Co-culture of organoids and stromal cells

Abstract

This invention relates to organoid co-cultures and their use in disease investigations.

Description

[Technical Field]

[0001] All references cited herein are incorporated in their entirety by reference.

[0002] Technical field This invention relates to organoid co-cultures and their use in disease investigations. [Background technology]

[0003] background Organoids are a promising tool for studying human physiological functions in vitro. Organoids are self-organized epithelial cell structures with physiological characteristics similar to in vivo tissues. They are widely used to model the onset and progression of disease. Therefore, stem cell-derived organoids provide advanced models for studying human development and disease.

[0004] Epithelial organoid models can be enhanced by incorporating non-epithelial cells to form "co-cultures." However, devising protocols and conditions that allow very different cell types to coexist in vitro is difficult, much less whether such coexistence promotes relevant intercellular interactions.

[0005] Stromal cells are a type of non-epithelial cell. As a component of the "interstitium" associated with the body's organs, stromal cells provide structural support to those organs. Traditionally considered an "inactive" (or purely structural) component, stromal cells have recently been identified as performing important active roles in metabolism, signaling, and disease—functions traditionally associated with non-stromal epithelial cell types.

[0006] For example, the interstitium is thought to play a crucial role in gastrointestinal disorders such as inflammatory bowel disease (IBD). In IBD patients, activation of the interstitial compartment, including an excess of pro-inflammatory fibroblasts, has been observed. At the molecular level, intestinal cells interacting with these pro-inflammatory fibroblasts are thought to exhibit increased apoptosis, thickening, or enlargement, and remodeling of the intestinal stem cell niche. These factors are thought to contribute to the chronicity and treatment resistance of IBD. However, the pathogenesis of IBD disorders remains unclear, and previous in vitro models cannot replicate in vivo physiological functions.

[0007] The present invention provides a co-culture formed by combining at least one organoid and at least one stromal cell. As described in the examples, such co-cultures have been validated and have therefore been shown to be able to reproduce and perform in vivo microstructures, intercellular interactions, and biochemical reactions (such as inflammatory responses) over long periods. Such co-cultures may be used in in vitro methods for the treatment, diagnosis, and / or prognosis prediction of diseases, particularly those related to inflammation and / or fibrosis.

[0008] For example, the inventors replicated an in vivo inflammatory process by exposing such co-cultures, which include at least one organoid and at least one stromal cell, to at least one pro-inflammatory stimulus to induce a pro-inflammatory profile in the co-culture, and then to at least one further inflammatory stimulus to induce an inflammatory response. These co-cultures express expected biomarkers of inflammation (such as IL-6 and CXCL2).

[0009] These co-cultures are useful for testing therapeutic agents, for example, by determining the reduction of inflammatory biomarkers when the therapeutic agent is applied to the co-culture. A reduction in inflammatory biomarkers indicates, for example, that the therapeutic agent is addressing a specific molecular pathway that contributes to the production of inflammatory biomarkers, and therefore, that the therapeutic agent may be able to treat the disease in vivo.

[0010] These co-cultures are also useful for diagnosis and / or prognosis prediction, for example, by determining whether a diagnostic agent known to induce inflammation via a specific molecular pathway can increase inflammation in a co-culture containing at least one PDO. If such a diagnostic agent does not increase inflammatory biomarkers, this may indicate that the specific molecular pathway targeted by this diagnostic agent is saturated (i.e., cannot be further activated), and therefore this specific molecular pathway may act in the patient's disease. Similar retrogrades may be used when a known therapeutic agent is used as a diagnostic agent. If this known therapeutic agent can decrease inflammatory biomarkers in a co-culture containing at least one PDO, then the molecular element targeted by this therapeutic agent may act in the patient's disease. [Overview of the Initiative]

[0011] The present invention provides a method for preparing a co-culture comprising at least one organoid and at least one stromal cell, the method being: To form a co-culture, combine at least one organoid with at least one stromal cell in the co-culture medium. Includes, Optionally, (a) The method comprises preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) The method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0012] The present invention further provides a method for determining the presence or absence of at least one change in a co-culture comprising at least one organoid and at least one stromal cell, the method being To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, To determine whether or not at least one change is present in the co-cultured material. Includes, Optionally, (a) The method comprises preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) The method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0013] The present invention further provides a method for determining the presence or absence of at least one change in at least one organoid, the method being To isolate a conditioned medium from at least one stromal cell and combine the conditioned medium with at least one organoid, To determine whether or not at least one change has occurred in the organoid. Includes, Optionally, (a) The method comprises preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) The method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0014] The present invention further provides a method for testing at least one therapeutic agent for a disease, the method being To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, Applying at least one therapeutic agent to the co-culture, To determine whether or not at least one change is present in the co-cultured material. Includes, Optionally, (a) The method comprises preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) The method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0015] The present invention further provides a method for testing at least one therapeutic agent for a disease, the method comprising: isolating a conditioned medium from at least one stromal cell and combining the conditioned medium with at least one organoid; applying at least one therapeutic agent to the organoid; determining the presence or absence of at least one change in the organoid; and optionally, (a) the method comprises preparing at least one stromal cell by culturing the stromal cell in a stromal cell medium, and / or (b) the method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0016] The present invention further provides a method for determining the presence or absence of a disease diagnosis and / or prognosis prediction in a subject, the method comprising: combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture; applying at least one diagnostic agent to the co-culture; determining the presence or absence of at least one change in the co-culture; and optionally, (a) the method comprises preparing at least one stromal cell by culturing the stromal cell in a stromal cell medium, and / or (b) the method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0017] The present invention further provides a method for determining the presence or absence of a disease diagnosis and / or prognosis prediction in a subject, the method comprising: isolating a conditioned medium from at least one stromal cell and combining the conditioned medium with at least one organoid; Applying at least one diagnostic agent to at least one organoid, To determine whether or not at least one change has occurred in at least one organoid. Includes, Optionally, (a) The method comprises preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) The method comprises preparing at least one organoid by culturing epithelial cells in an organoid medium.

[0018] The present invention further provides a co-culture product of the method of the present invention.

[0019] The present invention further provides a co-culture medium for the method of the present invention.

[0020] The present invention further provides an interstitial cell culture medium for the method of the present invention. [Brief explanation of the drawing]

