus, excellent enzyme processing can be achieved from the viewpoint of at least one of the following: improvement of the viable cell rate after processing, improvement of the number of cells after processing, or reduction of shear stress during processing. This embodiment of the cell enzyme processing apparatus can adopt any of the numerical values ​​and configurations listed in the above-described embodiments of the cell culture apparatus or the embodiment of the enzyme processing method.

[0194] One embodiment of the present disclosure provides a horizontally rotating cell enzyme processing container comprising a bottom surface, a top surface, and sides, wherein the bottom surface is square in shape and the top surface has 1 to 4 openings. This container is used to enzymatically process cells while the container is rotated horizontally. Using this container, excellent enzymatic processing can be achieved from the viewpoint of at least one of the following: improvement of the viability rate after processing, improvement of the number of cells after processing, or reduction of shear stress during processing. Embodiments of this horizontally rotating cell enzyme processing container can adopt any of the numerical values ​​and configurations listed in the above-described embodiments of the container or embodiments of the enzyme processing method.

[0195] The culture apparatus of this disclosure includes the following components: Culture chamber (10): The culture chamber is a container for housing stem cells and is made of a transparent material, allowing for external observation. Temperature control system (20): Equipped with a heater and a cooler to maintain the temperature inside the culture chamber at 37°C. The temperature is monitored by a temperature sensor and maintained at an appropriate temperature. Humidity control system (30): Equipped with a humidifier and a dehumidifier to maintain the humidity of the culture environment at 95% or higher. The humidity is monitored by a humidity sensor and maintained at an appropriate humidity. Gas supply system (40): Equipped with a gas supply device to control the concentration of carbon dioxide (CO2) and oxygen (O2). The gas concentration is monitored by a CO2 sensor and an O2 sensor and maintained at an appropriate concentration. Culture medium circulation system (50): Equipped with a pump and a filter for circulating the culture medium inside the culture chamber. This enables a uniform supply of nutrients and oxygen.

[0196] (Apparatus Operation) The apparatus that may be used in this disclosure operates as follows: Stem cells are placed in the culture chamber. The temperature control system sets the temperature inside the culture chamber to 37°C. The humidity control system sets the humidity to 95% or higher. The gas supply system supplies CO ２ Concentration 5%, O ２ The concentration is set to 20%. A culture medium circulation system is used to circulate the culture medium, uniformly supplying nutrients and oxygen to the stem cells.

[0197] (Applications) When using the culture apparatus of this disclosure, high-efficiency cell proliferation can be achieved: the culture environment can be designed to improve the proliferation efficiency of stem cells by optimally controlling temperature, humidity, and gas concentration. A stable culture environment can be maintained through a culture medium circulation system, enabling long-term culture. Ease of observation is possible because the transparent culture chamber allows for easy observation from the outside, and the state of the cells can be monitored in real time.

[0198] Examples of various stem cells, such as mesenchymal stem cells, or their derived progenitor cells include adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord blood-derived stem cells, or their derived progenitor cells. Examples of adipose-derived stem cells include vascular endothelial (progenitor) cells.

[0199] Adipose-derived stem cells are multipotent cells derived from fat that can differentiate into adipocytes, osteoblasts, chondrocytes, myofibroblasts, osteocytes, muscle cells, or nerve sheath cells. The proportion of adipose-derived stem cells can be identified as CD31-negative and CD90-positive cells. Adipose-derived stem cells can also be identified as CD45-negative, CD44-positive, CD29-positive, and CD13-positive cells. The above markers can be measured by FACS (fluorescence activated cell sorting).

[0200] Endothelial (progenitor) cells are cells that make up the inner surface of blood vessels and are in contact with the lumen through which blood circulates. The term "endothelial (progenitor) cells" encompasses both endothelial cells and endothelial progenitor cells. Endothelial (progenitor) cells retain the ability to divide and proliferate. Endothelial (progenitor) cells can be identified using CD45 negativity and CD31 positivity as indicators, and can also be identified as CD146 positivity and CD144 positivity.

[0201] (Manufacturing of cell-derived concentrates) This disclosure relates to a method for producing concentrates of culture supernatants of various stem cells, such as mesenchymal stem cells, or progenitor cells derived therefrom, comprising a culture supernatant acquisition step of obtaining culture supernatant by culturing various stem cells, such as mesenchymal stem cells, or progenitor cells derived therefrom in a culture medium; a concentration step of concentrating the culture supernatant; and a purification step of removing metabolic waste products from the concentrated culture supernatant.

[0202] Adipose-derived stem cells or their progenitor cells can be obtained from adipose tissue. Adipose tissue can be obtained, for example, by surgical excision from humans, or other mammals or birds. Examples of other mammals include dogs, cats, cattle, horses, pigs, goats, sheep, monkeys, ferrets, rabbits, mice, rats, gerbils, guinea pigs, and hamsters. Examples of birds include chickens. Local anesthesia may be used during surgical excision. Adipose tissue can also be obtained by aspiration by inserting a cannula into the subcutaneous adipose tissue of the abdomen, thighs, buttocks, or the entire body. The amount of adipose tissue obtained is, for example, 0.1 g to 1000 g, preferably 1 g to 500 g, 1 g to 100 g, 2 g to 50 g, or 2 g to 40 g, but is not limited to these amounts.

[0203] It is preferable to visually confirm that the obtained adipose tissue is free from neoplastic lesions and contamination. The adipose tissue may also be confirmed to be negative for HBV, HCV, HIV, HTLV-1, and TPHA / RPR. The adipose tissue may also be confirmed to have mycoplasma at a concentration of less than 128x (PA method) and herpes simplex at a concentration of less than 320x (CF method).

[0204] The method for preparing adipose-derived stem cells can be carried out by known methods and is not particularly limited. For example, aspirated adipose tissue can be allowed to stand, and after separating the oil layer, fat layer, and aqueous layer, the oil and aqueous layers can be removed to recover only the fat layer. The obtained fat layer can then be subjected to enzymatic treatment. Alternatively, the fragmented adipose tissue can be directly attached to a culture dish or the like to promote the proliferation and adherent culture of adipose-derived cells.

[0205] It is preferable to warm the adipose tissue before enzymatic treatment in a 37°C water bath at room temperature for 5 to 15 minutes.

[0206] Enzyme treatment can be carried out by adding an appropriate amount of enzyme reaction solution to the fat layer in the tube, fixing the tube to a constant temperature shaker, and shaking it. The temperature of the enzyme treatment is not particularly limited as long as the enzyme reaction proceeds, but is generally 25°C to 50°C, preferably 30°C to 45°C, for example 37°C. The shaking may be reciprocating or oscillating. In the case of reciprocating shaking, the reciprocating shaking speed is not particularly limited, but is generally 10 rpm to 300 rpm, preferably 50 rpm to 200 rpm, for example 120 rpm. The reaction time is not particularly limited, but is generally 10 minutes to 3 hours, preferably 15 minutes to 1 hour, for example 30 minutes.

[0207] As the enzyme, it is preferable to use at least collagenase. Collagenase is a recombinant protein produced by animal tissue cells, inflammatory cells, tumor cells, or bacteria such as Clostridium histolyticum, or artificially produced by genetic engineering technology, and is an enzyme that breaks down type I, type II, and type III collagen. Furthermore, neutral proteases, thermolysin, trypsin, dispase, etc. may also be added.

[0208] The final concentration of collagenase in the enzyme-treated solution is preferably 0.02% to 2%, more preferably 0.1% to 1%, even more preferably 0.1% to 0.5%, even more preferably 0.1% to 0.4%, even more preferably 0.1% to 0.3%, and most preferably 0.2%.

[0209] The enzyme treatment solution used for the enzyme treatment may also preferably contain DNaseI in addition to collagenase. When DNaseI is used, the concentration of DNaseI in the enzyme treatment solution is preferably 100 to 10,000 U / mL, more preferably 200 to 5,000 U / mL, and even more preferably 500 to 2,000 U / mL.

[0210] The enzyme treatment solution used for the enzyme treatment also contains CaCl ２ It is preferable to include CaCl in the enzyme treatment solution. ２ The concentration is preferably 0.01 mM to 10 mM, more preferably 0.1 mM to 5 mM, and even more preferably 1 mM to 4 mM, for example, 3 mM.

[0211] The enzyme treatment solution used for enzyme treatment is preferably a buffer solution, and more preferably HBSS (Hanks' Balanced Salt Solution).

[0212] The mixture of the fat layer and enzyme solution can be reacted by rotating it in a centrifuge tube at a temperature suitable for the enzymatic reaction (for example, 30°C to 45°C, preferably 35°C to 40°C). After the reaction, further centrifugation at 800G can separate the mixture into three layers: an oil layer, a fat layer, an aqueous layer, and a precipitate layer (cell layer). By removing the oil layer, fat layer, and aqueous layer, only the precipitate layer (cell layer) can be obtained. By suspending the precipitate layer in a suitable physiological saline solution, a suspension of adipose-derived cells (stromal vascular fraction, SVF) can be obtained.

[0213] In this disclosure, the culture supernatant is obtained by culturing various stem cells, such as mesenchymal stem cells, or precursor cells derived therefrom in a culture medium.

[0214] (Cell Preservation) This disclosure is applied to cell preservation techniques. In this specification, “cell preservation” or “cell storage” preferably includes cell cryopreservation or cryopreservation of cells. Cryopreservation is understood to mean the storage of cells, generally for a long period of time, at temperatures below 0°C, specifically between -20°C and -200°C. Freezing is generally carried out at 1°C / min.

[0215] The aqueous solutions for cell preservation according to this disclosure preferably further comprise cells to be preserved. These cells to be preserved are preferably mammalian cells. The term “cells” also includes cell aggregates.

[0216] Preferred mammalian cells include not only stem cells but also differentiated cells, such as lymphocytes, spleen cells, thymocytes, animal cells, somatic stem cells, mesenchymal stem cells, non-human embryonic stem cells, induced pluripotent stem cells, or cancer stem cells.

[0217] Particularly preferred mammalian cells include human induced pluripotent stem cells (hipSCs), various stem cells such as human bone marrow-derived mesenchymal stem cells (hMSC-BMs), human peripheral blood-derived mononuclear cells (hMNC-PB / PBMCs), human adult skin-derived dermal fibroblasts (NHDF-a), human adult skin-derived dermal melanocytes (NHEM-a), or human aortic-derived smooth muscle cells (HAoSMCs). The cells to be preserved may be either cells freshly isolated from tissue, or cells that have already been cultured or augmented in vitro.

