Improved culture method using integrin agonist

Abstract

To provide an improved method for culturing an epithelial stem cell or an organoid containing an epithelial stem cell.SOLUTION: Provided is a method for culturing an epithelial stem cell or an organoid containing an epithelial stem cell. The method includes culturing the epithelial stem cell in culture solution suitable for the epithelial stem cell, and furthermore, the culture method includes bringing a cell or an organoid in contact with an integrin agonist. The present invention also relates to a culture solution suitable for used in the method, an organoid that can be collected or is collected by using the method and the culture method, and a culture medium and organoid in creation of medicine and validation, toxicity assay, diagnosis and treatment.SELECTED DRAWING: None

Description

Technical Field

[0001] Field The present invention relates to an in vitro cell culture method for culturing stem cells or organoids. The present invention relates to a culture solution suitable for use in the above method, an organoid that can be collected or has been collected by the use of the above method and the above culture method, and a medium and an organoid in drug discovery and validation, toxicity assay, diagnosis and treatment.

Background Art

[0002] Background Current methods for culturing stem cells or organoids in culture generally require the use of an extracellular matrix. The extracellular matrix is composed of a multifunctional network structure of fibrous gel-like substances distributed throughout the body and provides a structural and biochemical support for all tissues. Matrix proteins are involved in many cellular processes such as cell adhesion, proliferation, differentiation and apoptosis. Examples of extracellular matrix proteins include laminin, collagen, glycoprotein, proteoglycan, and glycosaminoglycan, which self-organize in the interstitial space between cells or as a basement membrane. These extracellular matrix proteins may affect cell function via various cell receptors that may bind to a part of the matrix components [1].

[0003] Examples of commercially available extracellular matrices that may be used to culture epithelial stem cells include basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc) or Matrigel™ (BD Biosciences)). However, the exact composition of these extracellular matrices and the mechanisms by which they affect cell function remain unclear [2]. Since the exact components of these extracellular matrices are not clearly defined, they may cause variations when culturing stem cells.

[0004] Therefore, there is a need in the art for artificial substitutes of these extracellular matrices with controlled and reproducible matrix components. Artificial matrices can be made from a variety of materials, such as chemically treated culture dish plastics or layers of deposited proteins optionally supplemented with extracellular matrix proteins. However, artificial matrices in the art contain only the major extracellular matrix proteins and are not applicable to all cell types [2]. Furthermore, although these artificial matrices may support the growth of some stem cells, they are not yet as efficient as the extracellular matrix.

[0005] Therefore, there is a need in the art for improved methods of culturing stem cells. In particular, there is a need for a culture method that improves the growth of stem cells using an artificial matrix. SUMMARY OF THE INVENTION

[0006] The present invention provides a method for culturing epithelial stem cells or organoids containing epithelial stem cells, the method comprising culturing the epithelial stem cells in a culture medium suitable for epithelial stem cells, and further comprising contacting the cells or organoids with an integrin agonist. The inventors have advantageously identified that the use of an integrin agonist, such as an antibody, in a method for culturing epithelial stem cells or organoids results in improved cell growth in the presence or absence of an extracellular matrix.

[0007] The present invention further provides a culture medium suitable for epithelial stem cells and for use in the method of the present invention, the culture medium comprising one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor. The present invention also provides an organoid that can be harvested or has been harvested by any of the methods of the present invention.

[0008] Another aspect of the present invention provides a culture medium suitable for epithelial stem cells as defined in the present invention, and a composition comprising organoids that can be collected or have been collected by any of the methods of the present invention. The present invention also provides a composition comprising a culture medium suitable for epithelial stem cells as defined in the present invention, and an extracellular matrix or artificial matrix. The present invention also provides an extracellular matrix or artificial matrix as defined in the present invention, which further comprises an integrin agonist as defined in the present invention.

[0009] In a further aspect, the present invention provides the use of an integrin agonist as defined in the present invention for culturing cells. These include the use of an integrin agonist for pre-treating cells before transplantation into a patient and the use of an integrin agonist as a cell adhesion enhancer in cell transplantation methods.

[0010] The present invention also provides the use of the organoids of the present invention for drug screening, target validation, target discovery or toxicology. Further, the present invention provides the use of the organoids of the present invention for use in therapy or for use in diagnosis. [Invention 1001] A method for culturing epithelial stem cells or organoids containing epithelial stem cells, the method comprising culturing the epithelial stem cells in a culture medium suitable for epithelial stem cells, the culturing method further comprising contacting the cells or the organoids with an integrin agonist. [Invention 1002] The integrin agonist of the method of Invention 1001 interacts with the β subunit of integrin, and optionally the β subunit is β1, β2, β3 or β7. [Invention 1003] The integrin agonist of the method of Invention 1002 interacts with the β1 subunit. [Invention 1004] The integrin agonist of the method of Invention 1001 interacts with the α subunit of integrin. [The present invention 1005] The integrin agonist is selected from an anti-integrin antibody, talin, kindlin, dithiothreitol, and oxysterol 25-hydroxy cholesterol, and is any one of the methods of the present invention 1001 to 1004. [The present invention 1006] The agonist of integrin is an anti-integrin antibody, and optionally the anti-integrin antibody is JBS2, HP1 / 3, SNAKA51, PT25-2, PMI-1, MEM-83, NKI-L16, 496B, 12G10, 8A2, TS2 / 16, 15 / 7, HUTS-4, 8E3, N29, 9EG7, mAb 24, MEM-148, KIM127, CBR LFA-1 / 2, MEM-48, KIM185, AP3, AP5, LIBS6, LIBS2, 10F8, 2B8, 2G3, and is any one of the methods of the present invention 1001 to 1005. [The present invention 1007] The anti-integrin antibody is TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29 or 9EG7, and further optionally the anti-integrin antibody is TS2 / 16, 12G10, HUTS-4 or 8A2, and further optionally the anti-integrin antibody is TS2 / 16, 12G10, HUTS-4, and is the method of the present invention 1006. [The present invention 1008] The antibody is humanized, and is any one of the methods of the present invention 1006 or the present invention 1007. [The present invention 1009] The integrin agonist is a. Talin, optionally used in combination with kindlin, b. A reducing agent such as dithiothreitol, or c. A lipid such as oxysterol 25-hydroxy cholesterol, and is any one of the methods of the present invention 1001 to 1005. [The present invention 1010] The method is a. The growth of organoids, and / or b. An increase in epithelial stem cell proliferation of at least 10%, 20%, 50% over four days compared to the same method performed without the integrin agonist resulting from any of the methods of the present invention 1001 - 1009. [The present invention 1011] Any of the methods of the prior invention, wherein the method further comprises culturing the cells in contact with an extracellular matrix, optionally the extracellular matrix is a basement membrane extract or Matrigel. [The present invention 1012] Any of the methods of the prior invention, wherein the method further comprises culturing the cells in contact with an artificial matrix, optionally the artificial matrix comprises a polymer, optionally a polyester, polyethylene glycol or a hydrogel. [The present invention 1013] The artificial matrix is a. A crosslinked polyethylene glycol (PEG) hydrogel, and / or b. A biomaterial, preferably an extracellular matrix component comprising, optionally the biomaterial is one or more glycoproteins (optionally selected from collagen, laminin, perlecan, fibronectin, or the RGD adhesion ligand of fibronectin), and / or one or more carbohydrates (optionally hyaluronic acid), according to the method of the present invention 1012. [The present invention 1014] Any of the methods of the prior invention, wherein the extracellular matrix or the artificial matrix is three-dimensional and / or in a suspended state. [The present invention 1015] Any of the methods of the present invention 1001 - 1010 and 1012 - 1014, wherein the culturing method does not include contacting the cells with an exogenous extracellular matrix. [The present invention 1016] Any of the methods of the prior invention, wherein the epithelial stem cells are selected from colorectal, small intestine, stomach, pancreas, liver, lung, breast, prostate, kidney, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle and / or cochlear cells. [Invention 1017] The culture medium suitable for the epithelial stem cells is any method of the prior invention, including one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor. [Invention 1018] The culture medium as defined in Invention 1017, further comprising the integrin agonist as defined in Inventions 1002 to 1009. [Invention 1019] The extracellular matrix or artificial matrix as defined in any of Inventions 1011 to 1015, further comprising the integrin agonist as defined in Inventions 1002 to 1009. [Invention 1020] An organoid that can be collected or has been collected by any method of Inventions 1001 to 1017. [Invention 1021] A composition comprising the culture medium of Invention 1018 and optionally an extracellular matrix or artificial matrix as defined in any of Inventions 1011 to 1015. [Invention 1022] Use of an integrin agonist for culturing cells. [Invention 1023] Use of an integrin agonist for pre-treating cells before transplantation into a patient. [Invention 1024] An integrin agonist for use as a cell adhesion enhancer in a cell transplantation method. [Invention 1025] The organoid of Invention 1020 for use in treatment or for use in diagnosis. [Brief Description of the Drawings]

[0011]

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Mode for Carrying Out the Invention

[0012] Detailed Description As described in detail in the Examples section, the inventors hypothesized that anti-integrin antibodies might be useful in the context of epithelial stem cell culture in order to mimic the signaling and structural functions of the extracellular matrix. To test this hypothesis, the inventors generated integrin agonists and tested them in a method of culturing epithelial stem cells in the absence of the extracellular matrix. Surprisingly, the inventors found that this improved epithelial stem cell proliferation and organoid formation in the absence of the extracellular matrix, increased the proliferation efficiency, and thus was comparable to prior art methods involving extracellular matrices such as Matrigel or BME. Furthermore, the inventors also found that the addition of integrin agonists improved proliferation even in the case of culture methods involving extracellular matrices. Thus, surprisingly, the inventors have demonstrated that the addition of integrin agonists improves the method for culturing epithelial stem cells, whether in the presence or absence of the extracellular matrix. Without wishing to be bound by theory, the inventors hypothesize that integrin agonists activate signaling pathways and / or provide a structural support that promotes the adhesion of epithelial stem cells to the extracellular matrix or artificial matrix materials. In addition, the inventors hypothesized that integrin agonists can mimic the structural and signaling functions of the extracellular matrix because, surprisingly, the addition of integrin agonists enabled epithelial stem cell proliferation in the absence of the extracellular matrix. The ability of integrin agonists to promote epithelial stem cell proliferation in the absence of the extracellular matrix is particularly advantageous because it represents a further step towards a defined, controlled, and reproducible culture method.

[0013] In addition, when the proliferation rate of epithelial stem cells or organoids increases, it is advantageous because it enables the use of a large amount of cells for various applications that require a large amount of material to test various different drugs, for example, for drug screening. The ability to generate cells from a single starting material is advantageous for applications where it is necessary to compare the results between experiments. Similarly, it means that many cells can be used for transplantation and that multiple patients can be transplanted with cells harvested from a useful donor. By culturing cells in a culture medium, it becomes possible to proliferate the cells while maintaining their stem cell phenotype. Organoids containing these stem cells are formed. Therefore, the use of a culture medium is advantageous for increasing the number of these useful stem cells and harvesting the organoids containing these cells.

[0014] Accordingly, the present invention relates to a method for culturing epithelial stem cells or organoids containing epithelial stem cells using an integrin agonist. In particular, the present invention provides a method for culturing epithelial stem cells or organoids containing epithelial stem cells, the method including culturing the epithelial stem cells or organoids in a culture medium suitable for epithelial stem cells, and further including contacting the cells or organoids with an integrin agonist.

[0015] Integrin agonist Integrins are heterodimeric transmembrane adhesion receptors that support cell-cell interactions and interactions between cells and the extracellular matrix. They are formed by non-covalently associated α and β subunits and are present in all metazoans. Each subunit is a type I transmembrane glycoprotein having a relatively large extracellular domain that mediates ligand binding and a short cytoplasmic tail (except for the β4 subunit). Mammals have 18 types of α subunits and 8 types of β subunits, and their combinations can form at least 24 types of heterodimers, each having its own binding specificity [3, 9].

[0016] The structure of integrin is shown in Figure 1. This structure includes a "head" region (the main contact point between two subunits) supported by two rod-shaped "legs". Integrins adopt different conformations with different binding affinities for integrin ligands. Examples of integrin ligands include collagen, laminin, thrombospondin, and fibronectin [4]. In the bent-closed and extended-closed conformations, the lower "legs" and transmembrane regions maintain association, and ligand binding is closed. When adopting the high-affinity extended-open conformation, it is accompanied by a series of shape changes such as the bending-open of the receptor and movements between and within various modules. The shift from the bent-closed / extended-closed conformation to the extended-open conformation is termed "integrin activation". Integrin activation may occur via the α or β subunit. Many anti-integrin antibodies (including TS2 / 16) that stimulate integrin activation and ligand binding function allosterically. Antibody binding stabilizes the active integrin signaling conformational form, shifting the conformational equilibrium of integrin from the inactive form to the active form. Thereby, the proportion of active integrin molecules increases [5]. In studies investigating the β1 integrin subfamily, it has been shown that the TS2 / 16, 12G10, and HUTS-4 Fab antibodies induce nearly the same high affinity for cyclic RGD peptides, suggesting that they stabilize the same conformation of the ligand-binding site in the β1 domain [6].

[0017] Accordingly, an integrin agonist is an agent that activates integrin. In other words, an integrin agonist is an agent that induces a conformational change in integrin, thereby increasing its binding affinity to an integrin ligand. In some embodiments, the integrin agonist is not collagen, laminin, thrombospondin, or fibronectin. In some embodiments, the integrin agonist activates integrin and mimics the activity of an integrin ligand that binds to integrin. In some embodiments, the integrin agonist can activate integrin and mimic the activity of an integrin ligand that binds to integrin without depending on the conformational state of integrin.

[0018] These integrin agonists are expected to be particularly useful in the context of the present invention. Integrin agonists can be readily identified by those skilled in the art using cell adhesion assays such as those described in Example 3 and Reference

[17] . Also, integrin agonists that can mimic the activity of a ligand that binds to integrin can be identified by those skilled in the art using assays such as those described in Example 4.

[0019] In some embodiments, the integrin agonist has the same or similar affinity for cyclic RGD peptide as integrin agonist TS2 / 16. For example, the integrin agonist binds to FITC-cRGD with an FP max of 0.2 - 0.3, 0.20 - 0.25, or 0.21 - 0.23, or more preferably an FP max of 0.21 - 0.22 in a fluorescence polarization assay as described in [7]. In some embodiments, the integrin agonist can stabilize the open head conformation of the same integrin as TS2 / 16.

[0020] Integrin agonists having a similar or the same affinity as integrin agonist TS2 / 16 for cyclic RGD peptides can be identified by those skilled in the art using assays as described in [7]. Integrin agonists that can stabilize the same open-head conformation of integrin as TS2 / 16 can be identified by those skilled in the art using the electron microscopy assay described in reference [6]. Many integrin agonists are well known in the art, including anti-integrin antibodies, reducing agents such as dithiothreitol, and lipids such as 25-hydroxy cholesterol [8-11]. In a preferred embodiment, the integrin agonist activates integrin through its extracellular domain. Such integrin agonists include anti-integrin antibodies, reducing agents such as dithiothreitol, and lipids such as 25-hydroxy cholesterol. In contrast, talin and kindlin activate integrin through interaction with the intracellular domain of integrin.

[0021] Antibodies directed against integrins may have activating or inhibitory properties. The term "integrin agonist" encompasses only anti-integrin antibodies having a stimulatory or activating effect. All stimulatory or activation-specific anti-integrin monoclonal antibodies appear to enhance ligand binding affinity by reducing the dissociation rate and can be classified into two classes. The first subclass, such as the anti-β1 monoclonal antibody HUTS-4, recognizes epitopes (known as ligand-induced binding sites "LIBS") controlled by ligand and cation binding, while the second subclass (including the anti-β1 monoclonal antibody, TS2 / 16) is not affected by ligand or cation binding.

[0022] Examples of activating anti-integrin antibodies are shown below.

[0023] (Table 1) Summary of stimulatory anti-integrin antibodies TIFF2025090761000001.tif102128

[0024] The inventors have demonstrated that the addition of an integrin agonist to a cell culture medium can improve the proliferation of epithelial stem cells without the need for an extracellular matrix. In the examples, it is shown that the addition of a humanized TS2 / 16 anti-integrin antibody to the cell culture medium improved the proliferation of epithelial stem cells without the need for an extracellular matrix. Furthermore, the inventors have demonstrated that the addition of an integrin agonist can improve the proliferation of epithelial stem cells in combination with an extracellular matrix (basement membrane extract) that is Matrigel. Therefore, the integrin agonist described herein is particularly useful for culturing epithelial stem cells.

[0025] In some embodiments, the integrin agonist is selected from anti-integrin antibodies, talin, kindlin, dithiothreitol, and the oxysterol 25-hydroxy cholesterol.

[0026] While not wishing to be bound by any particular theory, the conformation state of integrin, which is usually associated with ligand binding, can be induced by integrin agonists, so that integrin agonists can advantageously improve the proliferation of epithelial stem cells without the need for the extracellular matrix. Integrin agonists can induce the high-affinity conformation of integrin and activate signal transduction pathways that are normally activated by ECM components. In the examples, both anti-integrin antibodies (e.g., HUTS-4) controlled by ligand and cation binding, and anti-integrin antibodies (e.g., TS2 / 16) not affected by ligand or cation binding are shown to be particularly useful for culturing epithelial stem cells. Integrin agonists can interact through the α or β subunits of integrin, for example, by interacting with α2, α4, α5, α11b, αL, αX, β1, β2, β3, β4 or β7. In some embodiments, the integrin agonist interacts with the β subunits β1, β2, β3, β4 or β7. In a preferred embodiment, the integrin agonist interacts with the β1 subunit.

[0027] The integrin agonist can be an anti-integrin antibody. To avoid ambiguity, the anti-integrin antibody of the present invention is a stimulatory anti-integrin antibody. Examples of anti-integrin antibodies include JBS2, HP1 / 3, SNAKA51, PTS25-2, PMI-1, MEM-83, NKI-L16, 496B, 12G10, 8A2, TS2 / 16, 15 / 7, HUTS-4, 8E3, N29, 9EG7, mAb 24, MEM-148, KIM127, CBR LFA-1 / 2, MEM-48, KIM185, AP3, AP5, LIBS6, LIBS2, 10F8, 2B8, and 2G3 antibodies. In some embodiments, anti-integrin antibodies such as the TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29, and 9EG7 antibodies interact with the β1 subunit. In one embodiment, the anti-integrin antibody interacts with the β1 subunit and is MAB1778 (available at the website https: / / www.rndsystems.com / products / human-integrin-beta1-cd29-antibody-4b7r_mab1778, catalog number: number MAB1778).

[0028] The TS2 / 16, 12G10 or 8A2 antibodies are known to interact with the βA domain. In the examples, the advantageous properties of the TS2 / 16 anti-integrin antibody for culturing epithelial stem cells are shown. Thus, the anti-integrin antibodies of the present invention can preferably interact with the βA domain. In a preferred embodiment, the anti-integrin antibody is TS2 / 16, 12G10 or 8A2. In other preferred embodiments, the anti-integrin antibody is TS2 / 16, 12G10 or HUTS-4. In a more preferred embodiment, the anti-integrin antibody is TS2 / 16 (or humanized TS2 / 16) or HUTS-4 (or humanized HUTS-4). In a more preferred embodiment, the anti-integrin antibody is TS2 / 16 or humanized TS2 / 16. In a more preferred embodiment, the anti-integrin antibody is HUTS-4 or humanized HUTS-4. An overview of the epitopes of known activating anti-integrin β1 chain antibodies is shown in Table 2. These epitopes are classified into two groups. The first group consists of residues of a very short sequence (residues 207 - 218) located in the predicted ligand-binding domain, and the others are epitopes within or near a cysteine-rich repeat structure (residues 442 - 629) located in the membrane-proximal stalk region of integrin. Some activating anti-integrin β1 chain antibodies are effective only when integrin is in a specific conformation, and these antibodies, such as HUTS-4, HUTS-7 and HUTS-21

[12] , are thought to bind to epitopes that are exposed only under specific physiological conditions

[13] .