[0021] As an example, embodiments of the present invention will be described with reference to the following drawings. [Figure 1] A schematic diagram of the process for preparing co-cultures and inducing pro-inflammatory profiles for analysis is shown, as described in the examples. Abbreviations: "FIB" fibroblasts; "org" organoids. [Figure 2A]Figure 2 shows imaging results of patient-derived organoids ("PDOs") cultured with and without fibroblasts ("Fib") and with and without pro-inflammatory stimuli ("OSM / IL-1b"), as described in Example 4. Figure 2A shows bright-field images. Figures 2B and 2C show the organoid area (μm2) under various conditions. Figure 2D shows bright-field images of different types of PDOs ("A", "B", and "C") with and without fibroblasts, with or without pro-inflammatory stimuli ("+OSM / IL1b"), or without pro-inflammatory stimuli ("Ctr"). Figures 2E and 2F show the organoid area (μm2) of different types of PDOs with and without different types of immortalized fibroblasts ("IM-CoF", "IM-SIF") or primary fibroblasts ("Clone 2", "SIF"). See also Table 1 below. Figures 2G and 2H show the measured area (μm2) of organoids ("Org") with and without a specific ratio ("F / O") of fibroblasts over a long period (up to day 3 (D3) or up to day 6 (D6)) with and without pro-inflammatory stimulation ("OSM + IL1β"). [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 2E] See the explanation in Figure 2A. [Figure 2F] See the explanation in Figure 2A. [Figure 2G] See the explanation in Figure 2A. [Figure 2H] See the explanation in Figure 2A. [Figure 3A]Figure 3 shows the measured secretion results of organoids cultured with and without fibroblasts, and with and without the pro-inflammatory stimulus ("OSM + IL-1β"), as described in Example 5. The following secretions are shown: (A) IL-6; (B) CXCL2; (C) IL-6 from different fibroblast cell lines, and (D) IL-6 from different fibroblast cell lines; (E) CXCL2 from different primary organoids ("A", "B", and "C") and different fibroblast cell lines; and (F) IL-6 and (G) CXCL2 (at different fibroblast-to-organoid ratios ("F / O") and different concentrations of IL-1β and OSM pro-inflammatory stimulus (0 ng / ml, 5 ng / ml, 15 ng / ml)). See also Table 1. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 3D] See the explanation in Figure 3A. [Figure 3E] See the explanation in Figure 3A. [Figure 3F] See the explanation in Figure 3A. [Figure 3G] See the explanation in Figure 3A. [Figure 4A] Figure 4 shows the measured values ​​of (A) caspase activity, (B) caspase activity in different fibroblast cell lines, and (C) caspase activity in different organoids, as described in Example 6. "PDO" = patient-derived organoid; "Org" = organoid; "A", "B", and "C" = specific organoid type; "IM-CoF", "IM-SIF" = specific immortalized fibroblast cell lines; "Clone 2", "SIF" = specific primary fibroblast cell lines. See also Table 1. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A] Figure 5 shows (A) organoid area measurements and (B) caspase activity measurements, as well as the effects of pro-inflammatory stimulation and / or tofacitinib administration on them, as described in Example 7. [Figure 5B]See the explanation in Figure 5A. [Figure 6] As described in Example 8, the images of organoids cultured in fibroblasts ("Fib"), fibroblast-conditioned medium ("Fib-CM"), with or without pro-inflammatory stimuli ("OSM / IL1b"), and in either growth medium ("CNM") or differentiation medium ("cCDM") are shown. [Figure 7A] Figure 7 shows the gene expression measurements of co-cultures when cultured in fibroblast-conditioned medium ("Fib-CM"), with or without pro-inflammatory stimulation ("OSM+IL1β"), and in either growth medium ("CNM") or differentiation medium ("cCDM"), as described in Example 9. (A) Expression of ALPI and MUC2, (B) Expression of LGR5 and KI67, and (C) Expression of KRT20 and OLMF4 are shown. [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 8A] Figure 8 shows imaging results of organoids ("Org") cultured in various media, as described in Example 10, with (A) fibroblasts ("Fib") and (B) fibroblasts ("Fib"), and with and without pro-inflammatory stimuli of OSM (1 ng / ml) and IL-1β (1 ng / ml) ("INF") ("Ctr"). "PD03" = PD0325901. [Figure 8B] See the explanation in Figure 8A. [Figure 9A] Figure 9 shows the measured secretion results of organoids cultured with and without fibroblasts ("Fib") in various media, with or without pro-inflammatory stimulation of OSM (1 ng / ml) and IL-1β (1 ng / ml), as described in Example 10. The following secretions are shown: (A) IL-6. "PD03" = PD0325901, and (B) CXCL2. "PD03" = PD0325901. [Figure 9B] See the explanation in Figure 9A. [Figure 10]As described in Example 10, the caspase activity measurements of organoids cultured in various media, either alone or with fibroblasts ("Fib"), with or without pro-inflammatory stimuli of OSM (1 ng / ml) and IL-1β (1 ng / ml) ("INF") or ("Ctr"), and with or without pro-inflammatory stimuli of TNF (15 ng / ml) and IFNγ (15 ng / ml). "PD03" = PD0325901. [Modes for carrying out the invention]

[0022] Detailed description of the invention definition Unless otherwise specified, the implementation of this invention will utilize conventional methods in chemistry, biochemistry, molecular biology, immunology, and pharmacology within the scope of the art. Such techniques are well described in the literature; see, for example, references [1-7].

[0023] In use herein, “approximately” or “about” are equivalent. Any numbers used herein, with or without “about” or “approximately,” are intended to encompass the normal variation understood by those skilled in the art. When used herein, the terms “approximately” or “about” applied to one or more target values ​​refer to values ​​similar to the stated reference values. In certain embodiments, unless otherwise specified or evident from the context (except when such numbers exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value. Values ​​may also be read as exact values, and therefore the term “about” may be omitted. For example, the term "approximately 100" encompasses the range from 90 to 110, and also includes 100.

[0024] A “co-culture” refers to two or more cell types maintained under conditions favorable for mutual proliferation. In the context of this disclosure, “organoid co-culture” refers to epithelial organoids as defined elsewhere in a culture with non-epithelial cell types, particularly stromal cell types. In some embodiments, the cell types in a co-culture are integrated in that they may exhibit structural, biochemical, and / or phenomenological relationships that they do not exhibit individually. In some embodiments, the cell types in a co-culture mimic structural, biochemical, and / or phenomenological relationships observed between cell types in vivo. In this application, the term “co-culture” may be used to refer to a normal (e.g., non-fibrous, non-inflammatory) co-culture or a disease co-culture. When a co-culture is described as a “disease” co-culture, this means that the co-culture has a disease phenotype, for example, typically because the co-culture is derived from one or more epithelial cells or one or more non-epithelial cells that have a disease phenotype, or, in some embodiments, because the organoids have been exposed to one or more pro-inflammatory stimuli that induce the characteristics of a disease phenotype.

[0025] The phrase "to comprise" and its conjugations are used in their non-restrictive sense to mean that the items following the word are included, but not excluded, even if they are not specifically mentioned. In addition, the verb "to consist of" may be replaced with "to essentially consist of" if necessary, meaning that the products defined herein may include additional components other than those specifically identified, and such additional components do not alter the inherent characteristics of the invention. In addition, the methods defined herein may include additional steps other than those specifically identified, and such additional steps do not alter the inherent characteristics of the invention. Furthermore, references to elements with the indefinite article "a" or "an" do not exclude the possibility of multiple elements being present unless the context clearly requires the presence of only one or one of the elements. Thus, the indefinite article "a" or "an" usually means "at least one."

[0026] Fibroblasts are the primary active cells in connective tissue, characterized by the secretion of collagen and other extracellular matrix proteins. Fibroblasts are one of the most abundant cell types in the stroma. Fibroblasts are a type of stromal cell.

[0027] "Fibrosis" or "fibrous disease" refers to a disorder or condition characterized by excessive, uncontrolled, and / or inappropriate deposition of collagen and other extracellular matrix components. Examples of fibrous diseases include intestinal fibrosis, solitary rectal ulcers, radiation enteropathy, and eosinophilic enteropathy.

[0028] The term "digestive system" includes the gastrointestinal tract as well as the liver, pancreas, and gallbladder.

[0029] The term "gastrointestinal tract" or "GI tract" encompasses the mouth, oral cavity, esophagus, stomach, intestines, rectum, and anus.