[0218] The number of cells per milliliter of the stored cell suspension is preferably 100,000 to 100 million. Alternatively, for example, 1,000,000 to 2,000,000 pieces, 2,000,000 to 3,000,000 pieces, 3,000,000 to 4,000,000 pieces, 4,000,000 to 5,000,000 pieces, 5,000,000 to 6,000,000 pieces, 6,000,000 to 7,000,000 pieces, 7,000,000 to 8,000,000 pieces, 8,000,000 to 9,000,000 pieces, 9,000,000 to 10,000,000 pieces, 1,000,000 to 2,000,000 pieces, 2,000,000 to 3,000,000 pieces 00 pieces, 3,000,000 to 4,000,000 pieces, 4,000,000 to 5,000,000 pieces, 5,000,000 to 6,0 00,000 pieces, 6,000,000 to 7,000,000 pieces, 7,000,000 to 8,000,000 pieces, 8,000,000 pieces ~9,000,000 pieces, 9,000,000~10,000,000 pieces, 10,000,000~50,000,000 pieces, 10 ,000,000 to 45,000,000 pieces, 10,000,000 to 40,000,000 pieces, 10,000,000 to 35,00 0,000 pieces, 10,000,000 to 30,000,000 pieces, 10,000,000 to 25,000,000 pieces, 10,000 ,000-20,000,000 pieces, 15,000,000-50,000,000 pieces, 15,000,000-45,000,0 00 pieces, 15,000,000 to 40,000,000 pieces, 15,000,000 to 35,000,000 pieces, 15,000,00 0 to 30,000,000 pieces, 15,000,000 to 25,000,000 pieces, 20,000,000 to 50,000,000 pieces, 20,000,000 to 45,000,000 pieces, 20,000,000 to 40,000,000 pieces, 20,000,000 to 35 ,000,000 pieces, 20,000,000 to 30,000,000 pieces, 25,000,000 to 50,000,000 pieces, 25, 000,000 to 45,000,000 pieces, 25,000,000 to 40,000,000 pieces, 25,000,000 to 35,00 0,000 pieces, 30,000,000 to 50,000,000 pieces, 30,000,000 to 45,000,000 pieces, 30,000,000 to 40,000,000 pieces, 35,000,000 to 50,000,000 pieces, 35,000,000 to 45,000,000 pieces, 40,000,000 to 50,000,000 pieces, 40,000,000 to 45,000,000 pieces, 45,000,000 to 50,000,000 pieces, 30,000,000 to 100,000,000 pieces, 35,000,000 to 95,000,000 pieces, 40,000,000 to 90,0 Examples include 00,000, 45,000,000 to 85,000,000, 50,000,000 to 80,000,000, 50,000,000 to 100,000,000, 55,000,000 to 95,000,000, 60,000,000 to 90,000,000, 65,000,000 to 85,000,000, 70,000,000 to 80,000,000, etc., but these are the most common.

[0219] Preferably, the cells are separated from the culture or isolation medium / buffer before being preserved with the help of the cell preservation aqueous solution according to this disclosure. All operations are preferably carried out under sterile conditions.

[0220] This disclosure further relates to the use of aqueous cell preservation solutions according to this disclosure as cryoprotective agents.

[0221] This disclosure also relates to aqueous solutions for cell preservation according to this disclosure, for use as pharmaceuticals or excipients.

[0222] Furthermore, this disclosure relates to a method for preserving cells, comprising the steps of (a) dispersing cells in an aqueous cell preservation solution according to this disclosure in a container; and (b) subjecting the container to cryopreservation.

[0223] Furthermore, the present disclosure relates to a method for thawing cells dispersed in an aqueous cell preservation solution and frozen according to the present disclosure, comprising the steps of (a) thawing a container in which frozen cells are dispersed in an aqueous cell preservation solution of the present disclosure; and (b) adding the thawed cells to a culture medium suitable for the cells.

[0224] Furthermore, the preservation solution of this disclosure may contain the essential protective components of this disclosure in an isotonic solution. The "isotonic solution" is not particularly limited as long as it is an isotonic solution whose salt concentration or sugar concentration, etc., is adjusted with sodium ions, potassium ions, calcium ions, etc., so that it is approximately the same as the osmotic pressure of body fluids or cell fluids. Specifically, examples include physiological saline, buffered physiological saline (phosphate-buffered saline [PBS], Tris-buffered saline [TBS], HEPES-buffered saline, etc.), Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, etc., but bicarbonate Ringer's solution or lactated Ringer's solution is preferred. The isotonic solution can be manufactured based on known compositions, but commercially available products can also be used. Examples of commercially available products include Otsuka Saline Injection (physiological saline solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Ringer's Solution "Otsuka" (Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Lactec® Injection (lactated Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), Veen® F Infusion (acetate Ringer's solution, manufactured by Fuso Pharmaceutical Industries Co., Ltd.), and Bicanate® Infusion (bicarbonate Ringer's solution, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.). In this specification, "isotonic" means that the osmotic pressure is in the range of 250 to 380 mOsm / L.

[0225] Furthermore, the preservation solution of this disclosure may contain albumin or sugar as an optional active ingredient for preserving mammalian cells. In this specification, "optional active ingredient" means an ingredient that may or may not be included. Albumin and / or sugar may also be referred to as "optional protective ingredient of this disclosure." Examples of the above-mentioned "albumin" include human serum albumin (HSA), bovine serum albumin (BSA), fetal bovine serum albumin (FBS), etc., but HSA is preferred. If the preservation solution of the present disclosure contains albumin, the albumin concentration may be within the range of 0.1 to 30 (w / v)%, for example, 1.0 to 20 (w / v)%, 1.0 to 15 (w / v)%, 1.0 to 10 (w / v)%, 1.25 to 10 (w / v)%, 1.25 to 7.25 (w / v)%, 1.5 to 5.0 (w / v)%, 1.5 to 2.5 (w / v)%, and 2.0 to 2.5 (w / v)%.

[0226] Examples of "sugars" used herein include glucose, trehalose, dextran, and hydroxyethyl starch. When glucose is included in the preservation solution of this disclosure, the glucose concentration may be in the range of 1 to 10,000 mg / L, for example, 10 to 8,000 mg / L, 20 to 6,000 mg / L, 30 to 6,000 mg / L, 40 to 6,000 mg / L, 50 to 6,000 mg / L, 100 to 6,000 mg / L, 200 to 6,000 mg / L, 500 to 6,000 mg / L, 1,000 to 6,000 mg / L, 2,000 to 6,000 mg / L, and 2,000 to 5,000 mg / L. If the preservation solution of the present disclosure contains trehalose, the concentrations of trehalose can be 0.1 to 100 g / L, 5 to 80 g / L, or 20 to 60 g / L. If the preservation solution of the present disclosure contains dextran, the concentrations of dextran can be 0.1 to 100 g / L, 5 to 80 g / L, or 40 to 70 g / L. If the preservation solution of the present disclosure contains hydroxyethyl starch, the concentrations of hydroxyethyl starch can be 1 to 500 g / L or 10 to 100 g / L.

[0227] The preservation solution of this disclosure may be used for the purpose of administering mammalian cells to mammals. That is, a mixture containing the preservation solution of this disclosure and mammalian cells can be stored under predetermined conditions and then administered directly into the vivo organism of a mammal (e.g., intravenously). For this reason, it is preferable that the preservation solution of this disclosure does not contain any components that may adversely affect the vivo organism of a mammal when administered into the vivo organism. Examples of such "components that may adversely affect" include polyvinylpyrrolidone, 2-mercaptoethanol, okadaic acid, sodium butyrate, G418, etc.

[0228] One aspect of this disclosure is a mammalian degradation inhibitor containing the essential protective component of this disclosure. Such a degradation inhibitor may further contain the optional protective component of this disclosure. The mammalian degradation inhibitor of this disclosure can be used to prepare the preservation solution of this disclosure by adding it to the isotonic solution, or it can be used to improve the preservation of a known mammalian preservation solution by adding it to the known mammalian preservation solution. This disclosure also relates to mammalian cell preservation containers containing the preservation solution or powder formulation of this disclosure. The preservation container of this disclosure may be in any form that can maintain sterility after the mammalian cell suspension is injected, and examples include blood bags, infusion bags, syringes, ampoules, vials, etc., but blood bags are preferred.

[0229] (Differentiation) This disclosure relates to the differentiation of various stem cells (MSCs), such as mesenchymal stem cells, and in particular provides a method for differentiating MSCs into various differentiated cells, tissues, and organs. Various stem cells, such as mesenchymal stem cells, are found in bone marrow, adipose tissue, umbilical cord blood, etc., and have the ability to differentiate into various cells, tissues, and organs, and are therefore widely used in the fields of regenerative medicine and tissue engineering.

[0230] In this disclosure, after proliferation, a different differentiation medium may be used, and the cells may be cultured while confirming their differentiation potential. Furthermore, the differentiation conditions commonly used for MSCs can be used. Preferably, the cells of this disclosure are less likely to become cartilage and / or have high cartilage differentiation potential.

[0231] The differentiation method for mesenchymal stem cells includes the following steps: Isolation and culture of mesenchymal stem cells: Mesenchymal stem cells are isolated from bone marrow or adipose tissue and grown under standard culture conditions. Culture conditions are 37°C and 5% CO2. ２ Use an incubator with 95% humidity. Addition of differentiation-inducing factors: Depending on the specific differentiation direction, add the following factors to the culture medium. Differentiation to osteocytes: Use a differentiation culture medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. Differentiation to chondrocytes: Use a differentiation culture medium containing TGF-β (transforming growth factor beta), dexamethasone, and ascorbic acid. Differentiation to adipocytes: Use a differentiation culture medium containing insulin, dexamethasone, and indomethacin. Control of culture conditions: Set culture conditions suitable for each differentiation and culture for a certain period of time. Differentiation to osteocytes: 21 days, 37°C, 5% CO2 ２ Humidity 95% Differentiation into chondrocytes: 14 days, 37°C, 5% CO2 ２ Humidity 95% Differentiation into adipocytes: 10 days, 37°C, 5% CO2 ２ Humidity 95% Confirmation of differentiation: Differentiated cells are stained with specific markers, and the success of differentiation is confirmed by microscopic observation and molecular biological techniques. Osteocytes: Alkaline phosphatase staining, measurement of osteocalcin expression Chondrocytes: Safranin O staining, measurement of collagen II expression Adipocytes: Oil Red O staining, measurement of adiponectin expression

[0232] The differentiation method disclosed herein has the following advantages: High differentiation efficiency: By using appropriate differentiation-inducing factors and culture conditions, differentiation efficiency is high. Diverse differentiation directions: Differentiation into various cells, tissues, and organs such as bone, cartilage, and fat is possible. Maintenance of cell function after differentiation: Differentiated cells retain normal function and can be applied to regenerative medicine and tissue engineering.