[0029] (Table 2) Overview of activating anti-integrin antibodies,

[13] and

[12] TIFF2025090761000002.tif110136

[0030] Thus, in some embodiments, the integrin agonist (optionally an anti-integrin antibody) interacts with at least a portion of residues 207-218 of the β1 subunit. In some embodiments, the integrin agonist (optionally an anti-integrin antibody) interacts with at least a portion of residues 442-629 of the β1 subunit. In some embodiments, the integrin agonist (optionally an anti-integrin antibody) interacts with at least a portion of residues 335-425 of the β1 subunit. In some embodiments, since their ability to activate integrins is not dependent on physiological conditions, integrin agonists that bind without depending on the conformation of integrins are preferred. In a preferred embodiment, the epitope for the integrin antibody is not the ligand-induced binding site. The anti-integrin antibody may comprise the heavy chain variable region (VH) of SEQ ID NO: 1 and the light chain variable region (VL) of SEQ ID NO: 2.

[0031] The anti-integrin antibody may also be humanized, for example, the anti-integrin antibody may be the humanized TS2 / 16 antibody as shown in the examples. The humanized TS2 / 16 antibody may comprise the heavy chain variable region (VH) of SEQ ID NO: 3 and the light chain variable region (VL) of SEQ ID NO: 4. In some embodiments, the anti-integrin antibody comprises HCDR1, HCDR2, and HCDR3 of the heavy chain CDRs of SEQ ID NOs: 5, 6, and 7, respectively, and LCDR1, LCDR2, and LCDR3 of the light chain CDRs of SEQ ID NOs: 8, 9, and 10, respectively.

[0032] In an embodiment, a combination of an agonist anti-β1 integrin antibody (TS2 / 16) and an agonist anti-β4 antibody (3E1) shows advantageous and synergistic properties for culturing epithelial stem cells. Thus, integrin agonists may be used in combination for culturing epithelial stem cells. For example, multiple anti-integrin antibodies may be used for culturing epithelial stem cells. In some embodiments, two anti-integrin antibodies target the same subunit of integrin. For example, two or more β1 integrin agonist antibodies are used for culturing epithelial stem cells. In some embodiments, the two or more β1 integrin agonist antibodies are selected from TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29, and 9EG7, or preferably selected from TS2 / 16, 12G10, and 8A2. In some embodiments, TS2 / 16 is used in combination with HUTS-4.

[0033] Alternatively, the two anti-integrin antibodies target different subunits of integrin. Possible combinations of anti-integrin antibodies include: (1) a combination of a β1 integrin agonist antibody and a β2 integrin agonist antibody, (2) a combination of a β1 integrin agonist antibody and a β3 integrin agonist antibody, (3) a combination of a β1 integrin agonist antibody and a β4 integrin agonist antibody, (4) a combination of a β1 integrin agonist antibody and a β7 integrin agonist antibody, (5) a combination of a β1 integrin agonist antibody and an α2 integrin agonist antibody, (6) a combination of a β1 integrin agonist antibody and an α4 integrin agonist antibody, (7) a combination of a β1 integrin agonist antibody and an α5 integrin agonist antibody, (8) a combination of a β1 integrin agonist antibody and an α11b integrin agonist antibody, (9) a combination of a β1 integrin agonist antibody and an αL integrin agonist antibody, (10) a combination of a β1 integrin agonist antibody and an αX integrin agonist antibody.

[0034] In a preferred embodiment, the β1 integrin agonist antibody is used in combination with a β4 integrin agonist antibody for culturing epithelial stem cells. In a preferred embodiment, the β1 integrin agonist antibody is TS2 / 16, 12G10 or 8A2. In a preferred embodiment, the β4 integrin agonist antibody is 3E1. In a more preferred embodiment, TS2 / 16 and 3E1 are used in combination for culturing epithelial stem cells.

[0035] In some embodiments, the β1 integrin agonist antibody is used in combination with a β4 integrin agonist antibody and an anti-integrin antibody of an agonist that binds to one of α2, α4, α5, α11b, αL, αX, β2, β3 and β7.

[0036] In some embodiments, the β1 integrin agonist antibody is used in combination with (i) a β4 integrin agonist antibody and (ii) two or more (e.g., 3, 4, 5, 6, 7, 8 or 9) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, αX, β2, β3 and β7.

[0037] In some embodiments, the β2 integrin agonist antibody is used in combination with an anti-integrin antibody of an agonist that binds to one of α2, α4, α5, α11b, αL, αX, β1, β3, β4 and β7. In some embodiments, the β2 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9 or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, αX, β1, β3, β4 and β7.

[0038] In some embodiments, the β3 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, α11b, αL, αX, β1, β2, β4, and β7. In some embodiments, the β3 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, αX, β1, β2, β4, and β7.

[0039] In some embodiments, the β4 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, α11b, αL, αX, β1, β2, β3, and β7. In some embodiments, the β4 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, αX, β1, β2, β3, and β7.

[0040] In some embodiments, the β7 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, α11b, αL, αX, β1, β2, β3, and β4. In some embodiments, the β7 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, αX, β1, β2, β3, and β4.

[0041] In some embodiments, the α2 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α4, α5, α11b, αL, αX, β1, β2, β3, β4, and β7. In some embodiments, the α2 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α4, α5, α11b, αL, αX, β1, β2, β3, β4, and β7.

[0042] In some embodiments, the α4 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α5, α11b, αL, αX, β1, β2, β3, β4, and β7. In some embodiments, the α4 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α5, α11b, αL, αX, β1, β2, β3, β4, and β7.

[0043] In some embodiments, the α5 integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α11b, αL, αX, β1, β2, β3, β4, and β7. In some embodiments, the α5 integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α11b, αL, αX, β1, β2, β3, β4, and β7.

[0044] In some embodiments, the α11b integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, αL, αX, β1, β2, β3, β4, and β7. In some embodiments, the α11b integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, αL, αX, β1, β2, β3, β4, and β7.

[0045] In some embodiments, the αL integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, α11b, αX, β1, β2, β3, β4, and β7. In some embodiments, the αL integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αX, β1, β2, β3, β4, and β7.

[0046] In some embodiments, the αX integrin agonist antibody is used in combination with an agonist anti-integrin antibody that binds to one of α2, α4, α5, α11b, αL, β1, β2, β3, β4, and β7. In some embodiments, the αX integrin agonist antibody is used in combination with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) integrin agonist antibodies that each bind to a different integrin subunit selected from α2, α4, α5, α11b, αL, β1, β2, β3, β4, and β7.

[0047] In other preferred embodiments, the binding of the integrin agonist is conformation-dependent of the integrin. In other embodiments, the epitope for the integrin antibody is the ligand-induced binding site. The anti-integrin antibody may be HUTS-4. In some embodiments, the anti-integrin antibody binds to the same epitope as the TS2 / 16 antibody. Thus, in some embodiments, the anti-integrin antibody binds to the same epitope as an antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 3 and the light chain variable region (VL) of SEQ ID NO: 4. In some embodiments, the anti-integrin antibody binds to the same epitope as the HUTS-4 antibody.

[0048] In some embodiments, the anti-integrin antibody competes with the TS2 / 16 antibody when binding to the β1 subunit. Thus, in some embodiments, the anti-integrin antibody binds to the same epitope as an antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 3 and the light chain variable region (VL) of SEQ ID NO: 4. In some embodiments, the anti-integrin antibody competes with the HUTS-4 antibody when binding to the β1 subunit. In some embodiments, the anti-integrin antibody competes with the 3E1 antibody when binding to the β4 subunit.

[0049] Conventional antibodies are composed of two identical heavy chains and two identical light chains linked by disulfide bonds. Each of the heavy and light chains is composed of a constant region and a variable region. Each of the variable regions is composed of three CDRs that mainly play a role in binding to the epitope of the target antigen. They are called CDR1, CDR2, and CDR3, numbered in order from the N-terminus, and the CDR3 region is the most variable region and usually provides most of the contact residues to the target. The most conserved part of the variable region is called the "framework region". In some embodiments, the antibody portion comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion is composed of one heavy chain and one light chain.

[0050] The term "antibody" is used herein in the broadest sense and includes, in particular, monoclonal antibodies of any isotype, such as IgG, IgM, IgA, IgD, and IgE (including full-length monoclonal antibodies), polyclonal antibodies including recombinant polyclonal antibodies, Oligoclonics, multispecific antibodies, chimeric antibodies, nanobodies, diabodies, BiTE, Tandabs, mimetobodies, bispecific antibodies, humanized antibodies, human antibodies, deimmunised antibodies, as well as antibody fragments, but is not limited thereto. Furthermore, scaffolds such as Anticalin, Ankarin, etc. are also included as belonging to this term. Antibodies that react with specific epitopes of a target molecule can be generated by recombinant methods such as the selection of a library of recombinant antibodies in a phage vector or a similar vector, or by immunizing an animal with a target nucleic acid encoding the target epitope.

[0051] In certain embodiments, the antibody or antigen-binding fragment of the invention is bispecific and comprises a first binding specificity to a first epitope of an integrin subunit and a second binding specificity to a second epitope of the integrin subunit, wherein the first and second epitopes are different and non-overlapping. In certain embodiments, the bispecific antibody binds to different epitopes on the same integrin subunit, such as the β1 subunit. In other embodiments, the bispecific antibody binds to epitopes on different integrin subunits, for example, the bispecific antibody has a first binding specificity to a first epitope of the β1 integrin subunit and a second binding specificity to a second epitope of the β3 integrin subunit.

[0052] In some embodiments, the antibodies according to the invention are composed of, or comprise, single domain antibodies (also called sdAbs or nanobodies), F(ab’)2, Fab, Fab’, Facb, or single chain Fv (scFv) fragments. The scFv fragment is an epitope-binding fragment and is composed of at least one fragment of the variable region of the heavy chain of the antibody (VH) linked to at least one fragment of the variable region of the light chain of the antibody (VL). The linker is a short flexible peptide selected to ensure that the appropriate three-dimensional folding of the VL and VH regions occurs when they are linked, so as to maintain the target molecule binding specificity of the whole antibody from which the single-chain antibody fragment is derived. The carboxyl terminus of the VL or VH sequence may be covalently linked by the linker to the amino terminus of the complementary VL or VH sequence.

[0053] In some embodiments, the antibody portion of the fusion protein comprises a constant region or an Fc region. The Fc region may be obtained from its native form isolated from humans and other animals such as cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be a recombinant or derivative thereof obtained from transformed animal cells or microorganisms. They can be obtained from native immunoglobulins by isolating all immunoglobulins from a human or animal organism and treating them with proteolytic enzymes. Papain digests native immunoglobulins into Fab and Fc regions, and pF’c and F(ab)2 fragments are generated by pepsin treatment. These fragments can be subjected to, for example, size exclusion chromatography to isolate the Fc.

[0054] In some embodiments, the Fc region is modified. For example, the immunoglobulin Fc region of the present invention may be in a form having natural sugar chains, more sugar chains compared to the natural form, or fewer sugar chains compared to the natural form, or may be in a deglycosylated form. The increase, decrease, or removal of immunoglobulin Fc sugar chains can be achieved by methods common in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removing sugar chains from the Fc region results in a sharp decrease in the binding affinity to complement (c1q), and a decrease or disappearance of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. In this context, a deglycosylated or aglycosylated form of the immunoglobulin Fc region (the latter being produced, for example, by prokaryotes, preferably E. coli) may be used according to the present invention.

[0055] Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or may be produced by its hybrid (a sequence encoding two or more immunoglobulin Fc regions of different origins present in a single-chain immunoglobulin Fc region). In the present invention, various types of hybrids are conceivable. That is, a domain hybrid may be composed of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc, and may include a hinge region. Therefore, in some embodiments, the Fc region is a hybrid. Preferably, the Fc region is derived from IgG or IgM, which are one of the most abundant proteins in human blood, and most preferably from IgG, which is known to extend the half-life of ligand-binding proteins. Furthermore, IgG is divided into IgG1, IgG2, IgG3, and IgG4 subclasses, and the present invention includes combinations or hybrids thereof. IgG2 and IgG4 subclasses are preferred.

[0056] In some embodiments, the Fc region is an isotype of IgG, such as IgG1, preferably human IgG or human IgG1.

[0057] In some embodiments, the Fc region is modified to abrogate or reduce ADCC and / or complement effector function. In some embodiments, the Fc region is modified to enhance FcRn affinity (which can extend the half-life). Suitable modifications are described in Monnet, Celine, et al. “Selection of IgG Variants with Increased FcRn Binding Using Random and Directed Mutagenesis: Impact on Effector Functions.” Frontiers in Immunology 6:39 (2015). In some embodiments, the antibody is composed of an Fc domain or a portion thereof that binds to the FcRn receptor. As a non-limiting example, suitable Fc domains may be derived from immunoglobulin subclasses such as IgA, IgE, IgG, or IgM. In some embodiments, suitable Fc domains are derived from IgG1, IgG2, IgG3, or IgG4. Particularly suitable Fc domains are those derived from human antibodies.

[0058] In some embodiments, the modified Fc region is an IgG variant that contains one or more, or preferably all, of the mutations: E294Del, T307P, and N434Y (see, e.g., Table 6, entry “C6A-66” of Monnet et al.). These specific mutations are expected to abrogate ADCC and complement effector function and extend the half-life.

[0059] In some embodiments, the antibodies of the invention are polyclonal, monoclonal, multispecific, murine, human, humanized, primatized or chimeric antibodies or single-chain antibodies. In some embodiments, the antibody may be a human antibody or a humanized antibody. In a preferred embodiment, the antibody is a humanized antibody.

[0060] In some embodiments, the antibody binds to integrin with a K -7 of 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 D M or less. For example, the antibody can bind to the α or β subunit of integrins such as α2, α4, α5, α11b, αL, αX, β1, β2, β3, β4, and β7. In some embodiments, the antibody binds to the β1, β2, β3, β4, or β7 subunit of integrin, and in a preferred embodiment, the antibody binds to the β1 subunit of integrin. Accordingly, in a preferred embodiment, the antibody binds to the β1 subunit of integrin with a K -7 of 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 D M or less. In a further embodiment, a first antibody that binds to the β1 subunit of integrin with a K -7 of 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 D M or less is combined with a second antibody that binds to the β4 subunit of integrin. For example, in one embodiment, a first antibody that binds to the β1 subunit of integrin with a K -7 of 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 D M or less is combined with a second antibody that binds to the β4 subunit of integrin with a K -8 of 10 D M or less. -8 M or less. D

[0061] ​In some embodiments, antibody binding affinity is determined using an Octet® RED96 system (ForteBio, Inc.). For example, a FLAG-tagged β1 subunit, FLAG-tagged β2 subunit, FLAG-tagged β3 subunit, or FLAG-tagged β7 subunit is immobilized on an Anti-FLAG biosensor and incubated with various concentrations of an antibody in solution, whereby binding data can be collected. In some embodiments, antibody binding affinity is determined by surface plasmon resonance.

[0062] In some embodiments, whether a test antibody competes with a control antibody when binding to an integrin is determined using an in vitro binding competition assay. For example, a FLAG-tagged β1 subunit, FLAG-tagged β2 subunit, FLAG-tagged β3 subunit, or FLAG-tagged β7 subunit is immobilized on an Anti-FLAG biosensor, and then the association of the control antibody to the immobilized FLAG-tagged β1, β2, β3, or β7 subunit is measured (e.g., using an Octet® RED96 system, ForteBio, Inc.), followed by assessing the extent of additional binding by exposing the immobilized FLAG-tagged β1, β2, β3, or β7 subunit to the test antibody in the presence of the control antibody.

[0063] In some embodiments, the anti-integrin antibody is a heavy-chain only antibody. The term "antibody" encompasses the entire tetrameric antibody and antigen-binding fragments thereof. In some embodiments, the antigen-binding fragment is selected from VH domain, Fab, Fab’, F(ab’)2, Fd, Fv, single-chain Fv (scFv), and disulfide-bonded Fv (sdFv).

[0064] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR adjacent to or within one or more framework sequences. V LIn an antigen-binding fragment having a VH domain that associates with a domain, the VH domain and the V L domains may be positioned in any suitable arrangement relative to each other. For example, the variable regions may be dimers, and may be VH-VH, VH-V L or V L -V L dimers. Alternatively, the antigen-binding fragment of an antibody may be composed of a monomeric VH or V L domain.

[0065] In certain embodiments, the antigen-binding fragment of an antibody may be composed of at least one variable domain covalently linked to at least one constant domain. Non-limiting and exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of the antibodies of the present invention include (i) VH-C H 1, (ii) VH-C H 2, (iii) VH-C H 3, (iv) VH-C H 1-C H 2, (V) VH-C H 1-C H 2-C H 3, (vi) VH-C H 2-C H 3, (vii) VH-C L , (viii) V L -C H 1, (ix) V L -C H 2, (x) V L -C H 3, (xi) V L -C H 1-C H 2, (xii) V L -C H 1-C H 2-C H 3, (xiii) V L -C H 2-C H 3, and (xiv) V L -C L are included.

[0066] In any configuration of the variable and constant domains, including any of the exemplary configurations described above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge or linker region may be composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Further, the antigen-binding fragments of the antibodies of the present invention may be non-covalently associated with each other and / or non-covalently associated with one or more monomeric VH or V L domains (e.g., by disulfide bond(s)) and may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations described above.

[0067] Similar to a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically includes at least two different variable domains, where each variable domain has the ability to specifically bind to a distinct antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present invention using routine techniques available in the art.

[0068] Methods and techniques for identifying CDRs from HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs from the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that may be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is intermediate between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences in antibodies.

[0069] In other embodiments, the integrin agonist is not an antibody. For example, the integrin agonist may be talin, or the integrin agonist may be kindlin. The integrin agonist may be talin used in combination with kindlin. In another example, the integrin agonist may be a reducing agent such as dithiothreitol, or a lipid such as the oxysterol 25-hydroxy cholesterol. In some embodiments, two or more integrin agonists are used, including any combination of integrin agonists described herein.

[0070] In some embodiments, the integrin agonist results in at least 50%, 60%, 70%, 80%, 90% or 100% cell growth in the claimed culture method as compared to the same method carried out using the humanized TS2 / 16 antibody of the examples (such as as tested by the method presented in Example 4). In some embodiments, the integrin agonist results in at least 50%, 60%, 70%, 80%, 90% or 100% cell growth in the claimed culture method as compared to the same method carried out using the mouse HUTS-4 antibody of the examples (such as as tested by the method presented in Example 6). In some embodiments, the method of the present invention results in at least a 10%, 20%, 50% increase in epithelial stem cell growth over 4 days as compared to the same method carried out without an integrin agonist. In some embodiments, the method of the present invention results in at least a 2-fold, 3-fold, 4-fold or 5-fold increase in epithelial stem cell growth over 4 days as compared to the same method carried out without an integrin agonist. Epithelial stem cell growth can be defined as the number of organoids and can be tested according to the methods of Examples 3, 4 or 5.

[0071] In some embodiments, the method of the present invention results in the growth of new organoids. In some embodiments, the method of the present invention results in the growth of existing organoids.

[0072] The integrin agonist can be brought into contact with epithelial stem cells or organoids containing epithelial stem cells as part of the culture medium, as a component of the extracellular matrix or artificial matrix, or as a separate component added to the culture vessel.

[0073] Extracellular matrix and artificial matrix Epithelial stem cells typically grow in culture with an exogenous extracellular matrix that is known to support cell proliferation (see, for example,

[20] which describes plating methods of cells with basement membrane extract (BME; Amsbio) or Matrigel (BD Biosciences)). Basement membrane extract (Amsbio) and Matrigel™ (BD Biosciences) are examples of commercially available extracellular matrices derived from basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. However, since the exact components of these extracellular matrices are not clearly defined, they can be a source of unwanted variability when culturing epithelial stem cells

[14] .

[0074] The component parts of the extracellular matrix and the mechanisms by which they act have not been fully confirmed. Artificial substitutes for the extracellular matrix, such as polymers and hydrogels, have not been able to faithfully reproduce the growth efficiency achieved by the extracellular matrix in epithelial stem cell culture. The use of extracellular matrix proteins such as laminin and fibronectin instead of the extracellular matrix has also not been able to achieve the reproduction of the growth efficiency achieved by the extracellular matrix in epithelial stem cell culture.

[0075] Unexpectedly, each example shows that a culture medium containing an integrin agonist can improve the growth of epithelial stem cells in the culture medium both in the presence and absence of an extracellular matrix. Without wishing to be bound by any particular theory, a functional extracellular matrix contains structural and signaling components. It is thought that artificial matrices alone provide only structural components. Therefore, it is proposed that integrin agonists may function as the missing and long-sought signaling components and also provide additional structural support.