[0030] "Inflammation" traditionally refers to a series of biochemical stimuli and cellular responses that result in physiological disorders characterized clinically by heat, pain, redness, and swelling at the site of inflammation. At the biochemical level, inflammation can be mediated by pro-inflammatory and inflammatory stimuli. "Inflammatory stimuli" are agents that elicit an "inflammatory response" in the cells and / or co-cultures on which they act, increasing caspase activity (among other types of cell damage) and ultimately increasing cell death (apoptosis). Examples of inflammatory stimuli include cytokines, such as TNF and IFNγ. Therefore, inflammatory stimuli include "damage-inducing cytokines." "Pro-inflammatory stimuli" are agents that do not directly increase apoptosis but induce a "pro-inflammatory profile" in the cells and / or co-cultures they act on, in which cells and / or co-cultures exhibit altered gene expression (such as upregulated PDPN and / or TGF-β in stromal cells), secretome alterations (including increased secretion of IL-6 and / or CXCL2), increased organoid size and / or altered aggregation, and / or increased sensitivity to inflammatory stimuli, cytokine-mediated damage, and / or increased caspase activity. Examples of pro-inflammatory stimuli include cytokines, such as oncostatin M (OSM) and IL-1β. Pro-inflammatory stimuli can be distinguished from inflammatory stimuli by typically signaling via molecular pathways including STAT, NF-κB, MAPK, JUNK, mTOR, NFAT, and PI3K-AKT, but by not directly inducing increased apoptosis themselves.

[0031] "Inflammatory disorders" refer to diseases in which inflammation is the symptom or cause. Examples of inflammatory disorders include intestinal diseases. In this specification, the terms "disease," "disorder," and "condition" are used interchangeably.

[0032] Inflammatory bowel disease ("IBD") refers to a condition characterized by chronic inflammation of the gastrointestinal tract ("GI"). Symptoms may include abdominal pain, diarrhea, weight loss, and bleeding in the GI tract. Ulcerative colitis ("UC") and Crohn's disease ("CD") are examples of IBD conditions. UC can manifest as persistent inflammation limited to the colon. CD can manifest as discontinuous inflammation affecting the entire GI tract.

[0033] The term "intestine" encompasses the colon and small intestine. The intestine is epithelial tissue and is part of the GI duct and digestive system. Therefore, those skilled in the art will recognize that the methods and uses described herein may be applied to other epithelial tissues, particularly other epithelial tissues of the digestive system.

[0034] “Organoids” refer to cellular structures obtained by the proliferation of adult (post-embryonic) epithelial stem cells, preferably characterized by Lgr5 expression, and consisting of tissue-specific cell types that self-organize through cell sorting and spatially restricted differentiation lineage determination (see, for example, [8], and in particular the section titled “Organoids derived from adult stem cells” starting on page 1590). Organoids may be “patient-derived organoids” (PDOs). In this application, the term “organoid” may be used to refer to normal (e.g., non-fibrous, non-inflammatory) organoids or disease organoids. When an organoid is described as a “disease” organoid, this means that the organoid has a disease phenotype, for example, typically because the organoid is derived from one or more epithelial stem cells that have a disease phenotype, or, in some embodiments, because the organoid has been genetically modified to exhibit specific characteristics of a disease phenotype.

[0035] "Safe" or "tolerable" refers to a treatment for a disease that, based on standard clinical practice, has no side effects or only side effects at tolerable levels.

[0036] "Side effects" or "adverse effects" refer to physiological responses resulting from treatment other than the desired effect.

[0037] The term "small intestine" includes the duodenum, jejunum, and ileum.

[0038] "Interstitium" (sometimes called "mesenchymal tissue" or "intercellular tissue") refers to the supporting structure associated with epithelial organs, glands, or tumors, including connective tissue. Interstitium is distinct from parenchyma, which refers to the part of an organ that performs organ-specific functions. "Interstitial cells" refer to cells found in or that produce interstitium, such as fibroblasts. Interstitium can refer to the gastrointestinal interstitium (particularly the intestinal interstitium, such as the colonic interstitium), the renal interstitium, or the pulmonary interstitium.

[0039] "Subject," "patient," or "individual" are used interchangeably herein and may refer to a human or any non-human animal (e.g., any mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). In preferred embodiments, the subject is a mammal, more preferably a human. "Human" may refer to a prenatal and / or postnatal form. The subject may be a human without disease. The subject may be a human visiting a healthcare provider for the diagnosis or treatment of a disease. The subject may have or be susceptible to a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0040] "To suffer from ~" refers to a person who has been diagnosed with a disease, disorder, and / or condition, or who exhibits one or more of its symptoms.

[0041] "Susceptible to ~" refers to an individual who has not been diagnosed with a disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, condition, or event may be characterized by one or more of the following: (1) a gene mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; and / or (5) having received, planning to receive, or needing a transplant. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0042] A "therapeutic dose" refers to the amount of therapeutic agent that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutic dose is usually administered via a drug regimen containing at least one unit dose.

[0043] "To treat," "to treat," and "treatment" refer to any method used to alleviate, improve, reduce, inhibit, prevent, delay the onset, decrease the severity, and / or decrease the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition, either partially or completely. Treatment may be administered to subjects who do not show signs of the disease, and / or who show only the initial signs of the disease, for the purpose of reducing the risk of developing a disease-related condition. Any reference to a treatment method that involves administering an agent to a subject also includes the agent for use in the treatment method described above, as well as the use of that agent in the treatment method described above, and the use of that agent in the manufacture of a pharmaceutical product.

[0044] overview The present invention relates to co-cultures (or “organoid co-cultures”) formed by combining at least one organoid and at least one stromal cell, methods for preparing them (sometimes referred to as “co-culturing”), and their use in in vitro methods for, for example, testing therapeutic agents for diseases and / or diagnosing and / or predicting the prognosis of diseases. Of particular interest are the use of co-cultures in drug screening, toxicity screening, clinical research, and drug discovery.

[0045] The present invention also provides co-culture products obtained by the method of the present invention.

[0046] While we do not wish to be bound by any theory, we believe that the organoid-stromal cell co-cultures described herein reproduce important in vivo interactions between the stroma and epithelium in healthy and diseased states at the biochemical level (e.g., caspase activity, secretion of soluble mediators such as IL-6 and CXCL2), the cellular level (e.g., apoptosis), and the physiological level (e.g., increased organoid area and changes in aggregation).

[0047] seed Cells (including epithelial cells and stromal cells), organoids, and / or co-cultures suitable for use in the present invention or in the methods of the present invention may primarily originate from any multicellular organism. In some embodiments, the cells, organoids, and / or co-cultures of the present invention are mammalian (i.e., derived from mammals), such as mouse, primate, non-human primate, rat, dog, miniature pig, or human cells, organoids, and / or co-cultures. In preferred embodiments, the cells, organoids, and / or co-cultures of the present invention are human (i.e., derived from humans).

[0048] sample The co-culture of the present invention may contain or consist of autologous cells, i.e., cells obtained from the same subject. For example, the co-culture can be obtained by preparing epithelial cells (e.g., colorectal cells) derived from the tissue of the subject and stromal cells (optionally, derived from the same tissue of the same subject) derived from the same subject, and combining them to form a co-culture. In some embodiments, the epithelial cells and stromal cells may be derived from the same sample from the same subject. The sample may be a tissue biopsy, e.g., a bowel tissue biopsy. The sample may be a healthy sample (e.g., without inflammatory or fibrous disease) or a diseased sample (e.g., with inflammatory or fibrous disease). In some embodiments, the epithelial cells and stromal cells may be obtained from different samples from the same subject.