[0233] Mesenchymal stem cells are isolated from cultured bone marrow or adipose tissue and grown under standard culture conditions. For culturing mesenchymal stem cells, a culture medium containing low-glucose DMEM (Dulbecco's Modified Eagle Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin is used. Culture conditions are 37°C and 5% CO2. ２ Use an incubator with 95% humidity.

[0234] Addition of Differentiation Inducing Factors Depending on the specific differentiation direction, the following factors are added to the culture medium:

[0235] Differentiation into osteocytes To differentiate into osteocytes, use the following differentiation culture medium: Dexamethasone: 100 nM Ascorbic acid: 50 μg / mL β-glycerophosphate: 10 mM Transfer mesenchymal stem cells to the differentiation culture medium and culture for 21 days at 37°C and 5% CO2. ２ The culture is performed under conditions of 95% humidity.

[0236] Differentiation into chondrocytes The following differentiation culture medium is used for differentiation into chondrocytes: TGF-β3 (Transforming Growth Factor Beta-3): 10 ng / mL Dexamethasone: 100 nM Ascorbic acid: 50 μg / mL Mesenchymal stem cells are transferred to the differentiation culture medium and cultured for 14 days at 37°C and 5% CO2. ２ The culture is performed under conditions of 95% humidity.

[0237] Differentiation into adipocytes For differentiation into adipocytes, the following differentiation culture medium is used: Insulin: 10 μg / mL Dexamethasone: 1 μM Indomethacin: 200 μM IBMX (3-isobutyl-1-methylxanthine): 0.5 mM Transfer mesenchymal stem cells to the differentiation culture medium and culture for 10 days at 37°C and 5% CO2 ２ The culture is performed under conditions of 95% humidity.

[0238] Controlling Culture Conditions: Set culture conditions suitable for various differentiation processes and culture for a certain period of time.

[0239] Confirmation of Differentiation: After differentiation, cells are stained with specific markers, and the success of differentiation is confirmed by microscopic observation and molecular biological techniques: Osteocytes: Alkaline phosphatase staining, measurement of osteocalcin expression Chondrocytes: Safranin O staining, measurement of collagen II expression Adipocytes: Oil Red O staining, measurement of adiponectin expression

[0240] The differentiation method disclosed herein has the following advantages: High differentiation efficiency: By using optimized culture medium components and appropriate differentiation-inducing factors and culture conditions, differentiation efficiency is high. Diverse differentiation directions: Differentiation into various cells, tissues, and organs such as bone, cartilage, and fat is possible. Maintenance of cell function after differentiation: Differentiated cells retain normal function and can be applied to regenerative medicine and tissue engineering.

[0241] In this specification, “cell” means any cell known in the art and refers to the constituent unit of an organism. Examples of organisms in this specification include humans, primates including chimpanzees, pet animals such as dogs, cats, and parakeets, domestic animals such as cattle, horses, sheep, goats, and chickens, rodents such as mice and rats, mammals and birds such as animals kept in zoos. In a broader sense, it can refer to a single cell or a collection of multiple cells, and in such cases, it is sometimes called a “cell population.” The broad sense of a cell encompasses both the narrow sense of a single cell and a cell population. Any type of cell can be used as long as the components of this disclosure are used, but stem cells may be advantageously targeted. Among stem cells, mesenchymal cells have shown efficacy in the examples and may be advantageously used. In this specification, “mesenchymal cells” include all cells that constitute mesodermal tissue, including, but not limited to, osteoblasts, adipocytes, muscle cells, and chondrocytes. The mesenchymal cells in this invention include mesenchymal stromal cells and mesenchymal stem cells. "Stem cells" refer to cells that have the ability to differentiate into various cell types and to self-renew. Examples include somatic stem cells and pluripotent stem cells.

[0242] In this specification, “Mesenchymal Stem Cell (MSC)” refers to cells that satisfy the following definition and are somatic stem cells with differentiation potential that are capable of differentiating into one or more types of cells belonging to mesodermal tissue. Mesenchymal stem cells in this specification include both mesenchymal stem cells obtained from any tissue and mesenchymal stem cells prepared in vitro. i) Adherence to plastic under standard culture conditions. Standard culture medium is a medium to which serum, serum substitute reagent or growth factor has been added to a basal medium (e.g., αMEM medium). ii) Positive for surface antigens CD73 and CD90, and negative for CD45 and CD326.

[0243] In this specification, “Subject” means the subject matter to which this disclosure applies. For example, a tissue, organ, or individual. Specifically, a tissue, organ, or individual of any of the organisms exemplified above.

[0244] Mesenchymal stromal cells (MSCs) are pluripotent stem cells that possess self-renewal capabilities and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes within the mesenchymal cell lineage. In recent years, focused research on the multi-lineage differentiation potential and immunomodulatory properties of human MSCs has demonstrated that these cells can be used to treat a variety of clinical conditions, including immunological disorders and degenerative diseases. Consequently, numerous clinical trials using MSCs are steadily increasing for a wide range of conditions, including graft-versus-host disease (GVHD), myocardial infarction and inflammation, and autoimmune diseases and disorders. Currently, clinical programs using MSCs rely on the isolation of these cells from adult sources and umbilical cord blood. The high cell counts required for clinical use of MSCs (up to several million cells per kg of patient body weight) demand a highly reliable, regenerative, effective, and active proliferation protocol capable of producing large numbers of cells from donor sources.

[0245] In certain embodiments, the Disclosure provides an organobud prepared from vascular cells, mesenchymal cells, and tissue or organ cells, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells.

[0246] In this disclosure, “organ bud” means a structure that can differentiate into an organ upon maturation and contains three types of cells: vascular cells, mesenchymal cells, and tissue or organ cells. Whether a structure is an organ bud can be confirmed, for example, by transplanting the structure into a living organism and examining whether it can differentiate into the target organ (if it differentiates into the target organ, it can be determined to be an organ bud), and / or by examining whether the structure contains all three types of cells mentioned above (if it contains all three types of cells, it can be determined to be an organ bud). Organ buds may be organ buds that differentiate into organs such as the brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, and lens; ectodermal organs such as the spleen, kidneys, ureters, heart, blood, gonads, adrenal cortex, muscles, skeleton, dermis, connective tissue, and mesothelium; and endodermal organs such as the liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestines), lungs, thyroid gland, parathyroid gland, urinary tract, and thymus. However, organ buds that differentiate into endodermal organs, such as organ buds that differentiate into the liver (hepatomegaly), organ buds that differentiate into the pancreas (pancreaticomegaly), and organ buds that differentiate into the intestines, are preferred. Whether a structure is an organ bud that differentiates into an endodermal organ can be confirmed by examining the expression of marker proteins (if one or more of the marker proteins described later are expressed, it can be determined to be an organ bud). For example, in liver buds, HHEX, SOX2, HNF4A, AFP, and ALB are used as markers; in pancreatic buds, PDX1, SOX17, and SOX9 are used as markers; and in organ buds that differentiate into the intestinal tract, CDX2 and SOX9 are used as markers. Among the terms used by those skilled in the art, liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, intestinal organoid (K. Matsumoto, et al. Science. 19;294(5542):559-63. (2001)) are included in the term "organoid" as used in this disclosure.

[0247] The organobuds of this disclosure are created from three types of cells—vascular cells, mesenchymal cells, and tissue or organ cells—all derived from pluripotent stem cells.

[0248] This disclosure may be applied to pluripotent cells. Pluripotent cells include pluripotent cells obtained from living organisms (e.g., ES cells), pluripotent cells induced from reprogramming (e.g., iPS cells), MUSE cells (Multilineage-differentiating stress-ending (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. PNAS, 2011), and iMPC cells (induced multipotent progenitor cells; Mouse liver population with hepatocytes generated). Examples of these can be found in "from human fibroblasts. Nature, 2014").

[0249] Pluripotent stem cells are preferably of human origin, but they may also be derived from animals other than humans (for example, animals used as laboratory animals, pets, working animals, racehorses, fighting dogs, etc., specifically mice, rats, rabbits, pigs, dogs, monkeys, cattle, horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, etc.).

[0250] The organobuds that can be produced by this disclosure can be prepared by co-culturing three types of cells derived from pluripotent stem cells (vascular cells, mesenchymal cells, and tissue or organ cells) in vitro. This disclosure provides a method for preparing organobuds, comprising culturing vascular cells, mesenchymal cells, and tissue or organ cells in vitro, wherein each of the vascular cells, mesenchymal cells, and tissue or organ cells is derived from pluripotent stem cells.