[0076] In an example, it is surprisingly shown that epithelial stem cell proliferation can be improved by culturing with a culture solution containing an integrin agonist in the absence of an exogenous extracellular matrix. Thus, in some embodiments, the culturing method does not include contacting the cells with an exogenous extracellular matrix. For example, the culturing method does not include contacting the cells with an exogenous extracellular matrix containing glycoprotein, and for example, the culturing method does not include contacting the cells with a basement membrane extract or Matrigel.

[0077] Integrin agonists are particularly useful for use with artificial matrices. Thus, in some embodiments, the culturing method of the present invention may include culturing epithelial stem cells in contact with an artificial matrix. The components of the artificial matrix can be controlled, thereby reducing the variability that can occur when culturing epithelial stem cells and facilitating the improvement of the proliferation rate, proliferation, and / or differentiation of epithelial stem cells during culture. The artificial matrix can include any polymer, such as polyester, polyethylene glycol, or hydrogel. In some embodiments, the artificial matrix includes polyethylene glycol and / or hydrogel. In some embodiments, the artificial matrix includes a crosslinked polyethylene glycol (PEG) hydrogel.

[0078] The artificial matrix can be provided in any suitable form, such as on a surface, on beads, or as a coating (e.g., on a culture plate, culture vessel, or beads). The beads are preferably microbeads. In the example, an ultra-low adhesion surface (Sigma Aldrich) is used. This surface is a covalently bonded hydrogel layer that is hydrophilic and electrically neutral. Thus, in some embodiments, the artificial matrix is a hydrogel layer.

[0079] In some embodiments, the artificial matrix comprises a biomaterial, preferably an extracellular matrix component. For example, the biomaterial may include one or more glycoproteins (optionally selected from collagen, laminin, perlecan, fibronectin, or the RGD adhesion ligand of fibronectin), and / or one or more carbohydrates (optionally hyaluronic acid). An example of an artificial matrix that can be used is Pronectin (e.g., Sigma Z378666). Pronectin contains a non-animal source polymer incorporating multiple copies of the RGD adhesion ligand of human fibronectin that occupies the space between the peptide units of the repeating structure. It can be provided in the form of beads coated with Pronectin-F. The core beads are solid copolymers sized 125 - 212 micrometers in diameter. Thus, in some embodiments, the artificial matrix contains the RGD adhesion ligand of fibronectin. In some embodiments, the PEG gel may be enriched with extracellular matrix components such as fibronectin, laminin-111, collagen IV, hyaluronic acid, and perlecan. In some embodiments, the fibronectin protein within the PEG gel can be replaced with an RGD (Arg-Gly-Asp) peptide

[15] .

[0080] Another example of an artificial matrix is an artificial hydrogel based on a 4-arm maleimide-terminated poly(ethylene glycol) macromer as described in

[16] . The macromer is functionalized with an adhesion peptide and crosslinked in the presence of cells to produce a PEG-4MAL hydrogel. The hydrogel polymer concentration can be 3.5 - 6.0% weight / volume, preferably the hydrogel polymer concentration is 4%. The hydrogel polymer can be prepared to include an RGD adhesion peptide or a GPQ-W crosslinking peptide.

[0081] Hydrogel polymers can be prepared with protein A / G. Protein A / G is a recombinant fusion protein that combines the IgG-binding domains of both protein A and protein G. Protein A / G is linked to the hydrogel by a linker, providing a framework to which antibodies can adhere. Then, by thoroughly soaking the hydrogel with anti-integrin antibodies, the antibodies can be present in a multivalent manner. This preparation of artificial ECM helps to reproduce the natural ECM environment found within cells because both the structural and signaling components of the ECM are provided together. The gel of the hydrogel can be made from dextran polymers or benzene-1,3,5-tricarboxamide polymers. In a preferred embodiment, the hydrogel is made from an elastin-like protein (ELP) having an antibody-binding unit (Z33 peptide) at its N-terminus.

[0082] Also, in the examples, it is shown that the integrin agonist results in an increase in the growth rate of epithelial stem cells in culture even in the presence of an exogenous extracellular matrix. Thus, in some embodiments, the method of the invention may include culturing epithelial stem cells in contact with an extracellular matrix (ECM). The ECM is secreted by epithelial cells, endothelial cells, cranial endoderm-like cells (e.g., Engelbreth-Holm-Swarm cranial endoderm-like cells as described in Hayashi et al. (2004) Matrix Biology 23:47-62), and connective tissue cells. It can be prepared by culturing ECM-secreting cells and isolating the ECM. Alternatively, commercial products such as Matrigel or BME are available. The ECM is composed of various polysaccharides, water, elastin, and glycoproteins, where the glycoproteins include collagen, entactin (nidogen), fibronectin, and laminin. The ECM referred to herein is naturally produced by ECM-secreting cells. In some embodiments, the ECM is a basement membrane preparation derived from Engelbreth-Holm-Swarm cranial endoderm-like cells (as described in Hayashi et al. (2004) Matrix Biology 23:47-62).

[0083] As used in the context of the present invention, the ECM is an exogenous ECM (which means not only any extracellular matrix protein naturally secreted by epithelial stem cells when contacting the culture medium of the present invention, but rather, produced by cells other than the epithelial stem cells that will be cultured by the method of the present invention).

[0084] In some embodiments, the ECM is a three-dimensional matrix. In some embodiments, the cells are embedded in the ECM. In some embodiments, the cells are adhered to the ECM. The culture medium of the present invention can be diffused into the three-dimensional ECM. In other embodiments, the ECM is contained in a suspension, i.e., the cells are in contact with the ECM within a suspension system. In some embodiments, the ECM is contained in the suspension at a concentration of at least 1%, at least 2% or at least 3%. In some embodiments, the ECM is contained in the suspension at a concentration of from 1% to about 10%, or from 1% to about 5%. The suspension method may be advantageous for improved methods.

[0085] In some embodiments, the culturing method of the present invention includes culturing epithelial stem cells in contact with an extracellular matrix and / or an artificial matrix. "In contact" means physical or mechanical or chemical contact, which means that force is required when separating the resulting organoids or population of epithelial cells from the above matrix. The culture medium and / or cells can be placed on, embedded in, or mixed with the extracellular matrix or artificial matrix.

[0086] In some embodiments, the culture medium is placed on top of the extracellular matrix or artificial matrix. Next, the culture medium may be removed and replenished as needed. In some embodiments, the culture medium is replenished every 1, 2, 3, 4, 5, 6 or 7 days. When a component is "added" to or "removed" from the medium, this may mean in some embodiments that the medium itself is removed from the extracellular matrix or artificial matrix and a new medium containing the "added" component or not containing the "removed" component is placed on the extracellular matrix or artificial matrix.

[0087] The three-dimensional matrix aids in the culture of three-dimensional epithelial organoids. Thus, in some embodiments, the extracellular matrix or artificial matrix is a three-dimensional matrix.

[0088] The extracellular matrix or artificial matrix may further contain an integrin agonist as described above.

[0089] Culture of epithelial stem cells or organoids When epithelial stem cells proliferate and / or differentiate to produce organoids, they are typically passaged (i.e., split) regularly according to methods known in the art. Passaging typically involves mechanically dissociating the organoids, optionally removing them from the extracellular matrix or artificial matrix, collecting, washing, and plating them at a suitable ratio (e.g., 1:5 to 1:20). The culture medium is also typically replenished regularly as needed.

[0090] Thus, in some embodiments, the method further comprises passaging the cells once every two weeks, once a week, once every ten days, once every two weeks, once every 5 - 20 days, preferably once every 7 - 14 days.

[0091] In some embodiments, the method further comprises plating the cells at a ratio between 1:5 and 1:20.

[0092] In some embodiments, the method further comprises replenishing the culture medium every 1 - 3 days, every 1 - 2 days, every two days, or daily. In preferred embodiments, the method further comprises replenishing the culture medium every 1 - 3 days or every 1 - 2 days.

[0093] In some embodiments, the method comprises culturing epithelial stem cells over at least 5 passages, for example, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60 passages, or over 6 - 40 passages, for example, about 8 - 35 passages, 10 - 30 passages, or 12 - 25 passages. In some embodiments, the method comprises culturing epithelial stem cells over 8 - 50, 10 - 50, 15 - 50, 20 - 50 or 20 - 40 passages. In some embodiments, the method comprises culturing epithelial stem cells for at least 2 weeks, at least 1 month, at least 2 months, more preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 24, at least 25, at least 30 months or more, for example 3 years or more.

[0094] Epithelial stem cells and their preparations for culture The epithelial stem cells can be a single cell or part of a population of epithelial stem cells. The epithelial stem cells can be included in organoids and / or epithelial tissue explants. In certain embodiments, the epithelial stem cells are mammalian epithelial stem cells, optionally human or mouse epithelial stem cells. In preferred embodiments, the epithelial stem cells are human epithelial stem cells. In some embodiments, the epithelial stem cells are characterized by Lgr5 expression.

[0095] The epithelial stem cells are obtained from adult tissue. That is, the epithelial stem cells are adult epithelial stem cells. In this context, "adult" means mature tissue, i.e., including neonates or infants, but not embryos or fetuses. Alternatively, the epithelial stem cells are not derived from, for example, embryonic stem cells or embryonic stem cell lines differentiated in vitro.

[0096] Epithelial stem cells may be derived from colorectal, small intestine, stomach, pancreas, liver, lung, breast, prostate, kidney, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle and / or cochlear tissue. In some embodiments, the epithelial stem cells are colorectal cells. Methods for culturing epithelial stem cells from various tissues have been previously described (e.g., WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613 and WO2016 / 083612, as well as, Clevers, Cell 165(7)1586-1597, (2016)).

[0097] Cells directly harvested from a tissue, i.e., newly isolated cells, are also referred to as primary cells. In some embodiments, the epithelial stem cells are primary epithelial stem cells.

[0098] Primary cell cultures can be passaged to form secondary cell cultures. Except for cancer cells, conventional secondary cell cultures have a limited lifespan. After a certain number of population doublings (e.g., 50 - 100 generations), the cells stop dividing through the process of senescence. Cells from secondary cultures may be immortalized to become continuous cell lines. Immortalization may occur spontaneously or be induced by viruses or chemically. Immortalized cell lines are also known as transformed cells. In contrast, the method of the present invention enables continuous passage of epithelial stem cells through organoid expansion without immortalization or transformation. Thus, in some embodiments, the epithelial stem cells are not immortalized cells or transformed cells, i.e., they do not derive from immortalized cell lines or transformed cell lines. An advantage of the present invention is that epithelial stem cells that have undergone multiple rounds of growth and passage retain the characteristics of primary cells and have few or no genetic or phenotypic changes. Thus, the starting population of epithelial stem cells (s) in the method of the present invention can be harvested from or derived from existing organoids and further cultured and expanded to generate new cells and organoids. Thus, in some embodiments, the epithelial stem cells of the population of epithelial stem cells are part of an organoid or isolated from an organoid, or the population of epithelial stem cells is an organoid, part of an organoid, or isolated from an organoid.

[0099] In some embodiments, the epithelial stem cells are normal cells. In alternative embodiments, the epithelial stem cells are cancer stem cells. Thus, the cells may be harvested from a tumor, if desired.

[0100] In some embodiments, the method includes culturing a tissue fragment containing epithelium. In some embodiments, the epithelial stem cells are isolated from the tissue fragment.

[0101] Organoids are preferably harvested using epithelial cells from adult tissue, optionally epithelial stem cells from adult tissue expressing Lgr5.

[0102] In some embodiments, the organoid is derived from a single cell that optionally expresses Lgr5. Advantageously, this allows for the formation of a homogeneous population of cells. A suspension of single cells containing epithelial stem cells can be generated mechanically. In some embodiments, a suspension of single cells containing epithelial stem cells is generated using mechanical processing and / or enzymatic digestion. Mechanical processing includes, but is not limited to, dissection, microdissection, and filtration.

[0103] In some embodiments, the starting culture is a cell aggregate or population, for example, a population of cells contained in a colorectal fragment. Thus, the method of the present invention is not limited to using a single cell as a starting point.

[0104] Epithelial stem cells can be obtained by any suitable isolation method known in the art. In some embodiments, the epithelial layer is microdissected from a tissue sample, such as a surgical specimen or biopsy material, digested with an enzyme, filtered, and the resulting cell suspension is plated. In some embodiments, microdissection involves removing other tissue types, such as fat and muscle. In some embodiments, the enzyme is trypsin, collagenase, or accutase. In some embodiments, the enzyme is trypsin, optionally 0.125% trypsin. In some embodiments, the sample is incubated at 37° C., and then optionally, the sample is disrupted at repeated time intervals, such as every 2, 5, 10, or 15 minutes, for example, using a pipette. In some embodiments, the sample is incubated with 0.125% trypsin at 37° C., and the sample is sheared approximately every 10 minutes using a pipette. In some embodiments, the enzymatic digestion is carried out for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, or about 60 minutes. In some embodiments, the enzymatic digestion is terminated by dilution with a suitable medium. In some embodiments, the filtration step is carried out using a 100 μm filter. In some embodiments, the cell suspension obtained from mechanical treatment or enzymatic digestion is contacted with ECM and culture medium.

[0105] One method for harvesting epithelial stem cells for culturing is based on the fact that epithelial stem cells express Lgr5 and / or Lgr6 on their surface and these proteins belong to the large G protein-coupled receptor (GPCR) superfamily (see, for example, WO2009 / 022907, the entire content of which is incorporated herein by reference). The Lgr subfamily is unique in having a large leucine-rich extracellular domain important for ligand binding. Thus, a preferred method includes preparing a cell suspension from the epithelial tissue as described above, contacting the cell suspension with an Lgr5 and / or 6-binding compound (such as an antibody, such as an anti-Lgr5 monoclonal antibody as described in WO2009 / 022907), isolating the Lgr5 and / or 6-binding compound, and isolating the stem cells from the binding compound.

[0106] After culturing, the method further includes harvesting and / or isolating one or more epithelial stem cells or organoids. For example, it may be useful for removing one or more stem cells and / or one or more organoids cultured in the culture medium from the culture medium for subsequent use in an application after culturing the stem cells. For example, it may be useful for isolating single cells for subsequent differentiation.

[0107] Organoids harvested and / or isolated by the methods of the present invention can recapitulate the properties of the starting epithelial stem cells. For example, cells cultured in a culture medium retain the same genotype and phenotype and include cells with stem-like properties (such as cells characterized by Lgr5 expression). This means that cells harvested from diseased tissue (such as cancerous tissue) faithfully model the disease in question. Alternatively, cells cultured in a differentiation medium may result in organoids containing more mature and differentiated cell types (compared to the starting cells).

[0108] Cells other than epithelial stem cells The method of the present invention may be advantageous not only for epithelial stem cells and epithelial organoids, but also for all cell types. In particular, integrin agonists may improve the cell culture of many cell types. For example, integrin agonists may be advantageous for other stem cells, particularly other stem cells that have been shown to benefit from culture with an extracellular matrix (such as Matrigel or BME) including embryonic stem cells or induced pluripotent stem cells (see, for example, Clevers, Cell 165(7)1586-1597, (2016)). The culture of embryonic stem cells and induced pluripotent stem cells often involves the differentiation of stem cells into more mature cell types. Thus, the culture of embryonic stem cells and induced pluripotent stem cells using integrin agonists may be particularly useful in relation to differentiation methods. Integrin agonists may also be particularly useful in hematopoietic stem cell culture (hematopoietic stem cells have been routinely used in many hospitals for decades, but the practical expansion of these cells during culture has not yet been achieved). The addition of integrin agonists is expected to improve hematopoietic stem cell culture.

[0109] Accordingly, this disclosure describes embodiments related to epithelial stem cells, which embodiments are also disclosed with respect to (general) stem cells, or embryonic stem cells or induced pluripotent stem cells. This disclosure describes embodiments related to epithelial stem cells, but the same embodiments are also disclosed with respect to hematopoietic stem cells.

[0110] Co-culture The method may further comprise co-culture with non-epithelial cell types, optionally with immune cells. The method includes the step of mixing the organoids described herein with immune cells in in vitro culture. In a preferred embodiment, the organoid co-culture is maintained in a co-culture medium as described herein.

[0111] In some embodiments, a method of preparing an organoid immune cell co-culture comprises one or more of the steps of preparing at least one organoid by culturing epithelial cells in an organoid culture medium and culturing immune cells in an immune cell growth medium. In some embodiments, the method further comprises the step of harvesting immune cells from an impure immune sample. Methods of isolating immune cells from an impure immune sample are known in the art.

[0112] The present invention also provides an organoid immune cell co-culture obtained by the above method. The present invention also provides the use of the above organoid immune cell co-culture in drug screening, toxicology screening, research and drug development.

[0113] Culture medium Culture media suitable for epithelial stem cells are well known in the art and are described, for example, in WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, WO2016 / 083612 and WO2017 / 149025. The culture media described in these documents are hereby incorporated by reference and any of them may be used in the context of the present invention.

[0114] In some embodiments, a culture medium suitable for culturing epithelial stem cells may contain one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor. For example, a culture medium suitable for epithelial stem cells contains a Wnt agonist. A culture medium suitable for epithelial stem cells may further contain a mitogenic growth factor and / or a BMP inhibitor.

[0115] In a preferred embodiment, the culture medium suitable for epithelial stem cells contains a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor. The culture medium suitable for epithelial stem cells may further contain one or more of a p38 inhibitor, a cAMP agonist, a prostaglandin pathway activator, nicotinamide, gastrin, B27, and N-acetylcysteine.

[0116] In some embodiments, the culture medium contains a basal medium for human or animal cells (e.g., DMEM / F12 optionally containing B27), an R-spondin family protein, a mitogenic growth factor (e.g., EGF), a BMP inhibitor (e.g., noggin), a TGFβ inhibitor (e.g., A83-01), a p38 inhibitor (e.g., SB202190), and optionally nicotinamide and N-acetylcysteine.

[0117] In some embodiments, the culture medium contains an advanced DMEM / F12 medium containing B27, nicotinamide, N-acetylcysteine, noggin, R-spondin 1, EGF, WNT-conditioned medium (50%, generated using stably transfected L cells), the TGF-β type I receptor inhibitor A83-01, and the P38 inhibitor SB202190.

[0118] In some embodiments, the culture medium suitable for epithelial stem cells is a culture medium suitable for growing epithelial stem cells. Those skilled in the art will understand that the above-described culture medium is particularly useful for growing epithelial stem cells.

[0119] In other embodiments, the culture medium suitable for epithelial stem cells is a culture medium suitable for differentiating epithelial stem cells.

[0120] The present invention provides a culture medium suitable for culturing epithelial stem cells as defined herein, where the culture medium further contains an integrin agonist as defined herein.

[0121] WNT agonist A culture medium suitable for culturing epithelial stem cells may contain one or more Wnt agonists. A Wnt agonist is defined herein as an agent that activates or enhances TCF / LEF-mediated transcription in cells.

[0122] The canonical Wnt signaling pathway is defined by a series of events that occur when a cell surface Wnt receptor complex, including Frizzled (FZD) receptors and LRP, is normally activated by an extracellular signaling molecule, such as a member of the Wnt family of secreted glycoproteins. This results in the activation of Dishevelled family proteins that inhibit a protein complex containing axin, GSK-3, and the protein APC, which degrades intracellular β-catenin. The resulting concentrated nuclear β-catenin promotes transcription by TCF / LEF family transcription factors (Driehuis & Clevers, British Journal of Pharmacology (2017) 174 4547-4563).

[0123] Furthermore, the R-spondin / Rnf43 / Lgr module controls canonical Wnt signaling. In the absence of R-spondin, the E3 ligase RNF43 / ZNRF3 adds ubiquitin to FZD, marks it for proteasomal degradation, and inhibits Wnt signaling. When extracellular R-spondin is present, it may interact via the Lgr receptor with the transmembrane E3 ligase RNF43 / ZNRF3 and interfere with the action of the E3 ligase. Lgr receptors, including Lgr4, Lgr5, and Lgr6, and especially Lgr5, are expressed in epithelial stem cells. R-spondin is mobilized by these stem cell markers, promoting Wnt signaling, and thus R-spondin and Lgr interact to promote proliferation and maintain stem cell pluripotency. For these reasons, R-spondin family proteins have been shown to be particularly useful in epithelial stem cell culture for obtaining long-lived organoid cultures.