[0049] Alternatively, the co-culture of the present invention may include, or consist of, non-self cells, i.e., cells obtained from different subjects. For example, a co-culture can be obtained by culturing epithelial cells derived from the tissue of a first subject (e.g., colorectal cells) and stromal cells derived from a second subject (optionally derived from the corresponding tissue of the second subject) and combining them to form a co-culture. The sample may be a tissue biopsy, for example, a bowel biopsy. The sample may be a healthy sample (e.g., without inflammatory or fibrotic disease) or a diseased sample (e.g., with inflammatory or fibrotic disease). The different subjects may have compatible antigens, for example, different patients may be blood relatives, for example, very close relatives.

[0050] In principle, epithelial cells suitable for organoid preparation can be derived from any epithelial tissue. For example, epithelial cells may be derived from the intestines, lungs, kidneys, pancreas, or liver. Particularly preferred are epithelial cells derived from the digestive system, such as the gastrointestinal tract. Most preferred are epithelial cells derived from the intestines. In some embodiments, the epithelial cells are not derived from the lungs, and not from the lungs, kidneys, pancreas, or liver, for example. The tissue from which the epithelial cells are derived may be healthy (e.g., without inflammatory or fibrotic disease) or diseased (e.g., with inflammatory or fibrotic disease).

[0051] Interstitial cells can, in principle, originate from any interstitium. For example, interstitial cells may originate from the lungs, kidneys, or intestines. Particularly preferred are interstitial cells originating from the digestive system, such as the gastrointestinal tract. Most preferred are interstitial cells originating from the intestines. In some embodiments, the interstitial cells do not originate from the lungs, and may not originate from the lungs, kidneys, pancreas, or liver, for example. The tissue from which the interstitial cells originate may be healthy (e.g., non-inflammatory, non-fibrous) or diseased (e.g., having an inflammatory disorder or a fibrous disorder).

[0052] Epithelial cells and / or stromal cells can be obtained during surgery, for example, from normal or diseased mucosa taken from the resected colon, rectum, small intestine, and / or ileum of a subject with or without disease.

[0053] stromal culture The present invention provides at least one stromal cell that can be combined with at least one organoid to form a co-culture. The present invention also provides at least one reference stromal cell that can be combined with at least one organoid to form a reference co-culture. The present invention also provides stromal cells that can be cultured to prepare at least one stromal cell. All stromal cells, including these stromal cells and other stromal cells discussed herein, may be collectively referred to as “stromal cells of the present invention” or “stromal cells,” and any description made to refer to “stromal cells” may include any of these stromal cell types.

[0054] cell type Features and methods relating to at least one stromal cell (combined with at least one organoid to form a co-culture) are described herein. These features and methods are generally applicable to stromal cells that can be cultured to prepare at least one stromal cell. Thus, unless the context indicates otherwise, features, methods and other disclosures herein apply to both at least one stromal cell or to multiple stromal cells from which said at least one stromal cell originates.

[0055] At least one stromal cell (or multiple stromal cells from which said at least one stromal cell originates) can be obtained from established cell lines available in the art (e.g., from a library of ATCC or similar cell lines). Alternatively, the stromal cells may be purified from an impure sample derived from the subject. There are advantages associated with obtaining stromal cells from the same subject as at least one organoid (or epithelial cells from which said at least one organoid originates), because for this reason the resulting co-culture is the most representative (and therefore the most faithful model) of the subject from which the cells originate. This is particularly useful from the perspective of personalized medicine.

[0056] The stromal cells may originate from the same histological type, the same organ, and / or the same specimen as the epithelial cells from which at least one organoid originates.

[0057] In some embodiments, at least one stromal cell is not a T cell, peripheral blood mononuclear cell (PBMC), natural killer (NK) cell, dendritic cell (DC), B cell, macrophage, neutrophil, basophil, eosinophil, monocyte, granulocyte, phagocyte, or mast cell. In some embodiments, at least one stromal cell is not a lymphocyte. In some embodiments, at least one stromal cell is not a leukocyte. In some embodiments, at least one stromal cell does not originate from lymphoid tissue. In some embodiments, at least one stromal cell is not part of any cell lineage that arises in lymphoid tissue. In some embodiments, at least one stromal cell does not originate from hematopoietic tissue. In some embodiments, at least one stromal cell is not part of any cell lineage that arises in hematopoietic tissue.

[0058] The preferred stromal cells are fibroblasts. These fibroblasts may also be intestinal fibroblasts (fibroblasts derived from the intestines), such as colonic fibroblasts or small intestinal fibroblasts.

[0059] Fibroblasts may be immortalized fibroblasts (derived from immortalized cell lines) or primary fibroblasts (such as patient-derived fibroblasts).

[0060] Any of these types of stromal cells could be human stromal cells, such as human fibroblasts.

[0061] Interstitial cell culture medium Stromal cell culture media may be used, for example, to prepare at least one stromal cell for co-culture by promoting the growth and division (proliferation) and / or differentiation of stromal cells to produce at least one stromal cell suitable for co-culture.

[0062] Any medium suitable for culturing stromal cells, such as a stromal cell medium containing fetal bovine serum suitable for culturing fibroblasts, may be used.

[0063] In some embodiments, the stromal cell medium includes a basic medium such as Advanced DMEM / F-12 medium ("Ad-DF") (optionally about 500 ml of Ad-DF).

[0064] In some embodiments, the interstitial cell medium includes an amino acid supplement, such as L-glutamine, or an L-glutamine substitute, preferably an L-glutamine substitute such as GlutaMax (optionally about 2 mM GlutaMax).

[0065] In some embodiments, the interstitial cell medium includes a medium buffer, such as HEPES (optionally about 25 mM HEPES).

[0066] In some embodiments, the interstitial cell medium contains one or more antibiotics, such as penicillin and / or streptomycin (optionally about 100 u / ml of penicillin and / or streptomycin).

[0067] In some embodiments, the interstitial cell medium contains serum, for example fetal bovine serum (FCS), for example, about 10% FCS.

[0068] In some embodiments, the interstitial cell medium comprises a basic medium, an L-glutamine substitute, an antibiotic, and optionally a medium buffer and / or serum.

[0069] In some embodiments, the stromal cell medium comprises Ad-DF, GlutaMax, HEPES, and antibiotics (optionally penicillin and / or streptomycin), and optionally serum (such as FCS). In some embodiments, the stromal cell medium comprises about 500 ml of Ad-DF, about 2 mM GlutaMax, about 25 mM HEPES, about 100 u / ml of penicillin and / or streptomycin, and about 10% FCS.

[0070] Preparation of stromal cells In some embodiments, combining at least one organoid and at least one stromal cell to form a co-culture involves preparing at least one stromal cell, which involves culturing the stromal cell in stromal cell medium.

[0071] Culturing stromal cells may include thawing the stromal cells and adding them to a stromal cell culture medium. Culturing stromal cells may also include incubating the stromal cells, for example, overnight.

[0072] Culturing stromal cells may involve aspirating the stromal cell medium, treating the stromal cells with trypsin, and subsequently resuspending the stromal cells in the stromal cell medium.