[0251] In this disclosure, "tissue or organ cells" is a concept that includes cells that have differentiated into functional cells constituting a tissue or organ, or undifferentiated cells that can differentiate into functional cells. Undifferentiated cells include stem cells, progenitor cells, endodermal cells, organoblasts, etc. Preferably, undifferentiated cells are cells whose differentiation fate into functional cells has been determined, but which have not yet differentiated into functional cells. "Undifferentiated tissue or organ cells" may include, for example, cells that can differentiate into organs such as the kidneys, heart, lungs, spleen, esophagus, stomach, thyroid gland, parathyroid gland, thymus, gonads, brain, and spinal cord. Examples include cells that can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, and lens; cells that can differentiate into mesodermal organs such as the spleen, kidneys, ureters, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesothelium; and cells that can differentiate into endodermal organs such as the liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestines), lungs, thyroid gland, parathyroid gland, urinary tract, and thymus. Whether a cell is capable of differentiating into an ectodermal, mesodermal, or endodermal organ can be determined by examining the expression of marker proteins (if one or more of these marker proteins are expressed, the cell can be determined to be capable of differentiating into an ectodermal, mesodermal, or endodermal organ). For example, markers for cells that can differentiate into the liver include HHEX, SOX2, HNF4A, AFP, and ALB; markers for cells that can differentiate into the pancreas include PDX1, SOX17, and SOX9; markers for cells that can differentiate into the intestinal tract include CDX2 and SOX9; markers for cells that can differentiate into the kidney include SIX2 and SALL1; markers for cells that can differentiate into the heart include NKX2-5, MYH6, ACTN2, MYL7, and HPPA; markers for cells that can differentiate into blood include C-KIT, SCA1, TER119, and HOXB4; and markers for cells that can differentiate into the brain and spinal cord include HNK1, AP2, and NESTIN.Among the terms used by those skilled in the art, hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, metanephric mesenchymal precursor cells, multipotent nephron progenitor,renal progenitor cell,cardiac mesoderm,cardiovascular progenitor cells,cardiac progenitor cells, (JR. Spence, et al. Nature.; 470(7332): 105-9. (2011), Self, et al. EMBO J., 25(21): 5214-5228. (2006), J. Zhang, et Circulation Research., 104: e30-e41 (2009), G. Lee, et al. Biotechnology 25, 1468-1475 (2007), etc., are included in the undifferentiated tissue or organ cells of this disclosure. Undifferentiated cells can be produced from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.For example, cells that can differentiate into liver cells can be prepared according to K. Si-Taiyeb, et al. Hepatology, 51 (1): 297-305 (2010), T. Touboul, et al. Hepatology. 51 (5): 1754-65 (2010), cells that can differentiate into pancreatic cells can be prepared according to D. Zhang, et al. Cell Res.; 19 (4): 429-38 (2009), and cells that can differentiate into intestinal cells can be prepared according to J. Cai, et al. J Mol Cell Biol.; 2 (1): 50-60 (2010), R. Spence, et al. Nature. Cells that can differentiate into heart cells can be prepared according to J. Zhang, et al. Circulation Research. ;, , 104: e30-e41 (2009), and cells that can differentiate into brain and spinal cord cells can be prepared according to G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007). Examples of functional cells that make up organs and tissues include endocrine cells of the pancreas, pancreatic ductal epithelial cells of the pancreas, hepatocytes of the liver, epithelial cells of the intestinal tract, renal tubular epithelial cells of the kidney, glomerular epithelial cells of the kidney, cardiomyocytes of the heart, lymphocytes and granulocytes of the blood, red blood cells, nerve cells and glial cells of the brain, and nerve cells and Schwann cells of the spinal cord.

[0252] In the preparation of organ buds according to this disclosure, the tissue or organ cells used are those produced (differentiated) from pluripotent stem cells.

[0253] <Cell Therapies> In one aspect of this disclosure, the disclosure provides cell therapy. The cell therapy of the disclosure comprises, as components, a non-freezing preservation solution for cell therapy and cells, which contain the components of the disclosure. According to the cell therapy of the disclosure, the cells in the cell therapy can be kept in a state that is available for use for a desired purpose.

[0254] (Composition) (Components) The components of the cell preparation of this disclosure are described below. The cell preparation of this disclosure includes a non-freezing preservation solution for cell preparations and cells containing the components of this disclosure, and includes a carrier as an optional component. Each component will be described in detail below.

[0255] (1) Components The cell preparations disclosed herein may appropriately contain dextran, albumin, sodium salt, potassium salt, calcium salt, etc.

[0256] The types of dextran that may be used in this disclosure are not particularly limited. For example, the dextran of the present invention may be a natural dextran, a processed natural dextran, or a synthetically synthesized dextran, and may include any modifications.

[0257] The types of sodium, potassium, and calcium salts that may be used in this disclosure are not particularly limited. Each salt may consist of one type of salt or multiple types of salts. In addition to these salts, the solutions may contain any other salts. Specifically, examples include lactates, acetates, carbonates, bicarbonates, phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfates, citrates, glucons, succinates, hydrochlorides, nitrates, oxalates, borates, magnesium salts, zinc salts and their hydrates.

[0258] The viscosity of the solution is not particularly limited as long as the cells being preserved can survive. For example, it is preferably 8 mPas or higher, and preferably 18 mPas or lower, at the temperature during cell suspension and / or storage. The viscosity of the non-freezing preservation solution can be measured, for example, using a TV-20 viscometer (Toki Sangyo Co., Ltd.) at a rotation speed of 10 rpm. The type of albumin used in this disclosure is not particularly limited. For example, it may be natural albumin, processed natural albumin, or artificially synthesized albumin. In addition to the components of this disclosure, the components contained in the non-freezing preservation solution for cell preparations may include any components used in the art. In this disclosure, any sugar may be further included. Specifically, examples include glucose, sucrose, fructose, sorbitol, maltose, trehalose, mixed sugars (e.g., GFX, etc.), or combinations thereof. In addition, the product may contain, as needed, additives such as stabilizers (e.g., polyethylene glycol), buffers (e.g., phosphate buffer, sodium acetate buffer), chelating agents (e.g., EDTA, EGTA, citric acid, salicylate), amino acids (e.g., glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, tyrosine, niacin, and other non-essential amino acids), vitamins (e.g., choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, biotin, inositol, etc.), solubilizers, preservatives, and antioxidants.

[0259] This disclosure may include cryoprotective agents. "Cryoprotective agent" means a compound that has the effect of suppressing the formation of ice crystals during cryopreservation. Cryoprotective agents as used herein include antifreeze and antifreeze agents. Specific examples of cryoprotective agents include dimethyl sulfoxide (DMSO), glycerin, polyethylene glycol (PEG), ethylene glycol, trimethylene glycol, dimethylacetamide, and polyvinylpyrrolidone.

[0260] The origin of the cells used in this disclosure is not particularly limited. The cells may be derived from any one or more organisms, such as mammals. For example, mammalian cells, human cells, cells from the same species as the subject of application, cells from the subject individual, or a combination thereof may be used.

[0261] The types of cells used in this disclosure are not particularly limited. Examples include cells derived from living tissue, cells derived from living tissue, stem cells, cells differentiated from stem cells, or combinations thereof.

[0262] For example, cells derived from living tissues include epithelial tissue cells, connective tissue cells, muscle tissue cells, nerve tissue cells, or combinations thereof. Furthermore, connective tissue cells and muscle tissue cells, for example, can be collectively referred to as mesenchymal cells.

[0263] When using mesenchymal cells, for example, cells derived from bone marrow, adipose tissue, umbilical cord, placenta, synovial membrane, synovial fluid, dental pulp, heart, etc., can be used. Specific examples of mesenchymal cells include dermal fibroblasts, osteoblasts, tendon and ligament fibroblasts, adipocytes, chondrocytes, tendinocytes, cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin-producing cells, and cells derived from endocrine glands (for example, insulin-producing cells such as β-islet cells). Furthermore, in the case of blood, which is a type of connective tissue, blood cells differ from other mesenchymal cells in that they are differentiated from hematopoietic stem cells, but in this specification, they are also treated as mesenchymal cells. Specifically, mesenchymal cells include, for example, dendritic cells, monocytes, natural killer (NK) cells, T cells (e.g., alpha-beta (αβ) T cells, gamma-delta (γδ) T cells, cytotoxic T cells (CTLs), helper T cells, etc.), B cells, macrophages, neutrophils, eosinophils, etc. Furthermore, these mesenchymal cells may be differentiated from pluripotent stem cells, as described later, and specifically may be iPS cell-derived cardiomyocytes, chondrocytes, nerve cells, etc.

[0264] When using stem cells, for example, somatic stem cells, pluripotent stem cells, or combinations thereof can be used. Examples of somatic stem cells include various stem cells such as mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hair follicle stem cells, mammary gland stem cells, and pigment stem cells. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells).

[0265] When using stem cells, particularly somatic stem cells, the tissue from which the cells originate is not particularly limited. For example, in the case of stem cells that exist in various tissues, such as mesenchymal stem cells, cells from any of these tissues may be used, or a combination of cells from multiple types of tissues may be used. Examples of tissues from which various stem cells, such as mesenchymal stem cells, may originate include those exemplified for mesenchymal cells. Specifically, for example, various stem cells, such as mesenchymal stem cells, may originate from connective tissue or adipose tissue.

[0266] When referring to a cell population by cell type, it is not necessary for all cells included to be of that type; the term simply refers to the inclusion of cells of that type. Furthermore, if a cell population includes cells derived from multiple types of tissue, the name of one of the tissues can be used as a representative designation for the cell population. For example, when referring to a cell population as adipose tissue-derived mesenchymal cells, the cell population only needs to include adipose tissue-derived mesenchymal cells; it may also include mesenchymal cells from other tissues or other cell types.

[0267] The cells used in cell preparations in this phase may be subjected to any treatment before contact with the preservation solution. Specific treatments are not limited to those described above, but examples include freezing, thawing, culturing, washing, sorting, transformation, genetic manipulation, or combinations thereof.

[0268] The state of cells in the cell preparation described herein may be single cells or cell aggregates such as spheroids. Preferably, the state is single cells. In this specification, "single cell state" means a state in which cells exist individually and are not aggregated. The method for bringing cells into a single cell state is not particularly limited. For example, the dispersion method described in Section 4 can be used. The percentage of single cells in the total number of cells in the cell preparation is, for example, 70% or more, 90% or more, 95% or more, 99% or more, or 100%. The percentage of single cells in the cell preparation can be measured using any method. For example, it can be measured by dispersing cells in a buffer solution (e.g., PBS) and observing several randomly selected cells under a microscope to check for aggregation.

[0269] The cells in the cell preparation may be suspended or in contact with the inner wall of the container, etc. Preferably, they are suspended. In this specification, "suspended" means that the cells are not fixed to the inner wall of the container containing the cell preparation by adhesion or the like. For example, the percentage of suspended cells among the total cells in the cell preparation may be, for example, 40% or more, 50% or more, 60% or more, 70% or more, 90% or more, 95% or more, 99% or more, or 100%.