[0124] Accordingly, one or more Wnt agonists in the culture medium can be selected from a Wnt ligand from the Wnt family of secreted glycoproteins, an inhibitor of intracellular β-catenin degradation, a GSK-3 inhibitor, a TCF / LEF activator, an RNF43 or ZNRF3 inhibitor, and an R-spondin family protein. In some embodiments, the Wnt agonist in the culture medium comprises an R-spondin family protein and a GSK-3 inhibitor, and further optionally comprises a Wnt ligand from the Wnt family of secreted glycoproteins.

[0125] The R-spondin family protein (also referred to herein as "R-spondin") can be selected from R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and their analogs, fragments, mutants and derivatives. In this regard, the above fragments, mutants or derivatives are capable of interfering with the action of the E3 ligases RNF43 / ZNRF3 on the Wnt receptor complex. All of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4 (also referred to herein as "R-spondin 1-4") are characterized by two amino-terminal furin-like repeat structures, which are necessary and sufficient for promoting Wnt signaling, and the thrombospondin domain is located towards the more carboxyl-terminal element (Lau et al. Genome Biol. 2012;13(3):242 (2012)). Examples of R-spondin fragments, mutants, and derivatives suitable for use in the present invention are known to those skilled in the art (see, for example, Example 2 of WO2012 / 140274, which is incorporated herein by reference and describes a furin domain fragment capable of promoting Wnt signaling). Examples of R-spondin family protein analogs include, for example, antibodies that interact with RNF43 / ZNRF3 / Lgr4. Agonist anti-Lgr5 antibodies capable of promoting Wnt signaling are known in the art (see, for example, antibody 1D9 described in Example 3 of WO2012 / 140274).

[0126] Many GSK-3 inhibitors are known in the art (see, for example, Greengard, P., and Meijer, L. (2004) Structural basis for the synthesis of indirubins as potent and selective inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinases. J Med Chem 47:935-946; and Thomas Kramer, Boris Schmidt, and Fabio Lo Monte, “Small-Molecule Inhibitors of GSK-3: Structural Insights and Their Application to Alzheimer’s Disease Models,” International Journal of Alzheimer’s Disease, vol. 2012, Article ID 381029, 32 pages, 2012. https: / / doi.org / 10.1155 / 2012 / 381029), and they are also commercially available (see, for example, the list available from Santa Cruz Biotechnology: https: / / www.scbt.com / scbt / browse / GSK-3-beta-Inhibitors / _ / N-x6oud). None of these GSK-3 inhibitors are suitable for use in the context of the present invention, and one of ordinary skill in the art would be able to determine a suitable concentration using the IC50 value.

[0127] It should be noted that there seems to be an error in the original text where it says "None of these GSK-3 inhibitors are suitable for use in the context of the present invention" which might be a misstatement as the previous part implies they could be relevant. The translation is done based on the provided text.CHIR-99021 (CAS: 252917-06-9; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CT99021) is a potent and selective inhibitor of GSK-3. Other aminopyrimidine inhibitors with IC50 values of 0.6 nM to 7 nM include CHIR98014 (Axon, Cat 1126), CHIR98023, CHIR99021 (see above), and TWS119 (Tocris, Cat 3835). Thus, in some embodiments, the GSK-3 inhibitor is an aminopyrimidine inhibitor and is optionally selected from CHIR98014, CHIR98023, CHIR99021, or TWS119. In some embodiments, the GSK-3 inhibitor is CHIR-99021.

[0128] The GSK-3 inhibitor (e.g., CHIR-99021) may be used at any suitable concentration, for example, from 10 nM to 500 μM, 10 nM to 400 μM, 10 nM to 300 μM, 10 nM to 200 μM, 10 nM to 100 μM, or 20 nM to 50 μM, or the final concentration is about 3 μM.

[0129] Wnt ligands from the Wnt family of secreted glycoproteins may be selected from Wnt-l / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM l and Wnt-16. An overview of human Wnt proteins is described in “THE WNT FAMILY OF SECRETED PROTEINS”, R&D Systems Catalog, 2004. In some embodiments, the Wnt ligand is Wnt-3a, Wnt-5 or Wnt-6a, or optionally Wnt-3a. The addition of soluble Wnt ligand has been shown to be particularly advantageous for the proliferation of human epithelial stem cells (as described, for example, in WO2012 / 168930).

[0130] A Wnt agonist is preferably added to the medium in an amount effective to stimulate intracellular Wnt activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably at least 100%, when compared to the level of Wnt activity in the absence of the above molecules when evaluated in the same cell type. As is known to those skilled in the art, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al., 1997. Science 275:1784-1787).

[0131] Soluble Wnt agonists such as Wnt-3a may be provided in the form of Wnt-conditioned medium. For example, Wnt-conditioned medium of about 10% to about 30%, such as about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml, may be used. In other embodiments, surrogate Wnt agonists (e.g., as described in

[19] and the Examples) may be used.

[0132] R-spondin may be provided in the form of R-spondin-conditioned medium or in the form of a recombinant protein. For example, R-spondin of about 10 ng / ml to about 500 ng / ml, about 10 ng / ml to about 400 ng / ml, about 10 ng / ml to about 300 ng / ml, about 10 ng / ml to about 250 ng / ml, about 50 ng / ml to about 250 ng / ml, about 100 ng / ml to about 250 ng / ml or about 150 ng / ml to about 250 ng / ml, preferably about 150 ng / ml to about 250 ng / ml, may be used. For example, the final concentration of R-spondin in the culture medium may be about 10 ng / ml, about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, about 150 ng / ml, about 175 ng / ml, about 200 ng / ml, about 225 ng / ml, about 250 ng / ml, about 275 ng / ml, about 300 ng / ml, about 325 ng / ml, about 350 ng / ml, about 375 ng / ml, about 400 ng / ml, about 450 ng / ml, or about 500 ng / ml.

[0133] One or more, e.g., 2, 3, 4 or more, Wnt agonists may be used in the culture medium.

[0134] Mitogenic growth factor A culture medium suitable for culturing epithelial stem cells may contain a mitogenic growth factor. The mitogenic growth factor typically induces cell division via the mitogen-activated protein kinase signaling pathway. Many receptor tyrosine kinase ligands are mitogenic growth factors. In some embodiments, the mitogenic growth factor may bind to a receptor tyrosine kinase. In some embodiments, the mitogenic growth factor may bind to two or more receptor tyrosine kinases. In some embodiments, one or more mitogenic growth factors bind to a receptor tyrosine kinase such as EGFR, FGFR, or HGFR, and optionally, one or more mitogenic growth factors are selected from EGF, FGF, and HGF.

[0135] The epidermal growth factor receptor (EGFR), also known as ErbB1 or HER1, is a cell surface receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. EGFR is a receptor tyrosine kinase and belongs to the HER family of receptors, which includes four related proteins (EGFR (HER1 / ErbB1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4)). The HER receptors are known to be activated by binding to different ligands, including EGF, TGFα, heparin-binding EGF-like growth factor, neuregulin, amphiregulin, betacellulin, and epiregulin. After the ligand binds to the extracellular domain of the receptor, the receptor functionally forms active dimers (EGFR-EGFR (homo-dimers) or EGFR-HER2, EGFR-HER3, EGFR-HER4 (hetero-dimers)). Dimerization involves activation of the tyrosine kinase domain, which results in autophosphorylation of the receptor on multiple tyrosine residues. This leads to the recruitment of classes of adapter proteins (e.g., SHC, GRB2) and activation of a series of intracellular signaling cascades that affect gene transcription. Thus, in some embodiments, the mitogenic growth factor binds to EGFR, HER1, HER2, HER3, or HER4. In some embodiments, the mitogenic growth factor binds to EGFR. In some embodiments, HER2-4 ligands are included in the culture medium in addition to EGFR ligands. For example, in some embodiments, neuregulin is included in the culture medium in addition to EGF. Neuregulin has been shown to be advantageous for culturing lung and breast tissues (see, e.g., WO2016 / 083613 and WO2016 / 083612). In some embodiments, one or more mitogenic growth factors in the culture medium is EGF. Any suitable EGF may be used, such as, for example, EGF obtained from Peprotech.EGF is preferably added to the basal culture medium at a final concentration of 0.1 ng / ml to 500 ng / ml, 0.1 ng / ml to 400 ng / ml, 0.1 ng / ml to 300 ng / ml, 0.1 ng / ml to 200 ng / ml, 0.1 ng / ml to 100 ng / ml, 1 ng / ml to 100 ng / ml, or the final concentration of the mitogenic growth factor is approximately 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 250 ng / ml or 500 ng / ml and below 500 ng / ml. A more preferred concentration is at least 50 ng / ml and 100 ng / ml or below. An even more preferred concentration is about 50 ng / ml. FGF stimulates cells by interacting with cell surface tyrosine kinase receptors (FGFRs). Four closely related receptors (FGFR1 - FGFR4) have been identified. Thus, in some embodiments, the mitogenic growth factor binds to an FGFR family member. FGFR family members include, but are not limited to, FGFR1, FGFR2, FGFR3 or FGFR4. The FGFR1 - FGFR3 genes have been shown to encode multiple isoforms, and these isoforms can be important in determining ligand specificity. There are several FGFs that bind to FGFR family members, including, but not limited to, FGF2, FGF4, FGF7 and FGF10. These are commercially available. Thus, in some embodiments, the mitogenic growth factor is FGF. In some embodiments, FGF is selected from FGF2, FGF4, FGF7 and FGF10. In a preferred embodiment, FGF is FGF2 and / or FGF10. In the most preferred embodiment, FGF is FGF2 and FGF10.

[0136] Most FGFs bind to two or more receptors (Ornitz J Biol Chem. 1998 Feb 27;273(9):5349-57). However, FGF10 and FGF7 are unique among FDFs in that they interact only with a specific isoform of FGFR2 called FGFR2b, which is expressed only in epithelial cells (Igarashi, J Biol Chem. 1998 273(21):13230-5). Thus, in some embodiments, the mitogenic growth factor binds to FGFR2b. FGF10 has been shown to be particularly useful in culture media. FGF10 can bind to FGFR2 or FGFR4. Thus, in some embodiments, the mitogenic growth factor binds to FGFR2 or FGFR4. FGF2 binds to all of FGFR1, FGFR2, FGFR3, and FGFR4. Thus, in some embodiments, the mitogenic growth factor binds to all of FGFR1, FGFR2, FGFR3, and FGFR4.

[0137] In some embodiments, the final concentration of FGF is 0.1 ng / ml to 500 ng / ml, 0.1 ng / ml to 400 ng / ml, 0.1 ng / ml to 300 ng / ml, 0.1 ng / ml to 200 ng / ml, 0.1 ng / ml to 100 ng / ml, 1 ng / ml to 100 ng / ml, or the final concentration of the mitogenic growth factor is about 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 250 ng / ml, or 500 ng / ml.

[0138] In some embodiments, one or more mitogenic growth factors in the culture medium is FGF10. The preferred concentration of FGF10 is approximately 0.1 ng / ml to 500 ng / ml, 0.1 ng / ml to 400 ng / ml, 0.1 ng / ml to 300 ng / ml, 0.1 ng / ml to 200 ng / ml, 0.1 ng / ml to 100 ng / ml, 1 ng / ml to 100 ng / ml, or the final concentration of the mitogenic growth factor is approximately 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 250 ng / ml or 500 ng / ml and 500 ng / ml or less. A more preferred concentration of FGF10 is about 10 ng / ml.

[0139] In some embodiments, one or more mitogenic growth factors in the culture medium is FGF2. The preferred concentration of FGF2 is approximately 0.1 ng / ml to 500 ng / ml, 0.1 ng / ml to 400 ng / ml, 0.1 ng / ml to 300 ng / ml, 0.1 ng / ml to 200 ng / ml, 0.1 ng / ml to 100 ng / ml, 1 ng / ml to 100 ng / ml, or the final concentration of the mitogenic growth factor is approximately 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 250 ng / ml or 500 ng / ml and 500 ng / ml or less. A more preferred concentration of FGF2 is about 5 ng / ml.

[0140] Hepatocyte growth factor / scatter factor (HGF / SF) is a morphogenetic factor that controls cell proliferation, cell motility, and morphogenesis by activating the tyrosine kinase signaling cascade after binding to the primary cancerous HGFR. HGFR is also known as the c-Met receptor. HGF has been shown to be useful in epithelial stem cell culture. Thus, in some embodiments, the mitogenic growth factor binds to HGFR. In some embodiments, the mitogenic growth factor is HGF. Any suitable HGF may be used, such as, for example, HGF obtained from Peprotech. Preferred concentrations of HGF are about 1, 10, 20, 25, 50 ng / ml, 50 ng / ml or less.

[0141] In some embodiments, two or more mitogenic growth factors, e.g., two or three mitogenic growth factors, are included in the culture medium. For example, in some embodiments, one or more mitogenic growth factors in the culture medium are EGF and FGF. In some embodiments, one or more mitogenic growth factors in the culture medium are EGF, FGF2, and FGF10. In some embodiments, one or more mitogenic growth factors in the culture medium are optionally EGF at a final concentration of about 50 ng / ml, optionally FGF2 at a final concentration of about 5 ng / ml, and optionally FGF10 at a final concentration of about 10 ng / ml.

[0142] In some embodiments, hepatocyte growth factor (HGF) is also present in the presence or absence of EGF and / or FGF.

[0143] In some embodiments, the respective final concentration of the mitogenic growth factor is 0.1 ng / ml to 500 ng / ml, 0.1 ng / ml to 400 ng / ml, 0.1 ng / ml to 300 ng / ml, 0.1 ng / ml to 200 ng / ml, 0.1 ng / ml to 100 ng / ml, 1 ng / ml to 100 ng / ml, or the final concentration of the mitogenic growth factor is about 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 250 ng / ml or 500 ng / ml.

[0144] BMP inhibitor The culture medium suitable for culturing epithelial stem cells may contain a BMP inhibitor. BMP, as a dimer ligand, binds to a receptor complex composed of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, and as a result, this receptor kinase is activated. The type I receptor then phosphorylates specific receptor substrates (SMAD), resulting in a signaling pathway leading to transcriptional activity.

[0145] A BMP inhibitor is defined as an agent that binds to a BMP molecule to form a complex, where BMP activity is neutralized, for example, by preventing or inhibiting the binding of the BMP molecule to the BMP receptor. Alternatively, the inhibitor is an agent that acts as an antagonist or reverse agonist. This type of inhibitor binds to the BMP receptor and prevents the binding of BMP to the receptor. An example of the latter agent is an antibody that binds to the BMP receptor and prevents the binding of the antibody-bound BMP to the receptor.

[0146] The BMP inhibitor may be added to the medium in an amount effective to inhibit BMP-dependent activity in the cells by up to 90%, more preferably up to 80%, more preferably up to 70%, more preferably up to 50%, more preferably up to 30%, more preferably up to 10%, more preferably 0%, when evaluated in the same cell type, compared to the level of BMP activity in the absence of the inhibitor. As is known to those skilled in the art, BMP activity can be determined, for example, by measuring the transcriptional activity of BMP as exemplified in Zilberberg et al., 2007. BMC Cell Biol. 8:41.

[0147] Several classes of natural BMP-binding proteins are known, for example, noggin (Peprotech), chordin and chordin-like proteins containing a cordon domain (R&D systems), follistatin and follistatin-related proteins containing a follistatin domain (R&D systems), DAN and DAN-like proteins containing a DAN cysteine knot domain (R&D systems), sclerostin / SOST (R&D systems), decorin (R&D systems), and α-2 macroglobulin (R&D systems).

[0148] Thus, in some embodiments, the BMP inhibitor is selected from noggin, DAN, and DAN-like proteins (R&D systems) including cerberus and gremlin. These diffusible proteins can bind to the BMP ligand with varying degrees of affinity and inhibit their access to the signaling receptor. Addition of any of these BMP inhibitors to the basal culture medium prevents the loss of stem cells. A preferred BMP inhibitor is noggin.

[0149] In some embodiments, the final concentration of the BMP inhibitor (e.g., noggin) is from about 10 ng / ml to about 500 ng / ml, from about 10 ng / ml to about 400 ng / ml, from about 10 ng / ml to about 300 ng / ml, from about 10 ng / ml to about 250 ng / ml, from about 50 ng / ml to about 250 ng / ml, from about 50 ng / ml to about 150 ng / ml, or the final concentration is about 100 ng / ml.

[0150] TGFβ inhibitor The culture medium suitable for culturing epithelial stem cells may contain a TGFβ inhibitor. The presence of the TGFβ inhibitor in the growth medium is particularly advantageous for enhancing the efficiency of human organoid formation. TGFβ signaling typically begins with the binding of a TGFβ superfamily ligand to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates SMAD, which functions as a transcription factor in the nucleus and controls target gene expression.

[0151] TGFβ superfamily ligands include bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Müllerian hormone (AMH), activin, nodal, and TGFβ. Generally, Smad2 and Smad3 are phosphorylated by the ALK4, 5, and 7 receptors of the TGFβ / activin pathway. In contrast, Smad1, Smad5, and Smad8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. However, in the context of the present invention, one of ordinary skill in the art will understand that a "TGFβ inhibitor" or "inhibitor of TGFβ signaling" is an inhibitor of the TGFβ pathway that includes ALK4, 5, and 7 and includes Smad2 and Smad3. It will be understood by one of ordinary skill in the art that a TGFβ inhibitor is not a BMP inhibitor, i.e., noggin is not a TGFβ inhibitor in the context of the present disclosure. In some embodiments, a BMP inhibitor is added to the culture medium in addition to the TGFβ inhibitor (see below).

[0152] Accordingly, a TGFβ inhibitor is any agent that reduces the activity of the TGFβ signaling pathway, also referred to herein as the ALK4, ALK5, or ALK7 signaling pathway. There are many methods of inhibiting the TGFβ signaling pathway that are known in the art and can be used in conjunction with the present invention. For example, TGFβ signaling can be inhibited by inhibition of TGFβ expression by small interfering RNA strategies; inhibition of furin (a TGFβ activating protease); inhibition of the pathway by physiological inhibitors; neutralization of TGFβ by monoclonal antibodies; inhibition by small molecule inhibitors of TGFβ receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK7; inhibition of Smad2 and Smad3 signaling, for example, by overexpression of their physiological inhibitor, Smad7, or by using thioredoxin as a Smad anchor that suppresses Smad activation (Fuchs, O. Inhibition of TGF-Signaling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy, Volume 6, Number 1, January 2011, pp. 29-43(15)).

[0153] Various methods are known for determining whether a substance is a TGFβ inhibitor and may be used in conjunction with the present invention. For example, a cell assay may be used in which cells are stably transfected with a reporter construct containing a human PAI-1 promoter or a Smad binding site and a luciferase reporter gene is driven. Inhibition of luciferase activity compared to a control group may be used as a measure of the degree of compound activity (De Gouville et al., Br J Pharmacol. 2005 May;145(2):166-177).

[0154] The TGFβ inhibitor according to the present invention may be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer or antibody. The inhibitor may be a naturally occurring one or an artificial one.

[0155] In some embodiments, the TGFβ inhibitor is a small molecule inhibitor such as A83-01. A83-01 is a commercially available selective inhibitor of ALK4, ALK5 and ALK7 (Tocris catalog number 2939). The inhibitor is a potent inhibitor of the TGFβ type I receptor ALK5 kinase, the type I activin / nodal receptor ALK4 and the type I nodal receptor ALK7 (IC 50 values are 12, 45 and 7.5 nM respectively), and is cataloged as blocking the phosphorylation of Smad2 and only slightly inhibiting ALK1, 2, 3, 6 and MAPK activities. Other commercially available inhibitors with similar properties include, but are not limited to, A77-01, LY2157299, LY2109761, LY3200882, GW788388, pirfenidone, RepSox, SB431542, SB505124, SB525334, LY364947, SD-208 and bactosertib. The IC50 values of these inhibitors are known in the art, and those skilled in the art will be able to select a suitable inhibitor at a suitable concentration based on the teachings provided in the examples of this application.