[0073] In some embodiments, culturing stromal cells involves culturing them in a container (such as a cell culture dish) coated with poly-L-lysine.

[0074] In some embodiments, culturing stromal cells involves subculturing them before they reach maximum threshold confluence and seeding them beyond minimum threshold confluence. The maximum and minimum threshold confluence values ​​can be determined empirically.

[0075] Single layer In some embodiments, at least one stromal cell is prepared as a monolayer. Monolayer culture generally involves a method comprising digesting or dissociating at least one stromal cell into a single-cell suspension, seeding the suspension onto a semipermeable membrane, and culturing the cell in the presence of growth medium until a monolayer is formed. Specific methods are disclosed in WO2023 / 281122, which is incorporated in its entirety by reference.

[0076] organoid cultures The present invention provides at least one organoid that can be combined with at least one stromal cell to form a co-culture. The present invention also provides a reference organoid that can be combined with at least one stromal cell to form a reference co-culture. These organoids, and other organoids discussed herein, may be collectively referred to as “the organoids of the present invention” or “organoids,” and any description made to refer to “organoids” may include at least one organoid that can be combined with at least one stromal cell to form a co-culture, and any reference organoid.

[0077] Preparation of organoids Organoids can be prepared by culturing epithelial cells in organoid medium as described herein. The organoids of the present invention may be characterized by Lgr5 expression.

[0078] Culturing epithelial cells may involve obtaining epithelial cells from a sample, such as a tissue biopsy.

[0079] Culture of epithelial cells may involve freeing foveals for organoid induction, which may include, for example, mechanical shearing of the tissue and resuspending the foveals in organoid medium.

[0080] In some embodiments, the organoids of the present invention are fragmented (divided) into organoid fragments, for example, in a 1:1 ratio, and then passaged. The fragmentation may occur by mechanical force (called "shearing") or enzymatically (for example, by TrypLE).

[0081] organoid cell type The organoids and / or organoid co-cultures of the present invention can be obtained from epithelial cells and may therefore be described as “epithelial organoids.” Any epithelial cells capable of generating organoids are suitable for use in the present invention. Preferred epithelial cells include intestinal cells, foveolar cells, rectal cells, lung cells, hepatocytes, mammary gland cells, skin cells, pancreatic cells, endocrine cells, exocrine cells, ductal cells, renal cells, adrenal cells, thyroid cells, pituitary cells, parathyroid cells, prostate cells, gastric cells, esophageal cells, ovarian cells, fallopian tube cells, and vaginal cells. Particularly preferred epithelial cells are intestinal cells, such as colorectal cells. The epithelial cells may preferably be epithelial stem cells characterized by Lgr5 expression.

[0082] Suitable specimens for obtaining epithelial cells include tissue biopsies, such as biopsies of resected colon and / or rectum tissue for colorectal epithelial cells, or urine for renal epithelial cells.

[0083] The organoids of the present invention may be gastrointestinal organoids, lung organoids, pancreatic organoids, tracheal organoids, skin organoids, or vaginal organoids—preferably gastrointestinal organoids. Furthermore, the organoids of the present invention may be gastrointestinal organoids, lung organoids, pancreatic organoids, tracheal organoids, skin organoids, endometrial organoids, or kidney organoids—preferably gastrointestinal organoids, lung organoids, or kidney organoids. In some embodiments, the organoids of the present invention are not liver organoids.

[0084] Organoids and / or organoid co-cultures can be obtained from normal epithelial cells or diseased epithelial cells (sometimes specifically referred to as “disease organoids” or “disease co-cultures”). In some embodiments, epithelial cells are obtained from samples derived from subjects with inflammatory diseases. In certain embodiments, diseased epithelial cells and normal epithelial cells are obtained from samples derived from the same subject, and optionally from the same sample.

[0085] Organoids can be obtained by culturing epithelial stem cells. Epithelial stem cells can be obtained from adult tissue; that is, epithelial stem cells are adult epithelial stem cells. In this context, “adult” means mature tissue, i.e., including neonates or children, but excluding embryos or fetuses. Alternatively, epithelial stem cells are not derived from, for example, in vitro differentiated embryonic stem cells or embryonic stem cell lines. Adult tissue can be healthy adult tissue. In other embodiments, adult tissue may be derived from patients with a disease, such as an inflammatory disease.

[0086] Epithelial stem cells may originate from colorectal, small intestine, lung, stomach, pancreas, liver, 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 to obtain organoids from various epithelial tissues have been described in the past (e.g., WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, and WO2016 / 083612, WO2017 / 149025, WO2020 / 234250, and [8]). Cells directly harvested from tissue, i.e., newly isolated cells, are also called "primary cells." In some embodiments, epithelial stem cells are primary epithelial stem cells. In some embodiments, primary epithelial cells are patient-derived epithelial cells.

[0087] In some embodiments, the organoids consist solely of epithelial cells; that is, non-epithelial cells are not present in the organoids. Even if other cell types are transiently present in the culture medium, for example in the tissue fragments used as starting material, these cells are unlikely to survive and are instead replaced by the long-term proliferation of stem cells that produce a population of pure epithelial cells.

[0088] Organoids obtained using a growth medium ("growth medium") may be called "growth organoids." Growth organoids contain at least one epithelial stem cell, which can divide to produce further epithelial stem cells or differentiated offspring. In preferred growing organoids, it will be understood that the majority of cells are growing cells (i.e., dividing cells) that retain an undifferentiated phenotype. While some spontaneous differentiation may occur, the cell population is generally a growing population. The length of time that the organoid can continue to grow while maintaining the presence of an epithelial stem cell core, as well as the integrity of the cell's genotype and phenotype, is a key characteristic of organoids. Organoids typically express Lgr5. Organoids also possess distinctive structures that rapidly develop as the cells grow and self-organize in vitro. These features are described in detail herein.

[0089] In some embodiments, the organoids and / or co-cultures of the present invention (e.g., derived from intestinal organoids) comprise one or more of the following cell types: Lgr5+ stem cells, intestinal cells, goblet cells, Paneth cells, and enteroendocrine cells.

[0090] The organoids of the present invention may include or be derived from organoid fragments. Examples of organoid fragments include any fragment of an organoid, such as an organoid-derived intestinal fovea. In some embodiments, the organoid fragment is a cell aggregate, preferably consisting of fewer than 10, fewer than 5, and preferably 2 to 4 cells. In preferred embodiments, one or more organoids are digested or dissociated to form a suspension containing single cells and cell aggregates.

[0091] Organoid structure In some embodiments, the organoid is a three-dimensional cellular structure. In some embodiments, the organoid includes a lumen surrounded by epithelial cells. In some embodiments, the epithelial cells surrounding the lumen are polarized. Polarization may be disrupted in disease organoids. The original epithelial cells from which the organoid is obtained are preferably primary epithelial cells, such as patient-derived epithelial cells. Preferably, the adult epithelial stem cells are not derived from induced pluripotent stem (iPS) cells.

[0092] In some embodiments, epithelial cells within the organoid surround a lumen. In some embodiments, the organoid does not have a lumen (in particular, disease organoids may not have a lumen). In some embodiments, the epithelial cells are polarized (i.e., proteins are differentially expressed at the apical or basolateral aspects of the epithelial cells). In some embodiments, the lumen is a sealed lumen (i.e., a continuous cell barrier separates the contents of the lumen from the culture medium surrounding the organoid). In some embodiments, the organoid contains stem cells that are actively dividing and preferably can differentiate into all major differentiated cell lineages present in the corresponding in vivo tissue (e.g., when the organoid or cells are transferred to a differentiation medium). In some embodiments, the organoid contains basal cells on the outside and more differentiated cells in the center.