[0270] The number of cells per unit dose contained in the cell preparation disclosed herein is not particularly limited. Generally, the number of cells varies depending on the type of cell, the route of administration, the purpose of administration, and the type of carrier, which is another component described later. Therefore, it should be determined appropriately taking each of these conditions into consideration. For example, it is sufficient that a sufficient number of cells is contained in a single dose of the cell preparation. The specific number of cells per unit dose is not particularly limited, but for example, 1 × 10⁻⁶ ３ ~1 x 10 １１ It is 1 x 10¹ / mL. Specifically, for example, 1 x 10¹⁶ ４ ~1 x 10 １０ pieces / mL, 1×10 ５ ~1 x 10 ９ pieces / mL, 1×10 ６ ~7.5 x 10 ８ pieces / mL, 2×10 ６ ~5 x 10 ８ pieces / mL, 4×10６ ~4 x 10 ８ pieces / mL, 5×10 ６ ~2 x 10 ８ pieces / mL, or 7.5 × 10 ６ ~1.5 x 10 ８ The concentration is cells / mL. When administered in multiple doses, it is sufficient that the total amount contains a sufficient number of cells. Furthermore, when the cell preparation of this disclosure is diluted before administration, it is sufficient that the diluted preparation contains a sufficient number of cells to achieve the desired effect.

[0271] (Carrier) The cell formulations of this disclosure may include a pharmaceutically acceptable carrier as necessary. "Pharmaceutically acceptable carrier" means an additive commonly used in the pharmaceutical technology field. Examples include excipients, binders, disintegrants, emulsifiers, flow modifiers, lubricants, etc.

[0272] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, Pluronic®, kaolin, silicic acid, or combinations thereof.

[0273] Examples of binders include starch paste made from plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0274] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0275] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0276] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0277] In addition to the above, if necessary, the composition may also contain solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, bulking agents, humectants, moisturizers, wetting agents, adsorbents, flavoring and deodorizing agents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, buffers, pH adjusters, isotonic agents, etc., which are commonly used in pharmaceutical compositions and cell preparations.

[0278] The carrier is used to avoid or inhibit the degradation of the active ingredient by enzymes, etc., within the target body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0279] The cell preparations of this disclosure are preferably sterile. The method for confirming sterility is described in Section 1. The method for achieving sterility is described in Section 4.

[0280] (Dosage Form) The dosage form of the cell preparation disclosed herein is a liquid. The liquid includes any formulation that has fluidity. Specifically, examples include creams, ointments, gels, injections, suspensions, etc. The specific volume and other details are not particularly limited and should be within the range known in the art for each dosage form. The method for manufacturing the cell preparation disclosed herein may be to formulate it according to a conventional method in the art.

[0281] (Method of Application) The method of application of the cell preparations disclosed herein is parenteral administration. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-sacral, intrathecal, intrafocal, intracranial, tissue, and organ administration. Systemic parenteral administration includes intracirculatory administration (e.g., intravenous, intra-arterial, and intra-lymphatic administration), intraperitoneal administration, rectal administration, intranasal administration, buccal administration, and vaginal administration. For example, the cell preparations disclosed herein can be administered locally, in which case they can be directly administered to the target site (including the oral cavity) by injection, for example. Alternatively, they can be used in the form of a cream, ointment, gel, suspension, or any other suitable substance at the time of application. For systemic administration, intracirculatory administration such as intravenous injection can be used. The dosage should be an effective amount for the cells to take effect. The effective dose is selected appropriately according to the information of the target, as described above. For example, when using cells with homing ability (e.g., various stem cells such as mesenchymal stem cells), even if the purpose is to administer to a specific site, it may be possible to deliver the cells to the target site through simple systemic administration.

[0282] The application method and dosage of the cell therapies disclosed herein may vary depending on the subject's information. In this specification, "subject's information" refers to various information about the subject's characteristics and condition. For example, if the subject is a human individual, this may include age, weight, sex, overall health status, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0283] The cell preparations described herein can be stored before application. The specific storage method, storage period, and storage temperature are not particularly limited as long as they are not frozen, but may be similar to those described in Section 4, for example.

[0284] Furthermore, the cell preparations disclosed herein do not need to be applied directly to the target organism. Specifically, they can be applied, for example, by transplanting a reservoir containing the cell preparations disclosed herein, or by transplanting tissue formed by further culturing the cell preparations. Further culturing can be performed for purposes such as increasing the number of cells, differentiating cells, transforming cells, or introducing genes into cells.

[0285] Furthermore, the cell therapy described herein can be used in combination with one or more known cell therapy products.

[0286] (Scope of Application) The scope of application of the cell preparations of this disclosure is not particularly limited. For example, they may be applied to the tissues, organs, or individuals of organisms as exemplified in the first section on definitions.

[0287] The cell therapies described herein may be applied to healthy individuals as well as those suffering from any disease or condition. Examples of diseases and conditions include cancer, leukemia, vascular disease, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, neurological disease, burns, chronic inflammation, heart disease such as ischemic cardiomyopathy and dilated cardiomyopathy, immunodeficiency, Crohn's disease, diabetes, arthritis, facial lipoatrophy, mastectomy, scarring, blemishes, wrinkles, and sagging skin.

[0288] Furthermore, the purpose of application is not particularly limited. For example, it can be used for the improvement, treatment and prevention of diseases and conditions, and cosmetic surgery. Specifically, examples include regenerative medicine for tissue augmentation such as tissue depression or for the treatment of osteoarthritis of the knee, immunotherapy such as T-cell therapy, NKT-cell therapy, and dendritic cell transfer therapy, gene therapy using genetically modified cells, breast augmentation, breast reconstruction, wrinkle removal, blemish removal, and other cell transplantation therapies. Specifically, for example, the cell preparation of this disclosure can also be used for breast reconstruction by mixing it with fat and administering it to a patient (for example, a patient who has undergone a mastectomy or a patient who requires breast reconstruction).

[0289] By applying the cell therapies of this disclosure, at least a portion of the applied cells may function similarly to endogenous cells in the subject.

[0290] <Cell Administration Device> In aspects of the cell administration device of this disclosure, the cell administration device of this disclosure includes a cell preparation and a container as essential components. According to the cell administration device of this disclosure, the cells in the cell preparation can be provided in a state ready for immediate use in cell administration.

[0291] The configuration of the cell delivery device of this disclosure will be described below. The cell delivery device of this disclosure includes a cell preparation and a container as essential components. Each component will be described in detail below.

[0292] The container is not particularly limited as long as it is suitable for cell administration. Specifically, examples include bottles, vials, test tubes, syringes, and plastic bags such as infusion bags. It is preferable that the cell preparation in the container be sterile. Furthermore, it is preferable that the container is one that prevents microorganisms from entering from the external environment. Specifically, examples include sealed containers and containers with filters and / or valves at the opening.

[0293] The container may have a catheter or sterile tube, a needle (sharp or blunt), or a cap attached to it.

[0294] Preferably, the container is labeled with cell information and / or subject information. Here, "cell information" refers to information about the type, strain, properties, origin, and source of the therapeutic cells contained in the cell administration syringe, which is useful for identifying the cells, and "subject information" refers to information about the subject receiving the suspension of therapeutic cells, such as their name, age, sex, and disease name, which is useful for identifying the subject. This ensures that the cell preparation is reliably administered to the subject to be administered.

[0295] The amount of cell preparation contained in the container is not particularly limited. For example, it can be determined according to the capacity of the container, or according to the target of application, usage situation, route of administration, etc. Specifically, for example, it may contain an amount of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the capacity of the container. Also, for example, it may contain an amount of 0.25 times or more, 0.5 times or more, 1 time or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 10 times or more of the single dose.

[0296] The cell delivery device described herein can be used as is for cell delivery, but additional operations may be performed. Examples of additional operations include removing gas from the device, equipping the device temperature, resuspending the cell preparation in the device, and adding solvents and / or components.

[0297] Cell administration can be used for any purpose. Specifically, it can be used for regenerative medicine, immunotherapy, gene therapy, cell transplantation therapy, and the like.

[0298] <Cell Maintenance> This disclosure also provides a method for maintaining cells. The method in this part includes a suspension step and a storage step as essential steps, and includes a dispersion step as an optional step. According to the method in this part, cells can be maintained under non-freezing conditions and with a high cell viability.

[0299] Constitutive Dispersion Step The "dispersion step" is an optional step, and is a step of detaching cells from the culture vessel, etc., after cell culture and / or dispersing cells that are bound to each other. The detachment method and dispersion method are not particularly limited. Any method known in the art may be used. For example, mechanical methods and chemical methods can be used, and either can be used. Examples of mechanical methods include methods that apply physical impact to the container and methods that use equipment such as cell scrapers. Examples of chemical methods include methods that use proteolytic enzymes such as trypsin and collagenase, chelating agents such as EDTA, and other compounds such as urea.

[0300] Suspension Step The suspension step is an essential step in which cells are suspended in the non-freezing storage solution described above. If a dispersion step is performed, this step can be performed simultaneously with or after the dispersion step.

[0301] The method used to suspend cells in a non-freezing storage solution is not particularly limited. For example, suspension can be achieved by aeration, fluid circulation, or other mechanical agitation. Specific methods include, for example, pipetting and tapping.

[0302] The state of the cells in the solution after suspension (whether they are single cells or not, and whether they are suspended or not, etc.) shall be in accordance with the description in Section 2.

[0303] The cell suspension prepared by this process can be used as the cell preparation described above. Therefore, this process can be used as a method for producing the cell preparation described above.

[0304] Other conditions such as temperature and humidity during this process are not particularly limited. Preferably, this process is carried out under sterile conditions or includes sterilization.

[0305] Sterile conditions refer to conditions under which an object can be kept sterile for a desired period of time. Furthermore, a sterile state means that the object is substantially free of viable microorganisms (including bacteria, viruses, etc.). The method for confirming a sterile state is as described above.

[0306] The method for achieving sterility is not particularly limited, as long as the target cells are not killed. For example, aseptic techniques can be used. Aseptic techniques are methods in which the product manufacturing process is carried out under sterile conditions, or the manufacturing process includes sterilization.

[0307] Sterile conditions can be achieved by any known method, and the method used is not particularly limited. Specifically, they can be achieved, for example, by a clean bench, isolator, sterile workroom, access restriction barrier system, or a combination thereof.