[0156] Thus, in some embodiments, the TGFβ inhibitor is an inhibitor of ALK4, ALK5, and ALK7, and optionally a selective inhibitor of ALK4, ALK5, or ALK7. For example, the TGFβ inhibitor can bind to ALK4, ALK5, and / or ALK7 and directly inhibit them. In some embodiments, the TGFβ inhibitor is an inhibitor that blocks the phosphorylation of Smad2. In some embodiments, the TGFβ inhibitor is selected from A83-01, A77-01 (Tocris catalog number 6712), LY2157299 (Selleckchem catalog number S2230), LY2109761 (Selleckchem catalog number S2704), LY3200882 (Selleckchem catalog number S8772), GW788388 (Tocris catalog number 3264), pirfenidone, RepSox (Tocris catalog number 3742), SB431542 (Tocris catalog number 1614), SB505124 (Tocris catalog number 3263), SB525334 (Tocris catalog number 3211), LY364947 (Tocris catalog number 2718), SD-208 (Tocris catalog number 3269), and bactosertib (Selleckchem catalog number S7530). In some embodiments, the TGFβ inhibitor is A83-01.

[0157] In some embodiments, only one TGFβ inhibitor is present in the culture medium. In other embodiments, two or more, such as two, three, four, or more TGFβ inhibitors are present in the culture medium.

[0158] In some embodiments, the final concentration of the TGFβ inhibitor is from 1 nM to 100 μM, from 10 nM to 100 μM, from 100 nM to 10 μM, or about 1 μM. For example, the final concentration of one or more inhibitors is from 10 nM to 100 μM, from 100 nM to 10 μM, or about 500 nM. In some embodiments, the final concentration of the TGFβ inhibitor is at least 5 nM, for example, at least 50 nM, at least 100 nM, at least 300 nM, at least 450 nM, at least 475 nM. For example, it is from 5 nM to 500 mM, from 10 nM to 100 mM, from 50 nM to 700 μM, from 50 nM to 10 μM, from 100 nM to 1000 nM, 350 to 650 nM or more, more preferably about 500 nM. In some embodiments, the TGFβ inhibitor is A83-01 with a final concentration of about 500 nM.

[0159] One of ordinary skill in the art will understand that the appropriate final concentration of the TGFβ inhibitor depends on the TGFβ inhibitor in question. Also, one of ordinary skill in the art will know methods for determining the concentration of other TGFβ inhibitors for use in the present invention.

[0160] Nicotinamide In some embodiments, the culture medium suitable for epithelial stem cells further contains nicotinamide. Nicotinamide is an amide derivative of vitamin B3, a poly(ADP-ribose) polymerase (PARP) inhibitor, and a major precursor of NAD+. It is commercially available (e.g., Stemcell Technologies catalog 07154). In some embodiments, nicotinamide is present at 7 to 15 mM, for example, about 10 mM.

[0161] Prostaglandin pathway activator An activator of the prostaglandin signaling pathway (also referred to as a prostaglandin pathway activator) may be any one or more of the compounds selected from the list including phospholipids, arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), and prostaglandin D2 (PGD2). In some embodiments, the activator of the prostaglandin signaling pathway is PGE2 and / or AA. In some embodiments, the activator of the prostaglandin signaling pathway is PGE2.

[0162] In some embodiments, the final concentration of the activator of the prostaglandin signaling pathway (e.g., PGE2) is 10 nM to 500 μM, 10 nM to 400 μM, 10 nM to 300 μM, 10 nM to 200 μM, 10 nM to 100 μM, or 20 nM to 50 μM, or the final concentration is about 1 μM.

[0163] One of ordinary skill in the art will understand that the appropriate final concentration of the activator of the prostaglandin signaling pathway depends on the activator of the prostaglandin signaling pathway in question. One of ordinary skill in the art will also know how to determine the concentration of other activators of the prostaglandin signaling pathway for use in the present invention.

[0164] CAMP activator The cAMP pathway activator may be any suitable activator that increases the intracellular cAMP level. The cAMP pathway is associated with the activation of many types of hormone and neurotransmitter G protein-coupled receptors. Binding of a hormone or neurotransmitter to its membrane-bound receptor induces a conformational change in the receptor, thereby activating the α subunit of the G protein. The activated G subunit stimulates adenylate cyclase, while the non-activated G subunit inhibits adenylate cyclase. Stimulation of adenylate cyclase catalyzes the conversion of cytoplasmic ATP to cAMP, thereby increasing the intracellular cAMP level.

[0165] Accordingly, in some embodiments, the cAMP pathway activator is an adenylate cyclase activator or a cAMP analog. Examples of suitable adenylate cyclase activators include forskolin, forskolin analogs, and cholera toxin. Examples of forskolin analogs are known in the art and include NKH477 (e.g., catalog number Tocris 1603). Examples of cAMP analogs are known in the art and include, for example, 8-bromo-cAMP. 8-Bromo-cAMP is a cell-permeable cAMP analog that is more resistant to hydrolysis by phosphodiesterase than cAMP. Accordingly, in some embodiments, the cAMP pathway activator is selected from forskolin, cholera toxin, NKH477, and 8-bromo-cAMP. In some embodiments, the cAMP pathway activator is forskolin. In some embodiments, the cAMP pathway activator is not cholera toxin.

[0166] cAMP pathway activators can be identified using methods known in the art, for example, using competitive immunoassays that measure cAMP levels. The CatchPoint® Cyclic AMP Fluorescent Assay Kit (Molecular Devices LLC) is an example of a commercially available kit for performing such immunoassays. Sample or standard cAMP competes with a horseradish peroxidase (HRP)-labeled cAMP complex for the binding site of the anti-cAMP antibody. In the absence of cAMP, most of the HRP-cAMP complex binds to the antibody. As the concentration of cAMP increases, the amount of bound complex decreases competitively, thereby reducing the measured HRP activity. A cAMP pathway activator will increase the level of cAMP and decrease the measured HRP activity compared to a control.

[0167] In some embodiments, the final concentration of the cAMP pathway activator (e.g., forskolin) is from 10 nM to 500 μM, 10 nM to 400 μM, 10 nM to 300 μM, 10 nM to 200 μM, 10 nM to 100 μM, or 20 nM to 50 μM, or the final concentration is about 1 μM. In some embodiments, the cAMP pathway activator is forskolin. In some embodiments, the final concentration of forskolin is about 1 μM.

[0168] The concentration selected can depend on the cAMP pathway activator used and can be determined by one of ordinary skill in the art depending on the potency of the cAMP pathway activator. For example, NKH477 is generally more potent than 8-bromo-cAMP and forskolin. To obtain the same effect, a more potent cAMP pathway activator can also be used at a lower concentration.

[0169] Additional components Basal media for cell culture typically contain a number of components necessary to support the maintenance of cultured cells. A suitable combination of components can be readily prepared by those skilled in the art taking into account the following disclosure. Basal media for use in the present invention generally contain a nutrient solution comprising standard cell culture components such as amino acids, vitamins, lipid supplements, inorganic salts, a carbon energy source, and buffers, as detailed in the literature and below. In some embodiments, for example, one or more standard cell culture components selected from amino acids, vitamins, lipid supplements, inorganic salts, a carbon energy source, and buffers are further supplemented to the culture medium. Suitable basal media are well-known to those skilled in the art and are also commercially available. Examples include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), high-glucose DMEM, Minimum Essential Medium (MEM), Knockout DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM / Ham's F12, high-glucose DMEM / Ham's F12, Iscove's Modified Dulbecco Medium and Minimum Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, and RPMI1640 Medium. For example, the basal medium may preferably be high-glucose DMEM supplemented with Glutamax, penicillin / streptomycin, and HEPES.

[0170] The culture medium suitable for epithelial stem cells may be supplemented with one or more of the compounds selected from the group consisting of gastrin, B27, N-acetylcysteine, and N2. Thus, in some embodiments, the aforementioned culture medium further contains one or more components selected from the group consisting of gastrin, B27, N2, and N-acetylcysteine. B27 (Invitrogen), N-acetylcysteine (Sigma), and N2 (Invitrogen), gastrin (Sigma) are thought to control cell growth and assist in DNA stability. In some embodiments, the culture medium further contains B27 and N-acetylcysteine.

[0171] In some embodiments, the B27 supplement is “B27 Supplement minus Vitamin A” (Invitrogen, Carlsbad, CA; www.invitrogen.com; currently catalog number 12587010, and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F01-002; available from Brewer et al., J Neurosci Res., 35(5):567-76, 1993), and using this, a culture medium containing biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin and transferrin can be prepared. The B27 supplement provided by PAA Laboratories GmbH is a 50-fold concentrated liquid and contains, among other things, the components of biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin and transferrin. Among these components, at least linolenic acid, retinol, retinyl acetate and triiodothyronine (T3) are nuclear hormone receptor agonists. The B27 supplement may be added to the culture medium as a concentrate or may be diluted before being added to the culture medium. It can be used at a final concentration of 1-fold or other final concentrations. The use of the B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin and transferrin into the culture medium of the present invention. It is also contemplated that some or all of these components may be added separately to the culture medium instead of using the B27 supplement. Thus, the culture medium may contain some or all of these components.In some embodiments, retinoic acid is not present in the B27 supplement used in the culture medium and / or is not present in the culture medium.

[0172] The "N2 supplement" is available from Invitrogen, Carlsbad, CA; www.invitrogen.com; catalog number 17502-048; and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. The N2 supplement provided by PAA Laboratories GmbH is a 100-fold concentrated liquid and contains 500 μg / ml human transferrin, 500 μg / ml bovine insulin, 0.63 μg / ml progesterone, 1611 μg / ml putrescine, and 0.52 μg / ml sodium selenite. The N2 supplement may be added to the culture medium as a concentrate or may be diluted before being added to the culture medium. It may be used at a final concentration of 1-fold or other final concentrations. The use of the N2 supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine, and sodium selenite into the culture medium of the present invention. It is of course contemplated that some or all of these components may be added separately to the culture medium instead of using the N2 supplement. Thus, the culture medium may contain some or all of these components. In some embodiments where the medium contains B27, the medium does not contain N2. In some embodiments, the final concentration of N-acetylcysteine is about 1 nM to about 100 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 30 nM, or about 25 nM.

[0173] In some embodiments, the culture medium further comprises a ROCK inhibitor (Rho kinase inhibitor). The ROCK inhibitor is particularly useful for cell adhesion when constructing a new culture and / or when dividing cells ( "subculturing"). Suitable ROCK inhibitors are known in the art and are also commercially available (including, but not limited to, GSK 269962, GSK 429286, H 1152 dihydrochloride, glycyl-H 1152 dihydrochloride, SR 3677 dihydrochloride, SB 772077B dihydrochloride and Y-27632 dihydrochloride, all of which are available from Tocris). In some embodiments, the final concentration of the ROCK inhibitor is 1 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 20 μM. In some embodiments, the ROCK inhibitor is Y-27632, and optionally its final concentration is about 10 μM.

[0174] In a preferred embodiment, the culture medium does not contain thrombopoietin.

[0175] The culture medium preferably does not contain undefined components such as fetal bovine serum or fetal bovine serum. Various different serum replacement formulations are commercially available and known to those skilled in the art. When a serum replacement is used, the replacement may be used at about 1% to about 30% by volume of the medium, according to the prior art. In some embodiments, the culture medium is serum-free.

[0176] A preferred culture method of the present invention is also advantageous in that feeder cells are not required. Feeder cell layers are often used to support the culture of stem cells and to inhibit their differentiation. The use of feeder cells is undesirable because it complicates cell subculturing (cells must be separated from the feeder cells with each subculture and new feeder cells are required with each subculture). The use of feeder cells can also contaminate the desired cells with feeder cells. This is clearly a problem for any medical use and also complicates the analysis of the results of any experiment performed on the cells, even at the research stage.

[0177] Thus, in some embodiments, the methods, media, and compositions of the invention are feeder cell-free. When the cells in the composition are cultured for at least one passage without the presence of a feeder cell layer, the composition is conventionally considered to be feeder cell-free. Feeder cell-free compositions of the invention typically contain less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% feeder cells (total number of cells in the composition is represented as ~%), or preferably contain no feeder cells at all.

[0178] The culture medium of the invention is typically prepared with deionized distilled water. The culture medium of the invention is typically sterilized prior to use, for example, by ultraviolet light, heating, radiation, or filtration, to prevent contamination. The culture medium may be frozen for storage or transportation (e.g., at -20°C or -80°C). The medium may contain one or more antibiotics to prevent contamination. The medium may have an endotoxin content of less than 0.1 endotoxin units per ml, or may have an endotoxin content of less than 0.05 endotoxin units per ml. Methods for determining the endotoxin content of the culture medium are known in the art.

[0179] A preferred cell culture medium is a defined artificial medium buffered with a carbonate-based buffer at pH 7.4 (preferably, pH 7.2 - 7.6, or at least pH 7.2 - maximum pH 7.6), and simultaneously, the cells are cultured in an atmosphere containing 5% - 10% CO2, or at least 5% - maximum 10% CO2, preferably 5% CO2.

[0180] The invention also provides a composition or cell culture vessel comprising cells and / or organoids according to any one of the aspects of the invention described above, and a culture medium according to any one of the aspects of the invention described above. For example, such a composition or cell culture vessel may contain any number of cells or organoids cultured according to the methods of the invention in a culture medium as described above.

[0181] According to yet another aspect of the present invention, there is provided a sealed container containing the culture solution of the present invention. In some embodiments, the culture solution is a growth medium. In some embodiments, the culture solution is a differentiation medium. A sealed container may be preferred for the transport or storage of the culture solution to prevent contamination. The container may be any suitable container such as a flask, plate, bottle, jar, vial, or bag.

[0182] Exemplary culture solutions for use in the present invention In some embodiments, the culture solution of the present invention comprises an integrin agonist, one or more receptor tyrosine kinase ligands (e.g., EGF and / or HGF), a BMP inhibitor (e.g., noggin), and a TGFβ inhibitor (e.g., A83-01). This culture solution optionally further comprises one or more Wnt agonists (e.g., Lgr5 agonist). These media are suitable for all tissues, such as the intestine, stomach, pancreas, liver, prostate, breast, and lung.

[0183] In some embodiments, the culture solution of the present invention comprises (i) FGF7 and / or FGF10, (ii) noggin, (iii) Lgr5 agonist, and (iv) integrin agonist. In some embodiments, the culture solution of the present invention comprises (i) FGF7 and / or FGF10, (ii) noggin, (iii) Lgr5 agonist, (iv) one or more additional receptor tyrosine kinase ligands (e.g., EGF), and (v) integrin agonist. In some embodiments, the culture solution further comprises an ErbB3 / 4 ligand (e.g., human neuregulin β-1). This is a culture solution particularly suitable for culturing breast stem cells, although not limited thereto.

[0184] In some embodiments, the culture medium of the present invention contains an integrin agonist, EGF, FGF (e.g., FGF10), HGF, a TGFβ inhibitor (e.g., A83-01), nicotinamide, one or more Wnt agonists (e.g., an Lgr5 agonist), a cAMP pathway activator (e.g., forskolin), and gastrin. This culture medium may optionally further contain (i) a BMP inhibitor (e.g., noggin), a Wnt agonist (e.g., Wnt-conditioned medium), and a ROCK inhibitor (e.g., Y27632), or (ii) a BMP activator (e.g., BMP7). These culture media are particularly suitable for culturing liver stem cells, although not limited thereto.

[0185] In some embodiments, the culture medium of the present invention contains an integrin agonist, one or more receptor tyrosine kinase ligands (e.g., EGF), a BMP inhibitor (e.g., noggin), and one or more Wnt agonists (e.g., an Lgr5 agonist). This culture medium may optionally further contain testosterone. These culture media are particularly suitable for culturing prostate stem cells, although not limited thereto.

[0186] In some embodiments, the culture medium of the present invention further contains one or more components selected from a p38 MAP kinase inhibitor (e.g., SB 202190), gastrin, and / or nicotinamide.

[0187] In some embodiments, the culture medium of the present invention further contains a ROCK inhibitor (e.g., Y27632). The addition of a ROCK inhibitor has been found to be useful for the start or splitting of culturing.

[0188] In some embodiments, the culture medium of the present invention further contains B27 and / or N-acetylcysteine. These additional components are often added to the culture medium as components of the basal medium.

[0189] In some embodiments, the culture medium of the present invention comprises an integrin agonist, an Lgr5 agonist, a BMP inhibitor (e.g., noggin), B27, N-acetylcysteine, nicotinamide, a ROCK inhibitor, a TGFβ inhibitor (e.g., A83-01), a p38 MAP kinase inhibitor (e.g., SB 202190), FGF7 and FGF10, and optionally one or more additional components selected from one or more receptor tyrosine kinase ligands (e.g., EGF, amphiregulin, TGFα, PDGF), a p53 stabilizer, and a Wnt agonist (e.g., Wnt3a).

[0190] In some embodiments, the culture medium of the present invention comprises (i) an integrin agonist, (ii) one or more receptor tyrosine kinase ligands (e.g., EGF and / or HGF), (iii) a BMP inhibitor (e.g., noggin) and (iv) a TGFβ inhibitor (e.g., A83-01), a p38 inhibitor (e.g., SB202190) and / or a ROCK inhibitor (e.g., Y-27632), and optionally further comprises one or more Wnt agonists (e.g., an Lgr5 agonist).

[0191] In some embodiments, the culture medium further comprises (i) gastrin and / or nicotinamide, (ii) a Notch inhibitor (e.g., DAPT and / or DBZ), and / or (iii) a prostaglandin pathway activator (e.g., PGE2 and / or AA). For example, in some embodiments, the culture medium of the present invention comprises (i) an integrin agonist, (ii) one or more receptor tyrosine kinase ligands (e.g., EGF and / or HGF), (iii) a BMP inhibitor (e.g., noggin) and (iv) gastrin, nicotinamide, a Notch inhibitor (e.g., DAPT and / or DBZ), and / or a prostaglandin pathway activator (e.g., PGE2 and / or AA).

[0192] In some embodiments, the culture medium further comprises a cAMP pathway activator (e.g., forskolin) and / or a BMP pathway activator (e.g., BMP7). In some embodiments, the culture medium comprises a BMP pathway activator (e.g., BMP7) but does not comprise a BMP pathway inhibitor (e.g., noggin). These culture media are suitable, without limitation, particularly for culturing liver stem cells or pancreatic stem cells.

[0193] In some embodiments, the one or more receptor tyrosine kinase ligands are EGF and / or one or more (e.g., 1, 2, 3, 4, or more than 4) ligands of FGFR2b, such as FGF7 and / or FGF10.

[0194] In some embodiments, the BMP inhibitor is noggin.

[0195] In some embodiments, the one or more Wnt agonists are Lgr5 agonist, Lgr4 agonist, Lgr6 agonist or Wnt3a. In some embodiments, the Lgr5 agonist is R-spondin, such as any one of R-spondin 1-4.

[0196] In some embodiments, the culture medium further comprises an ErbB3 / 4 ligand (e.g., human neuregulin β-1).

[0197] When culturing human epithelial stem cells, Wnt3a may be advantageously added to the culture medium.

[0198] In some embodiments, the culture medium comprises a basal medium and further comprises a differentiation medium containing an integrin agonist, one or more receptor tyrosine kinase ligands (e.g., selected from EGF, FGF, and HGF), a Notch inhibitor (e.g., DAPT), a glucocorticoid (e.g., dexamethasone), a TGFβ inhibitor (e.g., A83-01), and one or more Wnt inhibitors (e.g., (i) optionally a Porc inhibitor selected from IWP 2, LGK974, and IWP 1, and / or (ii) optionally an inhibitor of β-catenin target gene expression selected from iCRT3, CGP049090, PKF118310, PKF115 584, ZTM000990, PNU 74654, BC21, iCRT5, iCRT14, and FH535). In some embodiments, the differentiation medium further comprises a GSK-3 inhibitor (e.g., CHIR99201). In some embodiments, the differentiation medium further comprises an AP-1 stimulant (e.g., carbachol). Thus, in some embodiments, the culture medium contains EGF, FGF19, HGF, DAPT, IWP2, iCRT3, dexamethasone, CHIR99021, and carbachol.

[0199] In some embodiments, the culture medium comprises a basal medium and further comprises a differentiation medium containing an integrin agonist, one or more EGFR pathway inhibitors (e.g., gefitinib, afatinib, a MEK inhibitor (e.g., PD0325901) and / or an ERK inhibitor (e.g., SCH772984)), a Notch inhibitor (e.g., DAPT), and one or more Wnt inhibitors (e.g., (i) optionally a Porc inhibitor selected from IWP 2, LGK974, and IWP 1, and / or (ii) optionally an inhibitor of β-catenin target gene expression selected from iCRT3, CGP049090, PKF118310, PKF115 584, ZTM000990, PNU 74654, BC21, iCRT5, iCRT14, and FH535).