[0093] In terms of the structure of intestinal epithelial cells, the apical surface faces the lumen, modulates interactions with the lumen contents, including mediating nutrient absorption, detects microbial products, and secretes molecules that protect the epithelium from potentially harmful substances in the lumen. The basal lateral surface anchors the epithelial cells to the underlying basement membrane, delivers nutrients from the lumen into the bloodstream, and communicates with nearby cells.[9]

[0094] In some embodiments, organoids include stratified epithelium. “Stratified” means the presence of multiple (more than one) layers of cells. Such cells often tend to have their nuclei closer to the center of their structure, i.e., they are not polarized. Cells within the multilayered portion can organize themselves to contain gaps or lumens between cells.

[0095] In some embodiments, the organoid has a diameter at its widest point of at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 125 μm, at least about 150 μm, at least about 175 μm, at least about 200 μm, at least about 250 μm, or more.

[0096] In some embodiments, the organoid has a diameter at its widest point of approximately 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 250 μm, or more.

[0097] In some embodiments, the organoid has a diameter at its widest point of up to approximately 50 μm, up to approximately 60 μm, up to approximately 70 μm, up to approximately 80 μm, up to approximately 90 μm, up to approximately 100 μm, up to approximately 125 μm, up to approximately 150 μm, up to approximately 175 μm, up to approximately 200 μm, up to approximately 250 μm, or more.

[0098] In the context of this invention, tissue fragments are parts of adult tissue, preferably adult human tissue. In contrast, organoids are distinguished from tissue fragments because they develop structural features through in vitro proliferation.

[0099] Organoid culture medium Organoids for co-culture can be prepared using organoid media, for example, by promoting the growth, division (proliferation), structural organization, or other development of epithelial cells to produce organoids suitable for co-culture. Organoid media may be particularly suitable for tissues derived from the digestive system, such as gastrointestinal tissues like the intestine or colorectal tissue. Organoid media may also be called “proliferation media,” “organoid proliferation media,” or “normal colonic medium” (“CNM”).

[0100] The culture medium may include a basic medium. The basic medium is any suitable basic medium for animal or human cells, such as fully advanced DMEM / F12 medium, subject to any limitations set forth herein.

[0101] The culture medium may contain (a) WNT-acclimatized medium in an optional manner, approximately 50% of the final volume, or (b) a Wnt agonist such as NGS Wnt in an optional amount of approximately 0.5 nM. Particularly preferred is a Wnt-acclimatized medium such as Wnt3a-acclimatized medium (optionally, approximately 10% of the final volume).

[0102] The culture medium may contain R-spongin, for example, (a) R-spongin-1 conditioned medium at an optional rate of about 20% of the final volume, or (b) Rspo3 at about 250 ng / ml.

[0103] The culture medium may contain a BMP inhibitor, such as nogging, for example, (a) nogging-conditioned medium ("NCM") (optionally in amounts of about 1-4% of the final volume), or (b) recombinant nogging (optionally in amounts of about 100 ng / ml). Particularly preferred is nogging-conditioned medium ("NCM") (optionally in amounts of about 1-4% of the final volume).

[0104] The culture medium may contain B27, optionally about 1× of B27, based on the manufacturer's instructions.

[0105] The culture medium may contain N-acetylcysteine ​​("N-Ac") at, for example, about 1.25 mM.

[0106] The culture medium may contain, for example, about 10 mM nicotinamide.

[0107] The culture medium may optionally contain mitotic growth factors such as EGF at approximately 50 ng / mL.

[0108] The culture medium may optionally contain gastrin at approximately 10 mM or approximately 5 mM.

[0109] The culture medium may optionally contain a TGF-β inhibitor such as A-83-01 at approximately 500 nM.

[0110] The culture medium may optionally contain a p38 MAPK inhibitor, such as SB202190, at a concentration of approximately 3 μM or 10 μM.

[0111] The culture medium may contain a prostaglandin pathway activator, which may optionally be a prostaglandin such as prostaglandin E2 at a concentration of approximately 10 nM.

[0112] The culture medium may optionally contain antibiotics such as primosin at approximately 100 mg / mL or approximately 50 μg / mL.

[0113] Therefore, the culture medium may contain mitotic growth factor, a BMP inhibitor, and R-spondin. In some embodiments, the culture medium contains EGF, noggin, and R-spondin. In some embodiments, the culture medium contains about 50 ng / mL of EGF, about 1-4% of the final volume of noggin-conditioned medium, and about 20% of the final volume of R-spondin-1-conditioned medium. Such a culture medium may include a basic medium.

[0114] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-spondin. In some embodiments, the medium contains a Wnt agonist, EGF, noggin, and R-spondin. In some embodiments, the medium contains about 50% WNT3A conditioned medium by final volume, about 50 ng / mL EGF, about 1-4% noggin conditioned medium by final volume, and about 20% R-spondin-1 conditioned medium by final volume. Such a medium may include a basic medium.

[0115] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-spondin. In some embodiments, the medium contains a Wnt agonist, EGF, noggin, and R-spondin. In some embodiments, the medium contains about 50% WNT3A conditioned medium by final volume, about 50 ng / mL EGF, about 1-4% noggin conditioned medium by final volume, and about 20% R-spondin-1 conditioned medium by final volume. Such a medium may include a basic medium.

[0116] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, R-spongin, and a TGF-β inhibitor. In some embodiments, the medium contains a Wnt agonist, EGF, noggin, R-spongin, and A-83-01. In some embodiments, the medium contains about 50% WNT3A conditioned medium by final volume, about 50 ng / mL of EGF, about 1-4% of noggin conditioned medium by final volume, about 20% of R-spongin-1 conditioned medium by final volume, and about 500 nM of A-83-01. Such a medium may include a basic medium.

[0117] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, R-spongin, and a p38 MAPK inhibitor. In some embodiments, the medium contains a Wnt agonist, EGF, noggin, R-spongin, and SB202190. In some embodiments, the medium contains about 50% WNT3A conditioned medium by final volume, about 50 ng / mL of EGF, about 1-4% of noggin conditioned medium by final volume, about 20% of R-spongin-1 conditioned medium by final volume, and about 3 μM of SB202190. Such a medium may include a basic medium.

[0118] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, R-spongin, a TGF-β inhibitor, and a p38 MAPK inhibitor. In some embodiments, the medium contains a Wnt agonist, EGF, noggin, R-spongin, A-83-01, and SB202190. In some embodiments, the medium contains about 50% WNT3A conditioned medium by final volume, about 50 ng / mL of EGF, about 1-4% noggin conditioned medium by final volume, about 20% R-spongin-1 conditioned medium by final volume, about 500 nM of A-83-01, and about 3 μM of SB202190. Such a medium may include a basic medium.