[0308] The sterilization treatment used is not particularly limited, as long as it can kill or remove microorganisms in the object to the extent that the objectives of this disclosure are achieved. For example, chemical sterilization with ethanol and sodium hypochlorite, heat sterilization such as autoclaving and dry heat sterilization, gas sterilization with ozone gas, ethylene oxide gas and plasma hydrogen peroxide gas, radiation sterilization with ultraviolet light, gamma rays and electron beams, and sterile filtration can be used. Sterilization also includes, for example, germicidal sterilization. In general, the appropriate method and conditions for sterilization can be selected depending on the type of microorganism, the degree of contamination, and the properties and condition of the object to be sterilized. For example, the "Guidelines for the manufacture of sterile pharmaceuticals by final sterilization method" and the "Guidelines for the manufacture of sterile pharmaceuticals by aseptic operation method" provided by the Ministry of Health, Labour and Welfare of Japan, and the "WHO good manufacturing practices for sterile pharmaceutical products" provided by the World Health Organization (WHO) may be referenced.

[0309] Storage Process: The "storage process" is the process of storing the product at a temperature of 0°C to 37°C. This process can be performed simultaneously with or after the suspension process.

[0310] The storage temperature is not particularly limited as long as the cell suspension does not freeze and the cells can survive at that temperature. Specifically, for example, it can be stored at 0-37°C, 0-30°C, 0-25°C, 0-20°C, 0-17°C, 0-15°C, 0-14°C, 0-13°C, 0-12°C, 0-11°C, 0-10°C, 0.1-9°C, 0.2-8°C, 0.5-7°C, 1-6°C, 1.5-5°C, or 2-5°C. The storage temperature does not have to be constant. For example, the storage temperature may be changed intentionally during storage, or it may change naturally.

[0311] There are no specific limitations on the storage period. For example, it can be stored for 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 18 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more. The upper limit of the storage period is, for example, 30 days or less, 25 days or less, 20 days or less, 16 days or less, 15 days or less, 14 days or less, 10 days or less, 7 days or less, 6 days or less, or 5 days or less.

[0312] During storage, cells and / or cell suspensions can be subjected to any treatment. Specifically, this includes, but is not limited to, mixing, application of chemical and physical stimuli, transport, shipping, and storage. It is preferable that microorganisms do not enter the cell suspension during storage. For example, storage can be carried out using a container that prevents the introduction of microorganisms from the external environment, or under sterile conditions.

[0313] According to the preservation method of this disclosure, a high cell viability rate is maintained during cell storage. Cell viability can be counted using known methods capable of detecting cell death, such as trypan blue staining, TUNEL method, Nexin method, FLICA method, methods using cell counting instruments, and combinations thereof.

[0314] The cell viability is higher when using the non-freezing cell preservation solution of this disclosure compared to when cells are stored in known non-freezing cell preservation solutions (e.g., physiological saline), after a specific period of storage. Specific periods include, for example, 24 hours, 48 ​​hours, 72 hours, or 96 hours. Furthermore, the cell viability after a specific period of storage is, for example, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, or 97.5% or more, relative to the cell viability at the start of storage.

[0315] <Cell Delivery Device> This disclosure also provides a method for manufacturing a cell delivery device. The method in this section includes a suspension step and a filling step as essential steps, and includes a dispersion step and / or a storage step as optional steps. The method in this section can be used to produce the cell delivery device described in Section 3.

[0316] Dispersion step: The "dispersion step" is an optional step, and involves detaching cells from a culture vessel and / or dispersing cells that are bound to each other. This step may be carried out in accordance with other descriptions in this specification.

[0317] Suspension Step The "suspension step" is the process of suspending cells in the non-freezing preservation solution described above. If a dispersion step is performed, this step can be performed simultaneously with or after the dispersion step.

[0318] The basic method of this process is the same as the suspension process described above. Therefore, a detailed explanation is omitted here.

[0319] Filling Process: The "filling process" is the process of filling a container with the suspension. This process can be performed simultaneously with or after the suspension process.

[0320] The filling method is not particularly limited as long as it is a method that can contain the desired amount of suspension in the container. Depending on the shape of the container, any method known in the art can be used for the specific filling method. Specifically, for example, the container can be filled by pouring the liquid, by suction, by scooping, or a combination thereof. Preferably, this process is carried out under sterile conditions or includes a sterilization process. The details of the sterile conditions and sterilization process are as described above.

[0321] In this process, any necessary instruments can be connected to the container. These instruments may be connected, for example, for filling operations, for sterile operations, or to prevent leakage. Examples include inlets such as nozzles, pumps such as aspirators, tubes such as catheters or sterile tubes, needles such as sharp or blunt needles, or caps. Multiple instruments may be connected during this process; for example, different instruments may be connected for multiple purposes.

[0322] Storage Process: The "storage process" is an optional process that involves storing the product at a temperature between 0°C and 37°C. This process can be performed simultaneously with or after the suspension and filling processes.

[0323] The basic method of this process is the same as the preservation process described in Section 4. Therefore, a detailed explanation is omitted here.

[0324] <Quality Standards> In embodiments of this disclosure, the following inventions are provided according to this disclosure.

[0325] In other words, a method for quality control of stem cells is provided, which includes determining whether or not it has a coefficient C correlated with the culture medium of the present disclosure.

[0326] Here, the coefficient C can be determined, for example, by examining cell markers if it is on the cell side. For example, quality control is possible using common markers such as cell markers related to proliferation. Any of the markers described herein may be used, including those that enhance differentiation ability or those that exhibit long-term expression. If it is on the culture medium side, information regarding the identification of the components themselves can be used.

[0327] <1> A method for quality control of cells for therapeutic or drug discovery, comprising: controlling the behavior of one or more specific protein molecules essential for the expression of therapeutic effects by cells; and controlling the expression behavior of a group of related genes characteristic of the cells. <2> The method according to <1>, wherein the group of related genes characteristic of the cells consists of 10 to 2000 genes. <3> The method according to <1> or <2>, wherein the group of related genes characteristic of the cells is selected from at least one of the following: an immune-related gene group, an inflammation-related gene group, angiogenesis-related gene group, nerve regeneration-related gene group, chondrogenesis-related gene group, osteogenesis-related gene group, adipogenesis-related gene group, fibrosis-related gene group, immunogenicity-related gene group, migration-related gene group, adhesion-related gene group, aging-related gene group, various stem cell marker-related gene groups such as mesenchymal stem cells, surface marker-related gene group, growth factor-related gene group, and chemokine or cytokine-related gene group. <4> The method according to any one of <1> to <3>, wherein, in managing the expression behavior of the gene group characteristic of the above cells, the cells are deemed usable if the correlation between the expression pattern of the gene group obtained by measuring the expression status of the gene group characteristic of the above cells and a reference expression pattern is above a certain level. <5> The method according to <4>, wherein the above correlation is above a certain level, and the correlation coefficient or coefficient of determination, or the correlation coefficient and the coefficient of determination, is evaluated and the above correlation coefficient or the above coefficient of determination is above a certain level. <6> The method according to <5>, wherein, when evaluating the above correlation coefficient or the above coefficient of determination, the above correlation coefficient or the above coefficient of determination is calculated by doing any of the following: (i) substituting missing values ​​for which the gene expression level cannot be measured with the lower limit of measurement, (ii) substituting missing values ​​for which the gene expression level cannot be measured with the lowest value among the measured values, or (iii) processing missing values ​​for which the gene expression level cannot be measured with the following formula: read count of each gene + (1 × gene length / average gene length). <7> The method according to any one of <1> to <6>, wherein the specified protein molecule is a cell surface antigen. <8> The method according to any one of <1> to <7>, wherein the specified protein molecule contains one or more of CD29 and CD140b.<9> The method according to any one of <1> to <8>, wherein the behavior of the above-mentioned specific protein molecule is at the expression level. <10> The method according to any one of <1> to <9>, wherein the cells are at least one type of cell selected from the group consisting of somatic stem cells, induced pluripotent stem cells, embryonic stem cells, differentiated cells derived from somatic stem cells, differentiated cells derived from induced pluripotent stem cells, and differentiated cells derived from embryonic stem cells. <11> The method according to any one of <1> to <10>, wherein the cells are various types of stem cells such as mesenchymal stem cells. <12> The method according to any one of <1> to <11>, wherein the cells are cells used as a drug for treating arthritis. <13> The method according to any one of <1> to <12>, wherein the cells are cells used as a drug for treating meniscus disease or osteoarthritis. <14> The method according to any one of <1> to <11>, wherein the cells are cells for drug discovery. <15> A method for producing cells for therapeutic or drug discovery, comprising: obtaining a cell population containing candidate cells for therapeutic or drug discovery; and selecting cells that meet criteria from the cell population by the method of any one of <1> to <14>.

[0328] According to this disclosure, therapeutic efficacy can be ensured by identifying and managing molecules essential for drug efficacy, which are of paramount importance to therapeutic cells. Furthermore, this disclosure enables quality control that clearly demonstrates the equivalence and homogeneity of cells, which cannot be fully guaranteed by drug efficacy alone, by making it possible to distinguish between similar cells.

[0329] The method for quality control of cells for therapeutic or drug discovery described herein includes controlling the behavior of one or more specific protein molecules that are essential for the expression of therapeutic effects by the cells, and controlling the expression behavior of a group of related genes characteristic of the cells.

[0330] Controlling the behavior of one or more specific protein molecules essential for the expression of therapeutic effects by cells is a quality control method based on the identification and management of mechanism molecules essential for the direct drug efficacy of cells. Controlling the expression behavior of cell-specific related gene groups is a quality control method that identifies gene expression patterns specific to cell populations containing non-mechanism molecules that are not necessarily essential for the direct drug efficacy of cells, and identifies cells from crude populations based on these patterns.

[0331] The distinguishing feature of this disclosure lies in the combination of the two quality control methods described above. The first quality control method enables the production of cells that exhibit reliable therapeutic effects and allows for the control of equivalence and homogeneity of efficacy. The second quality control method enables the control of equivalence and homogeneity of cell populations. In this disclosure, the two quality control methods described above enable cell quality control that guarantees efficacy, safety, equivalence, and homogeneity, making it possible to provide cell therapy products.

[0332] When cells controlled in the method of this disclosure are used for therapeutic purposes, the cells may be autologous or allogeneic, but are preferably autologous.