[0200] In any case of the culture medium of the present invention, specific components may be omitted for cancer cells.

[0201] Organoid The present invention provides organoids that can be harvested or have been harvested by the culturing method of the present invention. Thus, in some embodiments, the method of culturing epithelial stem cells further comprises harvesting and / or isolating the organoids. As described above, in some embodiments, the organoids are tumor organoids. Organoids containing epithelial stem cells are also referred to herein as "epithelial organoids." Epithelial organoids have been described in the prior art (see, e.g., 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, WO2016 / 083612, and WO2017 / 149025).

[0202] Epithelial organoids that can be harvested or have been harvested, or are cultured, using a culture medium suitable for growth contain at least one epithelial stem cell, which can divide to produce another epithelial stem cell or generate differentiated progeny. Epithelial organoids also contain some differentiated (or more mature) cell types. These differentiated cell types occur naturally during organoid formation and provide structural features unique to the organoid, as described below. Epithelial organoids are advantageous in that they maintain a growing population of epithelial stem cells within the organoid while simultaneously containing more differentiated cell types characteristic of the originating epithelial tissue. The length of time that epithelial organoids can continue to grow while maintaining the presence of a core of epithelial stem cells and while maintaining the genetic and phenotypic integrity of the cells is an important feature that distinguishes epithelial organoids from many organoids in the prior art. Epithelial organoids also have a unique structure that develops rapidly as cells grow, differentiate, and self-organize in vitro. These features are described in detail below. Subsequently, epithelial organoids can also be cultured in a culture medium suitable for differentiation as described herein, which increases the proportion of differentiating cells within the epithelial organoid.

[0203] Image analysis methods may be used to evaluate cell characteristics during culture, such as cell morphology, cell structure, evidence of apoptosis or cytolysis, and organoid composition and structure. Many types of image analysis methods are well known in the art, such as electron microscopy methods including scanning electron microscopy, transmission electron microscopy, confocal microscopy, stereomicroscopy, and fluorescence microscopy. Histological analysis can reveal the basic structure and cell types.

[0204] In some embodiments, the epithelial organoids have a three-dimensional structure, i.e., the organoids are three-dimensional organoids. In some embodiments, the organoids contain only epithelial cells, i.e., non-epithelial cells are not present in the organoids. This is because the culture medium suitable for growing epithelial stem cells is specially designed to grow epithelial stem cells. Thus, even if other cell types are temporarily present in the culture medium, e.g., in the tissue fragments which are the starting materials of the present invention, these cells are likely to have a low survival rate, and instead, the stem cells are grown for a longer period, thereby generating a pure population of epithelial cells.

[0205] In some embodiments, the epithelial cells of the organoids surround a lumen. In some embodiments, the organoids do not contain a lumen (in particular, tumor organoids generally do not have a lumen). In some embodiments, the epithelial cells are polarized (i.e., it means that proteins are differentially expressed on the apical side or the basolateral side of the epithelial cells). In some embodiments, the lumen is a sealed lumen (i.e., it means that a continuous cell barrier separates the contents of the lumen from the medium surrounding the organoid). In some embodiments, the organoids can divide actively, for example when the organoids or cells are transferred to a differentiation medium, and preferably contain stem cells that can differentiate into all the major differentiated cell lineages present in the corresponding in vivo tissue. In some embodiments, the organoids contain basal cells on the outside and more differentiated cells in the center.

[0206] In some embodiments, the organoid comprises a stratified epithelium. "Stratified" means that there are multiple (two or more) layers of cells. Such cells often tend to have their nuclei more centrally located, i.e., are not polarized. Cells within the multilayer section may organize themselves to include intercellular spaces or lumens.

[0207] In some embodiments, the organoid comprises a single monolayer that is folded (or invaginated) to form a stem cell compartment and a differentiated cell compartment. In this embodiment, there need not be a central closed lumen surrounded by an epithelial layer; instead, the epithelium surrounding the three-dimensional organoid is substantially not folded and presents a monolayer. It can sometimes be difficult to distinguish between a folded (or invaginated) monolayer and a region of stratified cells. In some embodiments, the organoid comprises both a region of stratified cells and a region of folded monolayer. In some embodiments, the organoids of the invention have sections formed from multiple membranes and sections comprising a single monolayer of cells. In some embodiments, the organoids of the invention comprise or consist of a single monolayer of cells. In some embodiments, the organoid does not comprise a monolayer.

[0208] The organoids according to the invention may have a layer of cells comprising at least one bud and a central lumen.

[0209] In some embodiments, the organoids of the invention comprise or consist of epithelial cells. In some embodiments, the organoid comprises or consists of a single monolayer of epithelial cells. In some embodiments, non-epithelial cells are not present in the organoid.

[0210] In some embodiments, the organoids have been cultured, or can be cultured, in the growth medium of the present invention for at least 2 months, such as at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year.

[0211] In some embodiments, the organoids have been cultured, or can be cultured, for at least 5 passages, at least 10 passages, at least 15 passages, or at least 20 passages. In some embodiments, a population of organoids or epithelial stem cells is cultured for at least 10 passages.

[0212] In some embodiments, the number of cells in the organoids increases exponentially over 5 passages, 10 passages, 15 passages, or 20 passages. In preferred embodiments, the number of cells in a population of organoids or epithelial stem cells increases exponentially over 5 passages.

[0213] In some embodiments, the organoids have a diameter of at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 250 μm or more at the widest point.

[0214] In the context of the present invention, a tissue fragment is part of an adult tissue, preferably a human adult tissue. In contrast, organoids are distinguished from tissue fragments because they develop structural features through in vitro growth.

[0215] In preferred embodiments, the organoids may be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or more. In some embodiments, the organoids are grown or maintained during culturing for at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months or at least 12 months or more. Advantageously, use of the culturing method provided by the present invention results in the formation of organoids and / or cell populations in which the chromosome number is stably maintained when the cells or organoids are cultured for a long period. Thus, in some embodiments, the population of organoids or epithelial stem cells of the present invention has a stable chromosome number even after culturing in the culture medium of the present invention for 2, 4, 6, 8, 10, 12 or 14 weeks, or 4, 5, 6 months or more. Preferably, at least 65%, at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% of the cells have an appropriate chromosome number after culturing in the culture medium of the present invention for 2, 4, 6, 8, 10, 12 or 14 weeks, or 4, 5, 6 months or more. In the case of human epithelial cells, the appropriate chromosome number is 46. In some embodiments, the organoids have a normal karyotype. One method for determining the karyotype is by metaphase spread analysis. Of course, it should be understood that, for example, tumor cells within tumor organoids derived from tumor stem cells do not necessarily have an appropriate chromosome number since genomic instability is a characteristic of certain cancers.

[0216] In some embodiments, the organoids of the present invention are tumor organoids. In some embodiments, the tumor organoids are high-density structures similar to organoids resulting from the culture of non-cancerous epithelial stem cells. In other embodiments, the tumor organoids are cystic structures. In some embodiments, tumor organoids derived from different patients exhibit different morphologies. The organoid morphology can be evaluated using techniques such as image analysis including brightfield microscopy.

[0217] In some embodiments, the tumor organoids of the present invention retain tumor-specific histopathological changes or characteristics. Such changes can be identified by comparing the organoids derived from tumor tissue with the adjacent epithelium of the same patient and / or by comparing the tumor organoids with primary tissue specimens.

[0218] In some embodiments, the tumor organoids are high-density structures or cystic structures. In some embodiments, the tumor organoids contain transformed epithelial tumor cells. In some embodiments, a plurality of tumor organoids mainly contain a plurality of transformed epithelial tumor cells. In some embodiments, the tumor organoids contain only transformed epithelial tumor cells (i.e., non-transformed epithelial cells are not present in the tumor organoids). In some embodiments, the tumor organoids do not contain immune elements, connective tissue elements, and / or vascular elements. Those skilled in the art will understand that the presence of transformed epithelial tumor cells can be evaluated by various methods including keratin (KRT5) immunostaining.

[0219] In some embodiments, the tumor organoids of the present invention are selected by culturing in the culture medium of the present invention further containing an Mdm2 agonist such as Nutlin-3. The Mdm2 agonist can be present throughout or part of the culture process. In some embodiments, Nutlin-3 is present at a concentration of about 10 μM. Nutlin-3 blocks the growth of p53 wild-type cells. P53 is a tumor suppressor gene. Thus, for example, a decrease in the function of p53 caused by a mutation in the p53 gene or other misregulation of p53 is a common cause of tumor formation. Therefore, if organoids are cultured in the presence of Nutlin-3, organoids with reduced levels or activity of p53 are selected, and this culture can be used as a method for selecting tumor organoids.

[0220] In some embodiments, the organoids and tumor organoids of the present invention have different transcriptome characteristics. In some embodiments, based on principal component analysis of transcriptome analysis, multiple organoids of the present invention form clusters together, and multiple tumor organoids of the present invention form clusters together. In some embodiments, the organoids and tumor organoids of the present invention have differential gene expression. For example, KLK6 and / or EHF are downregulated in HNSCC tumor organoids. In contrast, SLCOB1, HOXC13, CALB1, NTS, and / or BCHE are upregulated in HNSCC tumor organoids. Further genes that may show differential expression between non-tumor organoids and tumor organoids include SDC2, HOXA1, NXPE3, and / or HOXC10. In some embodiments, when corresponding non-tumor organoids and tumor organoids are compared, there are more than 50 differentially expressed genes, more than 100 differentially expressed genes, more than 200 differentially expressed genes, or more than 300 differentially expressed genes.

[0221] In some embodiments, the tumor organoids of the present invention have a high variable allele frequency of mutations detected in relation to the tumor. The mutant allele frequency means the frequency at which a mutation is detected within a sample and may be represented, for example, as the proportion in which a specific mutation is included in the interpretation by sequencing analysis.

[0222] In some embodiments, the genetic modification of the tumor organoids of the present invention recapitulates the genetic modification of the tumor from which the organoids are derived. Genetic modifications include single nucleotide mutations, as well as small insertions and deletions. In some embodiments, the organoids of the present invention that are not derived from cancer cells contain fewer genetic modifications than the tumor organoids of the present invention that are derived from cancer cells.

[0223] Chromosomal missegregation is the cause of aneuploidy frequently observed in human tumors. Chromosome segregation errors include anaphase bridges and binucleated cells undergoing multipolar division. The increased rate of missegregation results in a phenotype known as chromosomal instability commonly observed in cancer. In some embodiments, tumor organoids exhibit chromosomal instability. In some embodiments, tumor organoids have a higher rate of chromosome segregation errors compared to organoids derived from non-cancer cells. In some embodiments, tumor organoids have a high rate of chromosome segregation errors including anaphase bridges and / or binucleated cells undergoing multipolar division.

[0224] In some embodiments, the tumor organoids of the present invention do not have a normal karyotype. In some embodiments, tumor organoids exhibit aneuploidy. The karyotype of the organoids, i.e., the number of chromosomes, can be identified by metaphase spread analysis.

[0225] In some embodiments, the tumor organoids of the present invention are tumorigenic when subcutaneously transplanted. The tumorigenic ability of human organoids may be determined by subcutaneous transplantation of the organoids into mice. Typically, subcutaneous transplantation of non-tumor organoids does not form tumors, or at least the organoids do not proliferate. In some embodiments, the tumor organoids of the present invention retain their tumorigenic ability during culture and can form tumors with characteristics similar to the parental tumor after subcutaneous transplantation. Such characteristics may include levels of atypia related to malignancy, tripolar mitotic figures, nuclear polymorphism, and / or muscle invasion.

[0226] The proliferating organoids of the present invention preferably contain at least 50% viable cells, more preferably at least 60% viable cells, more preferably at least 70% viable cells, more preferably at least 80% viable cells, more preferably at least 90% viable cells. The viability of the cells can be evaluated using Hoechst staining or propidium iodide staining by FACS. In some embodiments, one or more cryopreserved organoids of the present invention are provided. A method for preparing organoids for cryopreservation is also provided, which includes separating the organoid cultures, mixing them with a cryopreservation medium such as a regenerating cell culture cryopreservation medium (Gibco), and freezing them according to standard procedures. A method for thawing cryopreserved organoids is also provided, which includes thawing the cryopreserved organoids, embedding the thawed organoids in an extracellular matrix (e.g., Matrigel), and culturing the organoids in the culture medium of the present invention. Advantageously, after the first thawing, the culture medium may be supplemented with Y-27632, for example, about 10 μM of Y-27632. In some embodiments, after thawing, the culture medium is supplemented with Y-27632 by the first 1, 2, 3, 4, 5 days, preferably within the first 3 or 4 days. In some embodiments, Y-27632 is not present in the culture medium after the first 3, 4, 5, 6 days or more, preferably after the first 3 or 4 days. This cryopreservation method can be used for the proliferation of the organoids of the present invention.

[0227] In some embodiments, the organoids or tumor organoids of the present invention are cryopreserved.

[0228] Differentiation The organoids or cells from the organoids are transferred to a differentiation medium, and differentiation into all major differentiated cell lineages may be enabled or induced.

[0229] In some embodiments, the method of the present invention includes a first step of culturing epithelial stem cells or a population of stem cells in a growth medium, and a second step of culturing the proliferated cells or proliferated organoids in a differentiation medium. In some embodiments, the integrin agonist is included in the culture medium only in one of these steps. In other embodiments, the integrin agonist is included in the culture medium in both of these steps.

[0230] In some embodiments, the differentiation medium comprises a culture solution as disclosed herein that does not contain one or more of a mitogenic growth factor, a TGFβ inhibitor, an activator of the prostaglandin signaling pathway, a Wnt agonist, a cAMP pathway activator, a BMP inhibitor, and nicotinamide (e.g., as described in WO2012 / 168930). In some embodiments, the differentiation medium comprises a basal medium for animal or human cells. In some embodiments, the differentiation medium further comprises one or more of an EGFR pathway inhibitor, a Notch inhibitor, a Wnt inhibitor, or a BMP pathway activator (e.g., as described in WO2017220586).

[0231] In some embodiments, culturing a population of epithelial stem cells in a differentiation medium increases the expression level of mature epithelial cell markers. Those skilled in the art will recognize different techniques for measuring gene or protein expression, including quantitative PCR.

[0232] Use of organoids The organoids of the present invention faithfully represent the in vivo situation. This applies to both organoids grown from normal tissue and organoids grown from diseased tissue. Therefore, the organoids of the present invention are useful in medicine and diagnosis, as well as in research and drug development. In addition to providing a normal ex vivo cell / organ model, the organoids of the present invention can be used as an ex vivo model of disease and / or infection. Therefore, the organoids of the present invention can be used in drug screening, including drug discovery and validation, target discovery and validation, toxicology, infection models, and other research purposes. Diseases that may be studied by the organoids of the present invention include, but are not limited to, genetic diseases, metabolic diseases, pathogenic diseases, and inflammatory diseases. Organoids are also suitable for transplantation and may therefore be suitable for regenerative medicine. In addition, since organoids can be rapidly grown from the cells of any individual, they can be used, for example, in relation to personalized medicine to identify appropriate treatment methods. Some uses of organoids have been described in previous applications (e.g., in WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, and WO2016 / 083612), and these uses also apply to the organoids of the present invention.

[0233] Each of the above examples presents details regarding the characteristics of organoids suitable for use, and further specific examples are presented below.

[0234] Drug screening The present invention provides the use of organoids (or cells directly harvested from the above organoids) in drug screening, target validation, target discovery, toxicology, or toxicology screening.

[0235] The cells are preferably exposed to test agents at multiple concentrations for a defined period of time. At the end of the exposure period, the cultures are evaluated. Organoids can also be used to identify drugs that specifically target epithelial cancer cells. Those skilled in the art will understand that the organoids of the present invention are widely applicable as drug screening tools for infectious, inflammatory and neoplastic conditions. In some embodiments, the present invention provides for the use of organoids in drug screening, target validation, target discovery, toxicology, toxicology screens or ex vivo cell / organ models. In some embodiments, the present invention provides for the use of organoids in ex vivo methods for predicting clinical outcomes. In some embodiments, the organoids of the present invention can be used for screening anti-cancer agents.

[0236] In some embodiments, the organoids of the present invention can be used to test libraries of chemical substances, antibodies, natural products (plant extracts), etc. for their suitability for use as drugs, cosmetics and / or prophylactic agents. For example, in some embodiments, a cell biopsy material from a patient of interest, such as tumor cells from a cancer patient, can be cultured using the culture medium and methods of the present invention and then treated with a chemical compound or chemical library. Thereafter, it is possible to determine which compounds effectively modify, kill and / or treat the patient's cells. This makes it possible to test the responsiveness of a particular patient to a particular drug, so that the treatment can be adjusted according to the particular patient. Thus, this enables an individualized medicine approach. In some embodiments, the drug screening method is an ex vivo method that leads to an individualized theory and / or predicts clinical outcomes. In some embodiments, the present invention provides organoids for use in a method of leading to an individualized therapy. An additional advantage of using organoids to identify drugs in such a manner is that it is possible to screen normal organoids (organoids derived from healthy tissue) to identify which drugs and compounds have the least effect on healthy tissue. This enables the screening of drugs with minimal inappropriate activity or undesirable side effects.

[0237] In some embodiments, the present invention provides a method of testing the effect of a candidate compound, the method comprising culturing, according to the method of the present invention, an epithelial stem cell or a population of epithelial stem cells, optionally for less than 21 days, exposing the resulting cell population or resulting organoids to one or a library of candidate compounds, evaluating the above-grown organoids for any effects, identifying the candidate molecule that causes the above effect as a potential drug, and optionally, providing the above candidate molecule as a drug, for example and including.

[0238] In some embodiments, a method of testing the effect of a candidate compound includes exposing an organoid to radiation in the presence or absence of the candidate compound. In some embodiments, the effect evaluated by the method of testing the effect of a candidate compound is selected from a list including a decrease or loss of proliferation, a morphological change, cell death, or a change in gene or protein expression.

[0239] A library of candidate molecules includes two or more candidate molecules.

[0240] In some embodiments, the present invention culturing, optionally for less than 21 days, an epithelial stem cell or a population of epithelial stem cells according to the method of the present invention, exposing the obtained organoid or a population of cells derived from the obtained organoid to a treatment such as radiation and / or to one or a library of candidate molecules, evaluating the organoid or the population of cells for any effect of the candidate molecule, correlating the effect with the presence of one or more genetic mutations, such as mutations in the EGFR signaling pathway, including PIK3CA, KRAS, HRAS, or BRAF, which are characteristics of the organoid and provides a method comprising.

[0241] In some embodiments, the present invention culturing, optionally for less than 21 days, an epithelial stem cell or a population of epithelial stem cells according to the method of the present invention, exposing the obtained organoid or a population of cells derived from the obtained organoid to a treatment such as radiation and / or to one or a library of candidate molecules, evaluating the organoid or the population of cells for any effect of the candidate molecule, comparing the effect with a standard value and / or past observations, and optionally predicting clinical outcomes and / or selecting personalized medicine Provide a method including

[0242] In some embodiments, the organoid is derived from a patient's biopsy material. In some embodiments, a candidate molecule that provides a desired effect to the organoid or a population of cells derived from the organoid is administered to the patient. Thus, in one aspect, a method of treating a patient is provided, the method comprising a) obtaining a biopsy material from an affected tissue of a patient of interest; b) culturing the biopsy material obtained from the organoid, preferably using the method for culturing epithelial stem cells described herein; c) identifying a suitable drug using the screening method of the present invention; d) treating the patient with the drug obtained in step (c) and including.

[0243] In some embodiments, the present invention provides a method of selecting a treatment method for a patient, the method comprising optionally obtaining a biopsy material from the patient's tissue; culturing the biopsy material, a tissue fragment of the biopsy material, the epithelial stem cells of the biopsy material, or a population of epithelial stem cells of the biopsy material to obtain an organoid, preferably using the method for culturing epithelial stem cells described herein; exposing the obtained organoid to a treatment method including radiation and / or one or more candidate compounds; evaluating the organoid for any effect; identifying a treatment method that provides the effect; optionally providing the treatment method to the patient and including the steps of.

[0244] Transplantation and Medicine The present invention provides the use of organoids in regenerative medicine and / or transplantation. The present invention also provides a treatment method, the method including transplanting an organoid into an animal or a human.