[0119] B27 and N-acetylcysteine ​​are optional components of the culture medium. Therefore, the culture medium may include a basic medium, a Wnt agonist, R-spongin, noggin, B27, N-acetylcysteine, nicotinamide, mitotic growth factor, gastrin, a TGF-β inhibitor, a p38 MAPK inhibitor, a prostaglandin pathway activator, and primosine. In some embodiments, the basic medium is fully advanced DMEM / F12 medium, the Wnt agonist is WNT3A conditioned medium, the R-spongin is R-spongin-1 conditioned medium, the noggin is noggin conditioned medium, the mitotic growth factor is EGF, the TGF-β inhibitor is A-83-01, the p38 MAPK inhibitor is SB202190, and the prostaglandin pathway activator is prostaglandin E2.

[0120] The culture medium may contain approximately 50% WNT3A conditioned medium by final volume, approximately 20% R-spongin-1 conditioned medium by final volume, approximately 1-4% Noggin conditioned medium by final volume, and, according to the manufacturer's instructions, approximately 1× B27, approximately 1.25 mM N-acetylcysteine, approximately 10 mM nicotinamide, approximately 50 ng / mL EGF, approximately 10 mM gastrin, approximately 500 nM A-83-01, approximately 3 μM SB202190, approximately 10 nM prostaglandin E2, and approximately 100 mg / mL primosine.

[0121] More generally, those skilled in the art will recognize that some components of organoid media (such as B27 and N-acetylcysteine) are optional and / or may be replaced with suitable alternative components. Organoid media suitable for different tissues are known in the art (e.g., [8]). The medium may be any growth medium suitable for epithelial stem cells or progenitor cells, preferably a growth medium suitable for epithelial stem cells. Culture media suitable for culturing organoids are also described in WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613, WO2016 / 083612, WO2017 / 149025, and WO2020 / 234250. The culture media mentioned in these documents are incorporated herein by reference, and any of them may be used in connection with the present invention.

[0122] In some embodiments, the culture medium comprises a receptor tyrosine kinase ligand, a BMP inhibitor, and a Wnt agonist.

[0123] For example, in some embodiments, the culture medium includes EGF, noggin, and Wnt-acclimatized medium. In some embodiments, the culture medium includes EGF, noggin, Rspondin, and a Wnt substitute.

[0124] In some embodiments, the culture medium further comprises nicotinamide and a p38 inhibitor such as SB202190. In some embodiments, the culture medium further comprises a TGF-beta inhibitor.

[0125] For example, a preferred organoid medium comprises a Wnt agonist (e.g., one of R-spongin 1-4), mitotic growth factors (e.g., selected from EGF, FGF, HGF, and BDNF), and a BMP inhibitor (e.g., Noggin) (as described, for example, in WO2010 / 090513). In some embodiments, the organoid medium further comprises a TGF-beta inhibitor (e.g., A83-01, Tocris) (as described, for example, in WO2012 / 168930). The addition of a TGF-beta inhibitor is particularly suitable for culturing human cells. The TGF-beta inhibitor preferably inhibits the ALK4 / 5 / 7 signaling pathway.

[0126] A preferred organoid medium particularly suitable for culturing intestinal or colonic organoids comprises one or more (preferably all) of the following: a basic medium (e.g., advanced DMEM / F12 medium, Gibco), a Wnt ligand (e.g., Wnt-3a), a Wnt agonist (e.g., one of Rspondin1-4), a BMP inhibitor (e.g., Noggin), EGF, and a TGF-β inhibitor (e.g., A83-01, Tocris), and optionally further comprising one or more (or all) of the following: a p38 MAPK inhibitor, gastrin, nicotinamide, prostaglandin E2, N-acetylcysteine, B27, and / or an antimicrobial agent (e.g., primosine).

[0127] Specific organoid culture media In a preferred embodiment, the organoid medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml), (ii) noggin (e.g., at a concentration of about 100 ng / ml), (iii) Rspondin (e.g., at a concentration of about 250 ng / ml), (iv) Wnt substitute (e.g., NGS-Wnt at a concentration of about 0.5 nM), (v) p38 inhibitor (e.g., SB-203580 at a concentration of about 10 μM), (vi) TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), and (vii) nicotinamide (e.g., at a concentration of about 10 mM).

[0128] In another preferred embodiment, the organoid medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml), (ii) noggin (e.g., at a concentration of about 100 ng / ml), (iii) Wnt acclimatized medium (e.g., about 50% of the final volume), (iv) a p38 inhibitor (e.g., SB-203580 at a concentration of about 10 μM), (v) a TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), and (vi) nicotinamide (e.g., at a concentration of about 10 mM).

[0129] In some embodiments, particularly when the organoids are derived from the lungs, the organoid medium comprises one or more receptor tyrosine ligands, a Wnt agonist, a TGF-beta inhibitor, and a BMP inhibitor. In some embodiments, the organoid medium comprises FGF, Rspondin, a TGF-beta inhibitor, a BMP inhibitor, a Rho-kinase inhibitor, and a p38 inhibitor. In a preferred embodiment, the organoid medium comprises i) FGF (e.g., FGF-7 at a concentration of about 25 ng / ml and FGF-10 at a concentration of about 100 ng / ml), (ii) Rspondin (e.g., Rspondin-3 at a concentration of about 250 ng / ml), (iii) TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), (iv) BMP inhibitor (e.g., Noggin-Fc fusion protein conditioned medium at about 2% of the final volume), (v) Rho-kinase inhibitor (e.g., Y-27632 at a concentration of about 10 μM), and (vi) p38 kinase inhibitor (e.g., SB202190 at a concentration of about 500 nM).

[0130] In some embodiments, particularly when the organoids are derived from kidneys, the organoid medium comprises one or more receptor tyrosine ligands, a Wnt agonist, and a TGF-beta inhibitor. In some embodiments, the organoid medium comprises EGF, FGF, Rspondin, a TGF-beta inhibitor, and a Rho-kinase inhibitor. In preferred embodiments, the organoid medium comprises i) EGF (e.g., at a concentration of about 50 ng / ml), (ii) FGF (e.g., FGF-10 at a concentration of about 100 ng / ml), (iii) Rspondin (e.g., Rspo1-conditioned medium at about 10% of the final volume), (iv) a TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), and (v) a Rho-kinase inhibitor (e.g., Y-27632 at a concentration of about 10 μM).

[0131] Organoid culture period In some embodiments, epithelial cells are cultured in organoid medium for at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days to prepare at least one organoid. Preferably, epithelial cells are cultured in organoid medium for at least about 24 hours to prepare at least one organoid.

[0132] In some embodiments, at least one organoid of the present invention may be cultured or can be cultured in organoid medium for at least about two months, for example, at least about ten weeks, at least about twelve weeks, at least about fourteen weeks, at least about sixteen weeks, at least about four months, at least about five months, at least about six months, at least about nine months, or at least about one year.

[0133] In some embodiments, at least one organoid is cultured or can be cultured for at least about 5 passages, at least about 10 passages, at least about 15 passages, or at least about 20 passages, preferably at least about 10 passages.

[0134] In some embodiments, the number of organoid cells increases exponentially over approximately 5 passages, approximately 10 passages, approximately 15 passages, or approximately 20 passages, preferably over approximately 5 passages.

[0135] In preferred embodiments, organoids for use in the claimed method may be cultured for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or longer. In some embodiments, the organoids are grown or maintained in culture for at least about 3 months, preferably at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 9 months, or at least about 12 months, or longer.