[0333] <Effect Management> In one aspect, quality control involves managing the behavior of one or more specific protein molecules that are useful for confirming the state and effects of cells.

[0334] Specifically, controlling the behavior of a particular protein molecule involves controlling its expression level.

[0335] The expression level of a specific protein molecule refers to the expression level of the gene for that specific protein, or the expression level of that specific protein. The expression level of a specific protein molecule can be calculated as an absolute value or a relative value (such as a ratio or difference from a control or reference expression level).

[0336] The expression level of a specific protein molecule can be measured by any method known to those skilled in the art and can be carried out according to conventional methods. As a method for measuring the expression level of a specific protein molecule, the amount of mRNA, which is the transcript of the gene, may be measured. The method for measuring mRNA is not particularly limited as long as it can measure the desired amount of mRNA, and can be appropriately selected from known methods. For example, a gene amplification method using oligonucleotides that hybridize to the gene encoding the specific protein molecule as primers, or a hybridization method using oligo(poly)nucleotides that hybridize to the gene encoding the specific protein molecule as probes can be used. Specifically, examples include RT-PCR (reverse transcription polymerase chain reaction), real-time RT-PCR, DNA microarray, cell array, Northern blotting, dot blotting, and RNase protection assay.

[0337] The primers and probes used in the above measurement method can be labeled, and the amount of mRNA can be measured by examining the signal intensity of the label. Real-time RT-PCR is preferable because it allows the direct use of RNA in the sample and enables gene quantification from the number of temperature cycles required for amplification by optically measuring the gene amplification process. Furthermore, as a control, the expression levels of housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin mRNA can be used to standardize the expression levels of genes encoding specific protein molecules. Note that the primers and probes used in the above measurement method can be appropriately designed and prepared by those skilled in the art based on the nucleotide sequence information of genes encoding specific protein molecules.

[0338] To measure whether a protein expression level is present, immunological methods such as using an antibody or antibody fragment against a specific protein can be employed. Specifically, these methods include flow cytometry, Western blotting, enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunofluorescence assay, and cell array assay. These measurement methods can also be performed using standard protocols or protocols that have been appropriately modified or changed from standard protocols.

[0339] For example, when measuring the expression level of proteins in cells by flow cytometry, cells can be evaluated as being of high quality if the positive rate of a specific protein is preferably 70% or higher, more preferably 80% or higher, and even more preferably 90% or higher.

[0340] In this disclosure, quality control can be performed, for example, by comparing the expression level of a specific protein molecule in cells measured by the method described above with a predetermined reference expression level. The reference expression level may be, for example, the expression level of a specific protein molecule in cells that have already been confirmed to have a certain quality (positive control), or the expression level of cells that have already been confirmed not to have a certain quality (negative control).

[0341] By comparing the expression level of a specific protein molecule in a cell with its reference expression level, if the expression level of the specific protein molecule in the cell is equal to or higher than the expression level of the positive control, the cell quality can be evaluated as high. Conversely, if the expression level of the specific protein molecule in the cell is equal to or lower than the expression level of the negative control, the cell quality can be evaluated as low.

[0342] Alternatively, a cutoff value for the expression level of a specific protein molecule may be set in advance, and the expression level of the specific protein molecule measured for a cell may be compared with the cutoff value. The cutoff value can be, for example, determined based on a regression line showing the correlation between the expression level of the specific protein molecule and the therapeutic effect, and is set to the expression level of the specific protein molecule that gives the desired therapeutic effect. For example, if the expression level of the specific protein molecule in a cell is above the cutoff value, the cell quality can be evaluated as high, and if it is below the cutoff value, the cell quality can be evaluated as low.

[0343] <Managing the expression behavior of cell-specific related gene groups> In another aspect, quality control in this area involves managing the expression behavior of cell-specific related gene groups.

[0344] The group of genes characteristic of the cell preferably consists of 10 to 5000 genes. More preferably, it consists of 10 to 2000 genes. The lower limit of the number of genes may be 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, 300 or more, or 500 or more. The upper limit of the number of genes may be 4000 or less, 3000 or less, 2000 or less, or 1500 or less.

[0345] The characteristic gene groups for cells are not particularly limited and can be appropriately selected depending on the type of cell. For example, if the cells are various stem cells such as mesenchymal stem cells, then at least one of the following can be selected (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of the gene groups listed above, or even more gene groups not listed here) can be selected.

[0346] The above various gene groups can be collected, for example, using commercially available panels, literature information, microarray data, known databases (such as Ontology database (http: / / www.informatics.jax.org / vocab / gene_ontology / ), etc.). Also, genes with high appearance frequencies can be selected from the collected gene groups and used.

[0347] In the quality control of this aspect, it can be determined that the cell can be used based on the correlation between the expression pattern of the above gene group obtained by measuring the expression state of the related gene group characteristic of the cell and the reference expression pattern. Specifically, when the correlation between the expression pattern of the above gene group obtained by measuring the expression state of the related gene group characteristic of the cell and the reference expression pattern is above a certain level, it can be determined that the cell can be used. For example, the correlation coefficient or determination coefficient, or both the correlation coefficient and the determination coefficient, between the expression pattern of the above gene group obtained by measuring the expression state of the related gene group characteristic of the cell and the reference expression pattern is evaluated, and when the above correlation coefficient or the above determination coefficient is above a certain level, it can be determined that the cell can be used.

[0348] The method for measuring the expression state is not particularly limited, but the amount of mRNA can be measured. The measurement of whether it is the amount of mRNA is not particularly limited as long as it is a method capable of measuring the desired amount of mRNA, and it can be appropriately selected from known methods and used. For example, RNA Sequencing method (RNA-Seq), RT-PCR method, real-time RT-PCR method, etc. can be used.

[0349] As a method for calculating the coefficient of determination, various methods can be adopted and are not particularly limited. As an example, a method using the RSQ function adopted in Microsoft Excel (registered trademark) can be used. By the RSQ function, for the regression line using the (x, y) pairs of the total gene group consisting of the expression level y of a certain gene in the first target sample and the expression level x of the same gene in the second target sample, by returning the value of r squared (coefficient of determination), the coefficient of determination between the two target samples can be obtained.

[0350] As a method for calculating the correlation coefficient, various methods can be adopted and are not particularly limited. As an example, a method using the CORREL function adopted in Microsoft Excel (registered trademark) can be used. By the CORREL function, for the (x, y) pairs of the total gene group consisting of the expression level y of a certain gene in the first target sample and the expression level x of the same gene in the second target sample, by returning the value of the correlation coefficient, the correlation coefficient between the two target samples can be obtained.

[0351] When evaluating the correlation coefficient or the coefficient of determination, it is preferable to calculate the above correlation coefficient or the above coefficient of determination by performing any of the following: (i) substituting the missing value for which the gene expression level cannot be measured with the lower limit of measurement, (ii) substituting the missing value for which the gene expression level cannot be measured with the lowest value among the measured values, or (iii) processing the missing value for which the gene expression level cannot be measured with the following formula: read count of each gene + (1 × gene length / average gene length).

[0352] RNA sequencing (RNA-Seq) is a method for quantifying gene expression levels using a sequencer. The analysis process involves (1) performing quality control on the reads output from the sequencer, (2) mapping the reads to a reference sequence, and (3) counting how many reads were mapped to each gene region. The number of reads counted for each gene region in this way can be considered as the relative expression level of that gene. In other words, the read count for each gene is a result that is generally obtained from gene expression analysis and is a value that is counted for each gene region.

[0353] The threshold for the coefficient of determination or correlation coefficient can be selected from, for example, 0.70 or higher, 0.71 or higher, 0.72 or higher, 0.73 or higher, 0.74 or higher, 0.75 or higher, 0.76 or higher, 0.77 or higher, 0.78 or higher, 0.79 or higher, 0.80 or higher, 0.81 or higher, 0.82 or higher, 0.83 or higher, 0.84 or higher, 0.85 or higher, 0.86 or higher, 0.87 or higher, 0.88 or higher, 0.89 or higher, etc., depending on the cell types being distinguished.

[0354] <Method for producing cells for therapeutic or drug discovery, and use of cells> According to this disclosure, after obtaining a cell population containing candidate cells for therapeutic or drug discovery, cells for therapeutic or drug discovery can be produced by selecting cells that meet the criteria from the cell population using the cell quality control method described herein.

[0355] Cells whose quality is confirmed by this disclosure can be used for regenerative medicine and basic research (e.g., drug discovery). For example, cells whose quality is confirmed by this disclosure can be used as transplant material for the treatment of damage or impairment of living tissue or for cosmetic purposes. In one example of this disclosure, the cells are cells used as a treatment for arthritis. For example, the cells may be cells used as a treatment for meniscus or osteoarthritis.

[0356] As an example, the use of various stem cells, such as synovial mesenchymal stem cells, as treatments for arthritis (such as osteoarthritis) or meniscus disorders is described below.

[0357] Synovial tissue can be harvested from the joint under anesthesia. The excised synovial tissue is enzymatically treated with a protease such as collagenase or trypsin, and the treated cells are filtered through a mesh filter. The nucleated cells recovered by the above method are sorted by quality control in the method of this disclosure, and the sorted cells can be used for therapeutic purposes.

[0358] Synovial-derived mesenchymal stem cells can be cultured and proliferated in ex vivo without prior differentiation induction using differentiation media. These proliferated undifferentiated synovial-derived mesenchymal stem cells can then be transplanted into the patient. To achieve efficient treatment, a 10 cm translocation is necessary. ２ For cartilage defects or meniscal defects of a certain size, preferably 5 × 10 ７ One or more, more preferably 1 x 10 ８ One or more mesenchymal stem cells or other types of stem cells should be transplanted.

[0359] In another example of this disclosure, cells can be used for drug discovery. For example, cells quality-controlled by the methods of this disclosure can be used to evaluate the efficacy or toxicity of candidate drugs and screen for candidate drugs. For instance, the efficacy or toxicity of a candidate drug can be evaluated by comparing the properties of cells when they are in contact with the candidate drug with those when they are not.

[0360] (Description of Preferred Embodiments) <First Embodiment> In one aspect, the present disclosure provides a technology relating to the use of hallucinonides (1) as culture medium components. The hallucinonides of the present disclosure are shown below.