[0245] In some embodiments, the present invention provides organoids for use in diagnosis or medicine. In some embodiments, the present invention provides organoids for use in diagnosis or medicine, optionally for personalized medicine or diagnosis, or for regenerative medicine. In some embodiments, the present invention provides a method of treating a disease, comprising the step of administering an organoid of the present invention. In some embodiments, the present invention provides the use of an organoid of the present invention in the manufacture of a medicament for treating a disease.

[0246] Use of an integrin agonist The present invention provides the use of an integrin agonist as defined herein for culturing cells, optionally using any of the culturing methods and / or culture media described herein.

[0247] It may also be useful to contact cells in vitro with an integrin agonist as defined herein prior to transplanting the cells into a patient. Accordingly, the present invention provides the use of an integrin agonist for pretreating cells prior to transplantation into a patient.

[0248] It may also be useful to directly administer an integrin agonist to a patient to improve cell transplantation. Accordingly, the present invention also provides an integrin agonist as defined herein for use as a cell adhesion enhancer in a cell transplantation method. The present invention also provides a method of transplanting cells into a patient, the method comprising administering to the patient an integrin agonist as defined herein.

[0249] Pretreatment with an integrin agonist or administration of an integrin agonist in this context is expected to enhance cell adhesion and increase the success rate of transplantation therapy. This may be useful for the treatment of liver disease, diabetes or any other disease that may benefit from cell transplantation therapy, for example, in regenerative medicine.

[0250] These uses of the integrin agonist are expected to be beneficial not only for epithelial stem cells but also for all cell types. In particular, it is expected to be beneficial for stem cells including hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. The use of the integrin agonist is expected to be particularly useful in hematopoietic stem cell culture (hematopoietic stem cells have been routinely used in many hospitals for decades, but the practical expansion of these cells during culture has not yet been achieved). Thus, in some embodiments, the cells are hematopoietic stem cells. Thus, in some embodiments, the cells are stem cells and, optionally, are epithelial stem cells, hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells.

[0251] Definitions As used herein, the verb "comprise" and its conjugations are used in a non-limiting sense and mean to include the items that accompany that word and also to include items not specifically mentioned. In addition, the verb "consisting of" can, if necessary, be replaced by "consisting essentially of", which means that the product as defined herein may include additional component(s) other than those specifically identified, provided that the additional component(s) do not alter the unique features of the invention. Further, a method as defined herein may include additional step(s) other than those specifically identified, provided that the additional step(s) do not alter the unique features of the invention. Further, a reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly dictates that there is one and only one of that element. Thus, the indefinite article "a" or "an" typically means "at least one".

[0252] As used herein, the term "about" or "approximately" means that the recited value may vary by + / - 10%. Since the value may also be read as the exact value, the term "about" may be omitted. For example, the term "about 100" includes 90 to 110, and 100.

[0253] All patents and documents cited herein are hereby incorporated by reference in their entirety.

[0254] Any reference to a treatment method that includes administering a drug to a patient includes the drug used in the above treatment method, as well as the use of the drug in the above treatment method and the use of the drug in the manufacture of pharmaceuticals.

[0255] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0256] Embodiments The present invention includes the following numbered embodiments.

[0257] 1. A method of culturing epithelial stem cells or organoids containing epithelial stem cells, the method comprising culturing the epithelial stem cells in a culture medium suitable for epithelial stem cells, the culturing method further comprising contacting the cells or the organoids with an integrin agonist.

[0258] 2. The method according to embodiment 1, wherein the integrin agonist interacts with the β subunit of integrin.

[0259] 3. The method according to embodiment 2, wherein the β subunit is β1, β2, β3 or β7.

[0260] 4. The method according to embodiment 3, wherein the integrin agonist interacts with the β1 subunit.

[0261] 5. The method according to embodiment 1, wherein the integrin agonist interacts with the α subunit of integrin.

[0262] 6. The method according to any one of embodiments 1 to 5, wherein the integrin agonist is selected from anti-integrin antibodies, talin, kindlin, dithiothreitol, and oxysterol 25-hydroxy cholesterol.

[0263] 7. The method according to any one of embodiments 1 to 6, wherein the agonist of integrin is an anti-integrin antibody.

[0264] 8. The method according to embodiment 7, wherein the anti-integrin antibody is JBS2, HP1 / 3, SNAKA51, PT25-2, PMI-1, MEM-83, NKI-L16, 496B, 12G10, 8A2, TS2 / 16, 15 / 7, HUTS-4, 8E3, N29, 9EG7, mAb 24, MEM-148, KIM127, CBR LFA-1 / 2, MEM-48, KIM185, AP3, AP5, LIBS6, LIBS2, 10F8, 2B8, 2G3.

[0265] 9. The method according to embodiment 8, wherein the anti-integrin antibody is TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29 or 9EG7.

[0266] 10. The method according to embodiment 9, wherein the anti-integrin antibody is TS2 / 16, 12G10 or 8A2.

[0267] 11. The method according to any one of embodiments 7 to 10, wherein the antibody is humanized.

[0268] 12. The method according to any one of embodiments 1 to 6, wherein the integrin agonist is talin.

[0269] 13. The method according to embodiment 12, wherein the talin is used in combination with kindlin.

[0270] 14. The method according to any one of Embodiments 1 to 6, wherein the integrin agonist is a reducing agent such as dithiothreitol.

[0271] 15. The method according to any one of Embodiments 1 to 6, wherein the integrin agonist is a lipid such as oxysterol 25-hydroxy cholesterol.

[0272] 16. The method according to any one of Embodiments 1 to 15, which results in the growth of the organoid.

[0273] 17. The method according to any one of Embodiments 1 to 16, which results in an increase in epithelial stem cell proliferation of at least 10%, 20%, 50% over four days compared to the same method performed without the integrin agonist.

[0274] 18. The method according to any one of the preceding embodiments, further comprising culturing the cells in contact with an extracellular matrix.

[0275] 19. The method according to Embodiment 18, wherein the extracellular matrix is a basement membrane extract or Matrigel.

[0276] 20. The method according to any one of the preceding embodiments, further comprising culturing the cells in contact with an artificial matrix.

[0277] 21. The method according to Embodiment 20, wherein the artificial matrix comprises a polymer, optionally a polyester, polyethylene glycol or a hydrogel.

[0278] 22. The method according to Embodiment 20 or Embodiment 21, wherein the artificial matrix comprises a cross-linked polyethylene glycol (PEG) hydrogel.

[0279] 23. The method according to Embodiments 20 to 22, wherein the artificial matrix comprises a biomaterial, preferably an extracellular matrix component.

[0280] 24. The method according to embodiment 23, wherein the biological material is one or more glycoproteins (optionally selected from collagen, laminin, perlecan, fibronectin, or the RGD adhesion ligand of fibronectin), and / or one or more carbohydrates (optionally hyaluronic acid).

[0281] 25. The method according to any one of the preceding embodiments, wherein the extracellular matrix or the artificial matrix is three-dimensional.

[0282] 26. The method according to any one of the preceding embodiments, wherein the extracellular matrix or the artificial matrix is in a suspended state.

[0283] 27. The method according to any one of embodiments 1 to 17 and 20 to 26, wherein the culturing method does not include contacting the cells with an exogenous extracellular matrix.

[0284] 28. The method according to any one of the preceding embodiments, wherein the epithelial stem cells are part of a population of epithelial stem cells and are optionally included in organoids and / or epithelial tissue explants.

[0285] 29. The method according to any one of the preceding embodiments, wherein the method includes co-culturing with non-epithelial cell types, optionally immune cells.

[0286] 30. The method according to any one of the preceding embodiments, wherein the epithelial stem cells are mammalian cells.

[0287] 31. The method according to embodiment 30, wherein the epithelial stem cells are human cells.

[0288] 32. The method according to any one of the preceding embodiments, wherein the epithelial stem cells are selected from colorectal, small intestine, stomach, pancreas, liver, lung, breast, prostate, kidney, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle, and / or cochlear cells.

[0289] 33. The method according to any one of the preceding embodiments, wherein the epithelial stem cells are colorectal cells.

[0290] 34. The method according to any one of the preceding embodiments, wherein the culture medium suitable for the epithelial stem cells contains one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor.

[0291] 35. The method according to embodiment 34, wherein the culture medium suitable for the epithelial stem cells contains a Wnt agonist.

[0292] 36. The method according to embodiment 35, wherein the culture medium suitable for the epithelial stem cells further contains a mitogenic growth factor and / or a BMP inhibitor.

[0293] 37. The method according to embodiments 34 to 36, wherein the culture medium suitable for the epithelial stem cells contains a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor.

[0294] 38. The method according to embodiments 34 to 37, wherein the Wnt agonist is selected from one or more of an R-spondin family protein, a Wnt ligand from the Wnt family of secreted glycoproteins, an inhibitor of intracellular β-catenin degradation, a GSK-3 inhibitor, a TCF / LEF activator, an RNF43 or ZNRF3 inhibitor.

[0295] 39. The method according to embodiments 34 to 38, wherein the mitogenic growth factor binds to a receptor tyrosine kinase such as EGFR, FGFR, or HGFR.

[0296] 40. The method according to embodiments 34 to 39, wherein the mitogenic growth factor is one or more of EGF, FGF, and HGF.

[0297] 41. The method according to embodiments 34 to 40, wherein the BMP inhibitor is noggin.

[0298] 42. The method according to embodiments 34 to 41, wherein the TGFβ inhibitor is an inhibitor of the ALK4, ALK5 or ALK7 signal transduction pathway.

[0299] 43. The method according to embodiments 34 to 42, wherein the TGFβ inhibitor is a small molecule inhibitor such as A83-01.

[0300] 44. The method according to embodiments 34 to 43, wherein the culture medium suitable for the epithelial stem cells further comprises one or more of nicotinamide, gastrin, B27 and N-acetylcysteine.

[0301] 45. The method according to any one of the preceding embodiments, wherein the culture medium suitable for the epithelial stem cells is suitable for proliferating the epithelial stem cells.

[0302] 46. The method according to any one of the preceding embodiments, wherein the culture medium suitable for the epithelial stem cells is suitable for differentiating the epithelial stem cells.

[0303] 47. The culture medium according to any one of embodiments 34 to 46, further comprising the integrin agonist defined in embodiments 2 to 15.

[0304] 48. The extracellular matrix or artificial matrix according to any one of embodiments 18 to 27, further comprising the integrin agonist defined in embodiments 2 to 15.

[0305] 49. An organoid that can be collected or has been collected by the method according to any one of embodiments 1 to 46.

[0306] 50. The organoid according to embodiment 49, having a normal karyotype.

[0307] 51. The organoid according to embodiment 49 or embodiment 50, having a rosette-like structure.

[0308] 52. The organoid according to embodiment 49, wherein the organoid is a tumor organoid.

[0309] 53. A composition comprising the culture solution according to embodiment 47 and the organoid according to any one of embodiments 49 to 52.

[0310] 54. A composition comprising the culture solution of embodiment 47 and, optionally, an extracellular matrix or an artificial matrix defined in any one of embodiments 18 to 27.

[0311] 55. Use of an integrin agonist for culturing cells.

[0312] 56. Use of an integrin agonist for pretreating cells before transplantation into a patient.

[0313] 57. An integrin agonist for use as a cell adhesion enhancer in a cell transplantation method.

[0314] 58. The use according to embodiment 53 or 54, or the integrin agonist for the use according to embodiment 56, wherein the cell is a stem cell, optionally an epithelial stem cell, a hematopoietic stem cell, an induced pluripotent stem cell or an embryonic stem cell.

[0315] 59. Use of the organoid according to any one of embodiments 49 to 52 for drug screening, target validation, target discovery or toxicology.

[0316] 60. The organoid according to any one of embodiments 49 to 52 for use in treatment or for use in diagnosis.

Example

[0317] Example 1 - Preparation of a humanized TS2 / 16 antibody The humanized TS2 / 16 antibody was produced by U-Protein Express BV, Utrecht, The Netherlands. This also included the generation of the coding sequences of the antibody variable domains via artificial gene design and codon optimization. In a framework containing the constant regions of the heavy and light chains, an artificial fragment of the antibody variable domain was ligated into an antibody expression vector using BsmBI restriction enzyme recognition sites at the 5' and 3' ends to generate an expression vector. The antibody expression vector was transiently generated in HEK293 cells or CHO cells via the rPEx technology, and subsequently, the recombinant antibody was purified via affinity chromatography (Protein A), ion exchange chromatography, and / or gel filtration chromatography.

[0318] The humanized antibody was analyzed under non-reducing conditions using the NU-PAGE Tris-Acetate Gel / SDS buffer system (Invitrogen). The resulting NuPAGE gel is shown in Figure 2. Transfection of the expression plasmids of the heavy chain and L chain at ratios of 1:1 and 1:1.5 formed denser protein bands on the gel compared to those at a ratio of 1:3. The heavy and light chains of the humanized antibody were sequenced and found to be SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The CDR regions of the heavy and light chains were identified to be SEQ ID NOs: 5-7 and SEQ ID NOs: 8-10, respectively.

[0319] Example 2 - Test of Integrin β1 Recognition by the Humanized TS2 / 16 Antibody K562 cells (human immortalized myeloid leukemia cell line) were incubated with the humanized TS2 / 16 antibody in conditioned medium. The human erythroleukemia cell line K562 expresses only α5β1, an integrin of the β1 class, on its surface

[17] . Antibody binding was visualized by incubating the cells with goat anti-human IgG1 conjugated with Alexa488 (Life Technologies (A11013)) at a dilution of 1:250 in PBS (1% BSA) (Figure 3). The results showed that the antibody bound normally to the integrin on the cell surface.

[0320] Example 3 - Confirmation of the Functional Activity of the Humanized TS2 / 16 Antibody The ability of the humanized TS2 / 16 antibody to induce cell adhesion to fibronectin was tested as described in

[17] . Briefly, 96-well plates were coated overnight with BSA or fibronectin (5 μg / ml in PBS). Subsequently, non-specific binding sites were blocked with PBS (1% BSA) for 30 minutes. Human erythroleukemia cell line K562 cells (10 5 cells / well) were incubated at 37°C for 1 hour in the presence or absence of humanized TS2 / 16. The experiment was performed three times. Unbound cells were removed by rinsing the wells twice with PBS. Bound cells were quantified using the cell ATP-driven CellTiterGlo assay (Promega). Briefly, the CellTiter-Glo® assay measures the number of viable cells in culture based on the quantification of the presence of ATP, which indicates the presence of metabolically active cells.

[0321] Figure 4 shows that the humanized TS2 / 16 antibody can induce cell adhesion to fibronectin, as previously demonstrated with the non-humanized version of the antibody, but not to the control BSA.

[0322] A similar experiment was performed using the MAB1778 antibody, another anti-integrin agonist antibody that targets the β1 integrin subunit. MAB1778 was able to induce adhesion of these cells.

[0323] Example 4 - Cultivation of Epithelial Stem Cells in a Culture Medium Containing No Extracellular Matrix Components The inventors tested whether the addition of an integrin agonist would enable the proliferation of epithelial stem cells in a culture medium containing no exogenously added extracellular matrix components. As already mentioned, the presence of extracellular matrix in the culture medium is known to be a requirement for efficient epithelial stem cell proliferation.

[0324] A single human epithelial colon stem cell was used as described in

[18] . The single human epithelial colon stem cell was plated into a Corning ultra-low attachment 96-well flat-bottom plate containing culture medium as described below. The Corning ultra-low attachment surface is a hydrophilic and charge-neutral coating covalently bonded to the surface of a polystyrene container. The hydrogel inhibits specific and non-specific immobilization, keeps the cells in a floating state, and enables the formation of three-dimensional steroids. The coating is stable, non-cytotoxic, biologically inert, and non-degradable.

[0325] The culture medium contained advanced DMEM / F12 medium supplemented with B27, nicotinamide, N-acetylcysteine, noggin, R-spondin 1, EGF, WNT-conditioned medium

[19] , TGF-β type I receptor inhibitor A83-01, and P38 inhibitor SB202190. In particular, the culture medium did not contain an extracellular matrix.

[0326] Next, single human epithelial colon stem cells were cultured in human colon organoid medium supplemented with TS2 / 16-conditioned medium (UPE) at dilutions of 1:100, 1:200, and 1:400, or without antibody. Three replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured by counting the total number of organoids on the 4th and 7th days after treatment (Figure 5).

[0327] The addition of the integrin agonist increased epithelial stem cell proliferation compared to the culture medium without the humanized TS2 / 16 antibody (see Figure 5). The optimal effect of the integrin agonist was observed at the lowest dilution rate of 1:400, but an increase in the proliferation rate was seen at all dilution rates tested. An example of an organoid growing in the culture medium containing the humanized TS2 / 16 antibody is shown in Figure 6.

[0328] The inventors have surprisingly demonstrated that the addition of an integrin agonist to a culture medium containing no extracellular matrix components can successfully result in efficient epithelial stem cell proliferation. As observed in the negative control, in the absence of extracellular matrix components, epithelial stem cells do not significantly proliferate. Therefore, a culture method including contacting cells or organoids with an integrin agonist can be used for an improved method for culturing epithelial stem cells or organoids.

[0329] Since the inventors were able to enable epithelial stem cell proliferation in the absence of the extracellular matrix by the addition of an integrin agonist, it is hypothesized that the integrin agonist can mimic the structural and signaling functions of the extracellular matrix. Without wishing to be bound by any particular theory, the integrin agonist can activate integrin with respect to an integrin ligand by changing the conformational state of the integrin. Next, the integrin agonist also mimics the action of an integrin ligand that activates a signaling pathway and promotes epithelial stem cell proliferation.

[0330] Example 5 - Addition of a humanized TS2 / 16 antibody to a culture medium containing an extracellular matrix Colon epithelial stem cells were isolated as described in Example 4. Single cells were cultured in a medium containing high-glucose DMEM / F12 medium containing B27, nicotinamide, N-acetylcysteine, noggin, R-spondin 1, EGF, WNT-conditioned medium (50%, generated using stably transfected L cells), TGF-β type I receptor inhibitor A83-01, and P38 inhibitor SB202190 as described in

[20] . The cells were suspended in 10 μL drops of Matrigel / well in a round-bottom 96-well plate.

[0331] The cells were treated with the above medium as a control (bar 1 in Figure 7) or with the same human colonic organoid medium supplemented with humanized TS2 / 16 antibody by the two-fold dilution method (bars 2-4 in Figure 7). The proliferation of epithelial stem cells was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) as described above.

[0332] The results demonstrated that the addition of integrin agonists to the culture medium in combination with the cell-Matrigel suspension increased epithelial stem cell proliferation. Thus, integrin agonists can improve the proliferation of epithelial stem cells even in combination with extracellular matrix-derived signaling.

[0333] These results were confirmed in further experiments using Matrigel concentrated with Dyna beads. Colonic epithelial stem cells were isolated and cultured as described above. However, the Matrigel used was modified by adding Dynabeads (Invitrogen / Thermofischer Scientific Ref 10003D) coated with Protein G or Dynabeads coated with Protein G thoroughly soaked with humanized TS2 / 16 antibody. Figure 8 shows two results: the result of treatment with humanized TS2 / 16 antibody (bar 2) and the result of no treatment with humanized TS2 / 16 antibody (bar 1). These data also show that the addition of integrin agonists multivalently presented by Dynabeads coated with Protein G promotes classical Matrigel-driven epithelial cell proliferation.

[0334] In conclusion, integrin agonists can stimulate the proliferation of epithelial stem cells or organoids by adding them as a soluble component to the culture medium, regardless of the presence or absence of exogenously added extracellular matrix. Integrin agonists can stimulate proliferation when incorporated into the extracellular matrix, for example, when cross-linked with a carrier and directly mixed with Matrigel.

[0335] Example 6 - Effect of HUTS-4 on the culture of epithelial stem cells in a culture medium without extracellular matrix components As described above, each study has shown that the Fab integrin-activating antibodies TS2 / 16, 12G10, and HUTS-4 can induce almost the same high affinity for cyclic RGD peptides. This observation suggests that these antibodies stabilize the same conformation of the ligand-binding site in the β1 domain. The present inventors tested whether the addition of another integrin agonist, HUTS-4, would enable the growth of epithelial stem cells in a culture medium without exogenously added extracellular matrix components.

[0336] Fragments of human colon organoids were isolated and cultured in Corning ultra-low attachment 96-well flat-bottom plates as described in Example 4. Next, these human colon-derived organoid fragments were cultured in human colon organoid medium supplemented with TS2 / 16 or HUTS-4 at a final concentration of 1 μg / ml, or cultured without antibodies. Three replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured on day 7 after treatment by quantifying viable cells using the CellTiterGlo ATP-based assay (Figure 9).

[0337] The addition of two different integrin agonists increased epithelial stem cell proliferation compared to the control culture medium without integrin agonist antibodies (see Figure 9). Examples of growing organoids in all three culture medium tests are shown in Figure 11.

[0338] The inventors have surprisingly demonstrated that the addition of two different integrin agonists to a culture medium containing no extracellular matrix components can successfully result in efficient epithelial stem cell proliferation. Therefore, a culture method involving contacting cells or organoids with an integrin agonist can be used to improve the method for culturing epithelial stem cells or organoids. These data demonstrate that the culture methods described herein have an affinity for cyclic RGD peptides similar to integrin agonists TS2 / 16, 12G10, and HUTS-4, and / or can stabilize the head-open conformation of the same integrins as TS2 / 16, 12G10, and HUTS-4, and have been demonstrated to be particularly effective against integrin agonists.

[0339] Example 7 - Growth of the human colon in the absence of Matrigel starting from sheared organoids The inventors tested whether integrin agonists can improve the proliferation of epithelial stem cells starting from fragments of organoids (sheared organoids) rather than single cells as used in Example 4 in a culture medium containing no exogenously added extracellular matrix components.

[0340] Organoid fragments were mechanically harvested by repeatedly moving the organoids back and forth through a narrow pipette tip member. The degree of fragmentation was monitored under a microscope. Fragments of human epithelial colon organoids were plated in a Corning ultra-low attachment 96-well flat-bottom plate containing the culture medium as described in Example 4.

[0341] Next, fragments of human epithelial colon organoids were cultured in human colon organoid medium supplemented with humanized TS2 / 16 or human colon organoid medium without antibodies. Five replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured on the 7th day after treatment by counting the total number of organoids.

[0342] Addition of an integrin agonist to cultures initiated with organoid fragments increased epithelial stem cell proliferation compared to cultures without the humanized TS2 / 16 antibody (see Figure 10). Examples of organoids growing in cultures with and without the humanized TS2 / 16 antibody are shown in Figure 12. There are advantages associated with initiating epithelial stem cell cultures using organoid fragments rather than single epithelial stem cells. For example, organoid growth is typically improved in cultures initiated with organoid fragments rather than single cells. Additionally, single epithelial stem cells are typically harvested by digesting the organoids with trypsin, which can cleave integrin receptors from the cell surface. Thus, the cells require time to replace the integrin receptors on their surface before activity by an integrin agonist can occur.

[0343] The inventors have surprisingly demonstrated that addition of an integrin agonist to a culture medium containing no extracellular matrix components can successfully result in efficient epithelial stem cell proliferation from cultures initiated with both single epithelial stem cells and organoid fragments.

[0344] Example 8 - Human Colon Proliferation in the Presence of Matrigel and Antibody AIIB2 AIIB2 is a known integrin antagonist antibody that can bind to the β1 subunit of integrin. In contrast to the integrin agonists tested in Example 3, AIIB2 has been shown to inhibit cell adhesion to the ECM components fibronectin, laminin, and collagen IV

[21] .

[0345] Human colon-derived organoid fragments of P26N and STEM159N were isolated as described in Example 6. Next, the human colon-derived organoid fragments were cultured in the human colon organoid medium described in Example 5. In some of the conditions tested, the fragments were suspended in 10 μL drops of Matrigel / well. The following conditions were tested. a) P26N organoid fragments in the presence of Matrigel, with or without AIIB2 and without antibodies. b) P26N organoid fragments in the absence of Matrigel, with or without AIIB2 and without antibodies. c) STEM159N organoid fragments in the presence of Matrigel, with or without AIIB2 and without antibodies. Two replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured on day 7 after treatment by counting the total number of organoids.

[0346] Figures 13 - 15 show that the presence of AIIB2 in the culture medium inhibits epithelial stem cell proliferation. Without wishing to be bound by any particular theory, the lack of epithelial stem cell proliferation due to the presence of AIIB2 is attributed to the ability of AIIB2 to block the activation of integrins by ligands in Matrigel. As described above, without integrin activation by ligands or integrin agonists in the ECM, epithelial stem cells do not grow and proliferate in culture.

[0347] Epithelial stem cells in the organoids have a basolateral phenotype where the apical membrane of the cells faces the lumen of the organoid. In the case of organoid growth in the absence of Matrigel, this polarization turns apical - lateral due to a lack of ligands for the integrin β1 receptor, as described in reference

[22] . This apical - lateral phenotype is also observed in patients with the TTC7A mutation where integrin - related ROCK signaling is disrupted

[23] . This polarity reversal can also occur in Matrigel cultures containing AIIB2, as this antibody blocks the interaction between the ITGB1 receptor and extracellular matrix components in Matrigel. The principle of the polarity reversal caused by AIIB2 was first discovered in MDCK cysts

[24] .

[0348] In summary, the integrin β1 receptor of the extracellular matrix component is essential for ensuring the growth of organoids with a basolateral phenotype. The inventors have surprisingly demonstrated that the addition of an integrin agonist to a culture medium not containing extracellular matrix components can successfully lead to the growth of epithelial stem cells with a basolateral phenotype.

[0349] Example 9 - Culture of pancreatic epithelial stem cells in a culture medium not containing extracellular matrix components The inventors tested whether an integrin agonist could improve the growth of pancreatic epithelial stem cells in a culture medium not containing exogenously added extracellular matrix components.

[0350] Fragments of two independent pancreatic organoid strains, W15 - 50040 and W15 - 50020, were harvested using the method described in Example 6. Pancreatic cells were cultured in AdDMEM / F12 medium supplemented with HEPES (1x), Glutamax (1x), penicillin / streptomycin (1x), B27 (1x), Primocin (1 mg / ml), N - acetyl - L - cysteine (1 mM), Wnt3a - conditioned medium (50% volume / volume), RSPO1 - conditioned medium (10% volume / volume), Noggin - conditioned medium (10% volume / volume), or recombinant proteins (0.1 μg / mL) epidermal growth factor (EGF, 50 ng / ml), gastrin (10 nM), fibroblast growth factor 10 (FGF10, 100 ng / ml), nicotinamide (10 mM), and A83 - 01 (0.5 μM) as described in

[25] .

[0351] Next, these human pancreas - derived organoid fragments were cultured in human pancreatic organoid medium supplemented with TS2 / 16 at a final concentration of 1 μg / ml or cultured without an antibody. Three replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured on day 6 after treatment by quantifying live cells using the CellTiterGlo ATP - based assay (Figure 16B). An example of a growing pancreatic organoid is shown in Figure 16A.

[0352] The addition of integrin agonists increased pancreatic epithelial stem cell proliferation compared to the culture medium without the humanized TS2 / 16 antibody (see Figure 16).

[0353] Example 10 - Culture of lung epithelial stem cells in a culture medium containing extracellular matrix The inventors tested whether integrin agonists could improve the proliferation of lung epithelial stem cells in a culture medium containing extracellular matrix.

[0354] Fragments of lung organoids were collected using the method described in Example 6. The lung organoid fragments were cultured in a medium containing high-glucose DMEM / F12, HEPES, Primocin, penicillin / streptomycin, GlutaMax 100x, R-spondin 1, FGF7, FGF10, noggin, A83-01, Y-27632, SB202190, B27 supplement, N-acetylcysteine, and nicotinamide as described in

[26] . Cells were suspended in 10 μL of Matrigel / well in a round-bottom 96-well plate.

[0355] Next, these human lung-derived organoid fragments were cultured in human lung organoid medium supplemented with TS2 / 16 at a final concentration of 1 μg / ml or without the antibody. Three replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured on day 12 after treatment by quantifying live cells using a CellTiterGlo ATP-based assay (Figure 17A). An example of a growing lung organoid is shown in Figure 17B.

[0356] The addition of integrin agonists increased lung epithelial stem cell proliferation compared to the culture medium without the humanized TS2 / 16 antibody (see Figure 17).

[0357] Example 11 - Culture of head and neck epithelial stem cells in a culture medium containing extracellular matrix The inventors tested whether an integrin agonist could improve the proliferation of head and neck epithelial stem cells in a culture medium that does not contain extracellular matrix components added externally.

[0358] Fragments of head and neck organoids were collected using the method described in Example 6. The head and neck organoid fragments were cultured in a medium containing Advanced DMEM+ / + / + containing 1× B27 supplement, 1 mM N-acetyl-L-cysteine, 10 mM nicotinamide 505, 50 ng / ml human EGF, 500 nM A83-01, 10 ng / ml human FGF10, 5 ng / ml human FGF2, 1 μM prostaglandin E2, 3 μM CHIR-99021, 1 μM forskolin, 4% R-spondin, and 4% noggin. Cells were suspended in Matrigel / drop of 10 μL in a round-bottom 96-well plate.

[0359] Next, these human head and neck-derived organoid fragments were cultured in a human head and neck organoid medium supplemented with TS2 / 16 at a final concentration of 1 μg / ml or cultured without an antibody. Three replicate tests were performed for each treatment condition. The proliferation rate of epithelial stem cells was measured by quantifying live cells using a CellTiterGlo ATP-based assay on the 7th day after treatment (Figure 18B). An example of a proliferating head and neck organoid is shown in Figure 18A. The addition of the integrin agonist increased head and neck epithelial stem cell proliferation compared to the culture medium without the humanized TS2 / 16 antibody (see Figure 18).

[0360] Example 12 - Proliferation of human colon in the presence of antibody Asc8 Asc8 is a known integrin antagonist antibody that binds to the β4 subunit of integrin. Asc8 has been shown to block cell adhesion and inhibit wound closure

[27] .

[0361] Colon epithelial stem cells were isolated as described in Example 4 and cultured in the medium according to Example 5. The cells were suspended in Matrigel / well at a dropwise addition of 10 μL in a round-bottom 96-well plate. The cells were treated with the above medium as a control or with the following. (a) Asc8 at a final concentration of 0.3 μg / ml. (b) Asc8 at a final concentration of 1.2 μg / ml. (c) Asc8 at a final concentration of 5 μg / ml. (d) TS2 / 16 at a final concentration of 1 μg / ml.

[0362] The proliferation of epithelial stem cells was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) as described above.

[0363] Figure 19 shows that the presence of Asc8 in the culture medium inhibits epithelial stem cell proliferation. This observation is similar to that observed with AIIB2 described in Example 8. Without wishing to be bound by any particular theory, in this case too, the lack of epithelial stem cell proliferation due to the presence of Asc8 is thought to be due to the ability of this antibody to block the activation of integrin by the ligand in Matrigel.

[0364] Example 13 - Synergistic effect of multiple integrin agonists 3E1 is an antibody agonist of β4 integrin. When integrin β4 is involved with its natural ligand or activation antibody, tyrosine phosphorylation of the β4 cytoplasm occurs, thereby controlling multiple signal transduction pathways important for tumorigenesis

[28] . It has been revealed that phosphorylated Src associates with integrin β4 of A431 cells in response to laminin or 3E1 stimulation that increases Src kinase activity.

[0365] 3E1 was obtained from the Memorial Sloan Kettering Cancer Centre Antibody & Bioresource Core Facility. This antibody is sold by Sigma Aldrich under the catalog number MAB1964. Colonic epithelial stem cells were isolated as described in Example 4 and cultured in the medium according to Example 5. The cells were suspended in Matrigel / well at a drop of 10 μL in a round-bottom 96-well plate. The cells were treated with the above medium as a control (bar 1) or with the following. (a) TS2 / 16 at a final concentration of 0.1 μg / ml (bar 2). (b) TS2 / 16 at a final concentration of 0.1 μg / ml and 3E1 at a final concentration of 0.1 μg / ml (bar 3). (c) TS2 / 16 at a final concentration of 0.1 μg / ml and 3E1 at a final concentration of 1 μg / ml (bar 4).

[0366] The proliferation of epithelial stem cells was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) as described above. Figure 20 shows the synergistic effect of the β1 integrin agonist TS / 16 and the β4 integrin agonist 3E1 on epithelial stem cell proliferation. Therefore, multiple integrin agonists can improve the proliferation of epithelial stem cells in combination with signal transduction derived from the extracellular matrix.

[0367] The data presented in the above examples demonstrated the unexpected ability of integrin agonists to successfully achieve efficient epithelial stem cell proliferation in various cell types in the presence and absence of extracellular matrix components.

[0368] Sequence SEQ ID NO: 1 - TS2 / 16 H chain TIFF2025090761000003.tif50170 SEQ ID NO: 2 - TS2-16 kL chain TIFF2025090761000004.tif24170 SEQ ID NO: 3 - Humanized TS2 / 16 H chain TIFF2025090761000005.tif11170 Sequence number 4 - Humanized TS2 / 16 L chain TIFF2025090761000006.tif11170 Sequence number 5 - Humanized TS2 / 16 H chain HCDR1: GFTFSSYTMS Sequence number 6 - Humanized TS2 / 16 H chain HCDR2: TISSGGSYTYYPDSVKG Sequence number 7 - Humanized TS2 / 16 H chain HCDR3: IGYDEDYAMDH Sequence number 8 - Humanized TS2 / 16 L chain LCDR1: SVSSIISSNYLH Sequence number 9 - Humanized TS2 / 16 L chain LCDR2: RTSNLAS Sequence number 10 - Humanized TS2 / 16 L chain LCDR3: QQGSDIPLT

[0369] References TIFF2025090761000007.tif113150

[0370] Sequence information SEQUENCE LISTING <110> KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN <120> IMPROVED CULTURE METHOD USING INTEGRIN AGONIST <150> GB 1906978.0 <151> 2019-05-17 <160> 10 <170> SeqWin2010, version 1.0 <210> 1 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> TS2 / 16 H-chain <400> 1 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Met Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45 Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg 50 55 60 Leu Glu Trp Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr 65 70 75 80 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Gly Ser Leu Lys Ser Glu Asp Thr Ala 100 105 110 Met Tyr Tyr Cys Thr Arg Ile Gly Tyr Asp Glu Asp Tyr Ala Met Asp 115 120 125 His Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 2 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> TS2-16 kL-chain <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Ile Val Val Thr Gln Ser Pro Thr Thr Met Ala Ala 20 25 30 Ser Pro Gly Asp Lys Ile Thr Ile Thr Cys Ser Val Ser Ser Ile Ile 35 40 45 Ser Ser Asn Tyr Leu His Trp Tyr Ser Gln Lys Pro Gly Phe Ser Pro 50 55 60 Lys Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Pro 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly 85 90 95 Thr Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser 100 105 110 Asp Ile Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Asp Leu Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 H-chain <400> 3 Met Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp 35 40 45 Val Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Gly Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Thr Arg Ile Gly Tyr Asp Glu Asp Tyr Ala Met Asp His Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 4 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 L-chain <400> 4 Glu Ile Val Val Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 1 5 10 15 Asp Lys Ile Thr Ile Thr Cys Ser Val Ser Ser Ile Ile Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Ser Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Asp Ile Pro 85 90 95 Leu Thr Phe Gly Asp Gly Thr Lys Leu Asp Leu Lys 100 105 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 H-chain HCDR1 <400> 5 Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser 1 5 10 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 H-chain HCDR2 <400> 6 Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 H-chain HCDR3 <400> 7 Ile Gly Tyr Asp Glu Asp Tyr Ala Met Asp His 1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 L-chain LCDR1 <400> 8 Ser Val Ser Ser Ile Ile Ser Ser Asn Tyr Leu His 1 5 10 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Humanised TS2 / 16 L-chain LCDR2 <400> 9 Ser Val Ser Ser Ile Ile Ser Ser Asn Tyr Leu His 1 5 10 <210> 10 <211> 9 <212> PRT <213> Humanised TS2 / 16 L-chain LCDR3 <400> 10 Gln Gln Gly Ser Asp Ile Pro Leu Thr 1 5

Claims

1. A method for culturing epithelial stem cells or organoids comprising epithelial stem cells, the method comprising culturing the epithelial stem cells in a culture medium suitable for epithelial stem cells, the culture method further comprising contacting the cells or organoids with an integrin agonist.

2. The method of claim 1, wherein the integrin agonist interacts with a β subunit of an integrin, and optionally the β subunit is β1, β2, β3 or β7.

3. The method of claim 2 , wherein the integrin agonist interacts with the β1 subunit.

4. The method of claim 1 , wherein the integrin agonist interacts with the α subunit of an integrin.

5. The method of any one of claims 1 to 4, wherein the integrin agonist is selected from an anti-integrin antibody, talin, kindlin, dithiothreitol and the oxysterol 25-hydroxycholesterol.

6. 6. The method of any one of claims 1 to 5, wherein said agonist of an integrin is an anti-integrin antibody, optionally said anti-integrin antibody is JBS2, HP1 / 3, SNAKA51, PT25-2, PMI-1, MEM-83, NKI-L16, 496B, 12G10, 8A2, TS2 / 16, 15 / 7, HUTS-4, 8E3, N29, 9EG7, mAb 24, MEM-148, KIM127, CBR LFA-1 / 2, MEM-48, KIM185, AP3, AP5, LIBS6, LIBS2, 10F8, 2B8, 2G3.

7. 7. The method of claim 6, wherein the anti-integrin antibody is TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29 or 9EG7, and further optionally wherein the anti-integrin antibody is TS2 / 16, 12G10, HUTS-4 or 8A2, and further optionally wherein the anti-integrin antibody is TS2 / 16, 12G10, HUTS-4.

8. The method of claim 6 or claim 7, wherein the antibody is humanized.

9. The integrin agonist is a. Talin, optionally used in combination with kindlin; b. A reducing agent, such as dithiothreitol, or c. lipids, such as the oxysterol 25-hydroxycholesterol; The method according to any one of claims 1 to 5, wherein

10. The method comprises: a. Organoid proliferation, and / or b. An increase in epithelial stem cell proliferation of at least 10%, 20%, 50% over a four day period compared to the same method performed without the integrin agonist. The method according to any one of claims 1 to 9, wherein

11. 10. The method of any one of the preceding claims, wherein the method further comprises culturing the cells in contact with an extracellular matrix, optionally wherein the extracellular matrix is ​​a basement membrane extract or Matrigel.

12. 2. The method of any one of the preceding claims, wherein the method further comprises culturing the cells in contact with an artificial matrix, optionally the artificial matrix comprises a polymer, optionally a polyester, polyethylene glycol or a hydrogel.

13. The artificial matrix comprises: a. cross-linked polyethylene glycol (PEG) hydrogels, and / or b. Biomaterials, preferably extracellular matrix components 13. The method of claim 12, comprising: optionally the biomaterial is one or more glycoproteins (optionally selected from collagen, laminin, perlecan, fibronectin, or RGD adhesion ligand of fibronectin) and / or one or more carbohydrates (optionally hyaluronic acid).

14. 10. The method according to any one of the preceding claims, wherein the extracellular matrix or the artificial matrix is ​​three-dimensional and / or in suspension.

15. The method according to any one of claims 1 to 10 and 12 to 14, wherein the culture method does not include contacting the cells with an exogenous extracellular matrix.

16. 2. The method of any one of the preceding claims, wherein the epithelial stem cells are selected from colorectal, small intestine, stomach, pancreas, liver, lung, breast, prostate, kidney, mouth, nasopharynx, throat, hypopharynx, larynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle and / or cochlear cells.

17. 2. The method of any one of the preceding claims, wherein the culture medium suitable for epithelial stem cells comprises one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor, and a TGFβ inhibitor.

18. A culture medium as defined in claim 17, further comprising an integrin agonist as defined in claims 2 to 9.

19. An extracellular matrix or an artificial matrix as defined in any one of claims 11 to 15, further comprising an integrin agonist as defined in claims 2 to 9.

20. Organoids harvestable or harvested by the method according to any one of claims 1 to 17.

21. A composition comprising a culture medium according to claim 18 and optionally an extracellular matrix or an artificial matrix as defined in any one of claims 11 to 15.

22. Use of an integrin agonist for culturing cells.

23. Use of integrin agonists to pre-treat cells prior to transplantation into a patient.

24. An integrin agonist for use as a cell adhesion enhancer in cell transplantation methods.

25. 21. The organoid of claim 20 for use in therapy or for use in diagnosis.

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