[0361] Here, R １ and R ２ Each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), or R １ and R ２ They come together to form a ring, R ３ is a halo or hydrogen, and R ４A culture medium comprising at least one component selected from the group consisting of a halo (preferably chloro) or an optionally substituted hydroxyl, or a pharmaceutically acceptable salt or solvate thereof. While we do not wish to be bound by theory, hallucinonides (1) are thought to bind to various factors related to cell proliferation and support the proliferation of cells such as stem cells, either directly or indirectly via cytokines, etc.

[0362] In one embodiment, the present disclosure includes (1) and (1')R. １ and R ２ Each independently comprises at least one component selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl).

[0363] In one embodiment, (1")R １ and R ２ It contains at least one component that, together, forms a ring (such as dioxolane).

[0364] In one embodiment, the components of the present disclosure are (1A) or or a pharmaceutically acceptable salt or solvate thereof.

[0365] In one embodiment, the components of the present disclosure include (1B) hallucinonide or a pharmaceutically acceptable salt or solvate thereof. Such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; aliphatic monocarboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid; aromatic monocarboxylic acids such as benzoic acid and salicylic acid; aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; and aliphatic tricarboxylic acids such as citric acid; methanesulfonic acid, etc. Examples include organic sulfonic acids such as aliphatic sulfonic acids like tansulfonic acid and 2-hydroxyethanesulfonic acid, and aromatic sulfonic acids like benzenesulfonic acid and p-toluenesulfonic acid; acid addition salts with acidic amino acids such as aspartic acid and glutamic acid; salts with metals such as alkali metals or alkaline earth metals such as sodium, potassium, magnesium, and calcium; salts with organic bases such as methylamine, ethylamine, ethanolamine, pyridine, lysine, arginine, and ornithine; and ammonium salts. Propionic acid is preferred, but the invention is not limited to these.

[0366] In one embodiment of the disclosure relating to the various components of the hallucinonides (1) of this disclosure, the components of this disclosure or the culture medium containing them are for culturing various stem cells, such as mesenchymal stem cells. In one embodiment, the components of this disclosure or the culture medium containing them are preferably for culturing mesenchymal stem cells.

[0367] In another embodiment of the disclosure relating to the various components of the hallucinonides (1) of this disclosure, the culture medium of this disclosure is a serum-free medium. In another embodiment, the culture medium of this disclosure is a medium that does not contain biologically derived materials.

[0368] In one aspect of the disclosure relating to the various components of the hallucinonides (1) of this disclosure, the disclosure provides a method for producing such cells, comprising the step of culturing cells using the components of the disclosure or a culture medium containing them.

[0369] Methods for producing cells according to the disclosure of various components of the hallucinonides (1) of this disclosure can be carried out as follows, but are not limited thereto. For example, first, prepare the components to be used for culture or a culture medium containing them. Next, inoculate the cells into the medium and grow the cells under appropriate culture conditions. Culture conditions include temperature, pH, oxygen concentration, carbon dioxide concentration, etc. Change the medium periodically and replenish the necessary nutrients. As the culture progresses, observe the growth of the cells and take samples as appropriate. Once the desired cell volume is reached, collect the cells and wash and purify them. Finally, use the harvested cells as intended. Specifically, any combination of the general descriptions given elsewhere in this specification may be used. Particularly noteworthy is the significant enhancement of growth.

[0370] In one embodiment of the disclosure relating to the various components of the hallucinonides (1) of this disclosure, the culturing step in this disclosure includes culturing under conditions of a temperature of 25 to 40°C, a pH of 6 to 8, an oxygen concentration of 15 to 25%, and a carbon dioxide concentration of 3 to 7%. Examples of the conditions for the culturing step in this disclosure include, but are not limited to, a temperature of 37°C, a pH of 7.4, an oxygen concentration of 21%, and a carbon dioxide concentration of 5%. The culturing period is usually 48 to 144 hours, preferably 48 to 120 hours. It is important that the culture medium contains appropriate amounts of nutrients such as glucose, amino acids, and vitamins. Furthermore, it is recommended to add growth factors to promote cell proliferation. During culturing, the medium should be changed regularly to maintain the health of the cells. Meeting these conditions enables efficient cell proliferation and harvesting of high-quality cells.

[0371] In one embodiment of the disclosure relating to the various components of the hallucinonides (1) of this disclosure, the culturing step in this disclosure includes culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. Specifically, a...

Claims

(1) Hallucinonides (Here, R １ and R ２ Each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl), or R １ and R ２ They come together to form a ring, R ３ is a halo or hydrogen, and R ４ (where is a halo or a substituted hydroxyl) or a pharmaceutically acceptable salt or solvate thereof (2) ALK4 / 7 inhibitor or TGFβ receptor type 1 kinase inhibitor (3) BRD7 / 9 dual degradation factor or BRD7 / 9 binding molecule (degradation factor) (4) LATS1 / 2 inhibitor, PKA inhibitor, or AKT1 inhibitor (5) CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor or PKC inhibitor or adenosine kinase inhibitor (6) E-selectin inhibitor, VCAM1 inhibitor or ICAM1 inhibitor; and (7) LATS1 inhibitor + LATS2 inhibitor A culture medium comprising at least one component selected from the group consisting of the following. (1')R １ and R ２ Each is independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, and OR (where R is hydrogen or alkyl). (2') TGFβ receptor type 1 kinase inhibitor (3') BRD7 / 9 binding molecule (degradation factor) (4') AKT1 inhibitor (5') Adenosine kinase inhibitor (6') ICAM1 inhibitor; and (7') LATS1 Inhibitor The culture medium according to claim 1, comprising at least one component selected from the group consisting of the following. 　 The above (1) to (7) are each independently as follows: (1")R １ and R ２ combine to form a ring (2") ALK4 / 7 inhibitor (3”) BRD7 / 9 double decomposition factor (4") LATS1 / 2 inhibitor or PKA inhibitor (5") CK1 / 2 inhibitor, insulin receptor Tyr kinase inhibitor, PKA inhibitor, or PKC inhibitor (6") E-selectin inhibitor or VCAM1 inhibitor; and (7") LATS2 inhibitor The culture medium according to claim 1, comprising at least one component selected from the group consisting of the following. (1A) or or a pharmaceutically acceptable salt or solvate thereof (2A) TGFβ receptor type 1 kinase inhibitor, which is also an ALK4 / 7 inhibitor. (3A) BRD7 / 9 binding molecule (degradation factor) which is also a BRD7 / 9 dual degradation factor. (4A) AKT1 inhibitor, which is also a LATS1 / 2 inhibitor or a PKA inhibitor. (5A) Adenosine kinase inhibitors that are also CK1 / 2 inhibitors, insulin receptor Tyr kinase inhibitors, PKA inhibitors, or PKC inhibitors. (6A) ICAM1 inhibitor which is also an E-selectin inhibitor or a VCAM1 inhibitor; and (7A) LATS1 / 2 Inhibitor The culture medium according to claim 1, comprising at least one component selected from the group consisting of the following. 　 The above (1) to (7) are, respectively (1B) Hallucinonide or a pharmaceutically acceptable salt or solvate (2B) AZ12601011 or a pharmaceutically acceptable salt or solvate thereof (3B) VZ185 or a pharmaceutically acceptable salt or solvate thereof (4B) A-674563 or a pharmaceutically acceptable salt or solvate thereof (5B) 5-iodotubercidin or a pharmaceutically acceptable salt or solvate thereof (6B) ICAM-1-IN-1 or a pharmaceutically acceptable salt or solvate thereof; and (7B) LATS-IN-1 or a pharmaceutically acceptable salt or solvate thereof The culture medium according to claim 1. 　 The culture medium according to any one of claims 1 to 5, wherein the culture medium is for culturing mesenchymal stem cells. 　 A method for producing cells, comprising the step of culturing cells using a culture medium according to any one of claims 1 to 6. 　 The method according to claim 7, wherein the culturing step comprises culturing under conditions that satisfy at least one selected from the group consisting of a specific range of cell numbers, a specific container, and a specific temperature. 　 A cell culture apparatus comprising a culture medium according to any one of claims 1 to 6 or means for providing the culture medium, a container capable of containing the culture medium, and means for providing conditions for culturing cells as necessary. 　 A method for preserving cells, comprising the step of preserving the cells using a culture medium according to any one of claims 1 to 6. 　 A composition for preserving cells, comprising the components described in any one of claims 1 to 6. 　 A cell-containing composition comprising the components and cells described in any one of claims 1 to 6. 　 Cells cultured using the component described in any one of claims 1 to 6. 　 The cell according to claim 13, wherein the properties of the cell are improved. 　 A cell population comprising the cells described in claim 13, wherein the expression of at least one selected from the group consisting of CD44, CD73, CD90, and CD105 is not significantly reduced even after long-term passage. 　 A cell population comprising 50% or more of the cells described in any of claims 13 to 14, or a cell population according to claim 15 comprising 50% or more of the said cells. 　 A pharmaceutical product comprising the cells described in claim 13 or 14, or the cell population described in claim 15 or 16. 　 A method for producing differentiated cells, comprising the step of culturing cells obtained using the culture medium described in any one of claims 1 to 6 under conditions that induce differentiation. 　 Differentiated cells produced by the method described in claim 18. 　 A method for treating or preventing a subject, comprising the step of administering an effective amount of the cells described in claim 13, 14, or 19, the cell population described in claim 15 or 16, or the pharmacopoeia described in claim 17 to a subject in need thereof. 　 A method for quality control of stem cells, comprising determining whether the culture medium described in any one of claims 1 to 6 has a coefficient C that correlates with it. 　 A system for culturing cells, 1) A cell culture section for containing cells intended for culture, 2) A culture medium providing unit that provides the culture medium or raw materials according to any one of claims 1 to 6, 3) A cell culture condition adjustment unit that adjusts the culture conditions as needed. A system that includes this. A culture medium suitable for transporting cells, comprising the culture medium components described in any one of claims 1 to 6. 　 A method for managing cell culture for cells suitable for cultivation using the culture medium described in any one of claims 1 to 6, 1) A step of providing information regarding the culture medium for the cells provided by the user, 2) A step of assigning an identifiable label to the cells and the culture medium, 3) A step of checking whether the cells and the culture medium are compatible by referring to the label. A method of including.

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