[0136] Single layer In some embodiments, the organoid is prepared as a monolayer. In some embodiments, the organoid includes a single monolayer folded (or invaginated) to form two or more layers. It may be difficult to distinguish between the folded (or invaginated) monolayer and the lamellar cell region. In some embodiments, the organoid includes both the lamellar cell region and the folded monolayer region. In some embodiments, the organoid has a portion formed from multiple layers and a portion containing a single monolayer of cells. In some embodiments, the organoid includes or consists of a single monolayer of cells. In some embodiments, the organoid does not include a monolayer. The organoid may have at least one budding and a layer of cells having a central lumen.

[0137] Monolayer culture of organoids generally involves a method comprising digesting or dissociating one or more organoids into a suspension of single cells and / or organoid fragments, seeding the suspension onto a semipermeable membrane, and culturing the cells and / or organoid fragments in the presence of a culture medium until a monolayer is formed. Specific methods are disclosed in WO2023 / 281122, which is incorporated in its entirety by reference.

[0138] In particular, the following examples from WO2023 / 281122 have been incorporated by reference, and these are especially relevant to intestinal organoids. Example 1 describes the preparation of an epithelial monolayer from a normal human intestinal organoid (for example, the intestinal organoid of the present invention). Example 2 describes the establishment, differentiation, and characterization of the epithelial monolayer of human GI tubules. Example 3 describes the development of an in vitro biological system (for example, usable in the context of the present invention and IBD) that mimics components of IBD pathophysiology and has a robust readout for barrier function pathways. Example 4 describes the verification of the robustness of a barrier function assay using a monolayer derived from intestinal organoids. Example 5 describes the establishment of a human GI tubule organoid epithelial monolayer. Example 6 illustrates the polarization of the human GI tubule organoid epithelial monolayer.

[0139] co-culture The present invention provides the co-culture described herein, and uses the co-culture described herein in the method of the present invention.

[0140] combination The method of the present invention may include combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture product.

[0141] The methods of the present invention, for example, a method for determining the presence or absence of at least one change in a co-culture (including a method for testing one or more therapeutic agents, and a method for determining the presence or absence of a diagnosis and / or prognosis prediction), may be performed on a co-culture that has already been prepared, i.e., the step of combining at least one organoid with at least one stromal cell is not an essential step in all of the methods of the present invention.

[0142] In a preferred embodiment, organoid medium (optionally including any extracellular matrix such as basement membrane matrix "BME" or Matrigel) is removed from at least one organoid, and then at least one organoid is combined with at least one stromal cell. Removal may be carried out using a protease such as dispase. The extracellular matrix may be destroyed using a commercially available kit such as Cell Recovery Solution® (Corning). An alternative matrix such as collagen may be used in place of the removed matrix.

[0143] In some embodiments, at least one organoid and at least one stromal cell are combined using a multi-drop dispenser.

[0144] In some embodiments, at least one stromal cell and at least one organoid are combined in an equal ratio, i.e., one stromal cell per organoid ("1:1"). In some embodiments, at least one stromal cell is combined with at least one organoid in a ratio of about 0.5. In some embodiments, at least one stromal cell is combined with at least one organoid in a ratio of about 2. In some embodiments, at least one stromal cell is combined with at least one organoid in a ratio of about 2.5. A higher fibroblast / organoid ratio may result in an increased pro-inflammatory profile (e.g., increased secretion of IL-6 and CXCL2) and / or an increased inflammatory response.

[0145] In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of at least about 0.5. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of at least about 1:1. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of at least about 2. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of at least about 2.5.

[0146] In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of up to about 0.5. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of up to about 1:1. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of up to about 2. In some embodiments, at least one stromal cell is paired with at least one organoid in a ratio of up to about 2.5.

[0147] In some embodiments, combining at least one organoid with at least one stromal cell involves seeding the at least one organoid and the at least one stromal cell in a co-culture medium. In some embodiments, the at least one organoid and the at least one stromal cell are seeded simultaneously.

[0148] In some embodiments, combining at least one organoid with at least one stromal cell includes the hanging drop method.

[0149] In some embodiments, the combination of at least one organoid with at least one stromal cell includes a multiwell plate format, where the multiwell plate optionally includes an ultra-low adhesion plate.

[0150] In some embodiments, at least one organoid may be prepared from epithelial cells at the same time as combining epithelial cells with at least one stromal cell, so that co-culture and organoid formation occur together.

[0151] Co-culture medium The present invention provides a culture medium for co-culturing organoids and stromal cells (for example, a medium as described in the examples). The co-culture medium may also be described as a “differentiation medium” or “combined-colon differentiation medium” (“cCDM”). The co-culture medium may also be described as an “enterocyte-colon differentiation medium” (“eCDM”).

[0152] The co-culture medium of the present invention advantageously enables the co-culture of stromal cells and organoids. Such co-culture would be difficult or even impossible without the medium adaptation used in the co-culture medium of the present invention. The inventors have for the first time observed that the function of stromal cells is preserved in co-cultures with organoids according to the present invention.

[0153] For example, while not wishing to be bound by any theory, the co-culture medium was selected to identify organoid responses to factors released by stromal cells in the form of one or more changes in the co-culture. Stromal cells are known to secrete ligands that support the presence of stem cells and thus potentially prevent differentiation. The inventors theorized that such responses may be determined based on morphological differences between differentiated organoids (small lumen and thick epithelium) and undifferentiated organoids (large lumen and thin epithelium). The selected co-culture medium also contains an ERK inhibitor (PD0325901), which the inventors theorized to enhance the observability of the co-culture's sensitivity to cytokine damage. The method of the present invention reveals active communication between organoids and stroma. Surprisingly, the inclusion of serum (such as fetal calf serum (FCS), also known as fetal bovine serum (FBS)) in the co-culture medium was found to support the long-term culture of fibroblasts and organoids without disrupting organoid structure.

[0154] The co-culture medium may be particularly suitable for tissues derived from the digestive system, such as gastrointestinal tissues like the intestine or colorectal tissue, and stromal tissue.

[0155] To form a co-culture, at least one organoid and at least one stromal cell can be combined in cCDM.

[0156] In some embodiments, the culture medium includes a basic medium. The basic medium is any suitable basic medium for animal or human cells, such as fully advanced DMEM / F12 medium, subject to any limitations set forth herein.

[0157] In some embodiments, the culture medium optionally contains N-acetylcysteine ​​("N-Ac") at approximately 1.25 mM.

[0158] In some embodiments, the culture medium contains a TGF-β inhibitor, optionally A83-01, optionally at about 500 nM.

[0159] In some embodiments, the culture medium optionally contains B27 at approximately 1×, based on the manufacturer's instructions.

[0160] In some embodiments, the culture medium optionally contains a mitotic growth factor such as EGF at approximately 50 ng / mL.

[0161] In some embodiments, the culture medium optionally contains gastrin at approximately 5 nM.

[0162] In some embodiments, the culture medium contains a BMP inhibitor, for example, nogging, e.g., (a) nogging-conditioned medium ("NCM") (optionally in amounts of about 1-2% of the final volume), or (b) recombinant nogging (optionally in amounts of about 100 ng / ml). Particularly preferred is nogging-conditioned medium ("NCM") (optionally in amounts of about 1-2% of the final volume).

[0163] In some embodiments, the culture medium optionally contains an antibiotic such as primosin at approximately 50 μg / mL.

[0164] In some embodiments, the culture medium optionally contains R-spongin, for example Rspo3, at approximately 250 ng / mL.

[0165] In some embodiments, the culture medium contains a Notch pathway inhibitor. In some embodiments, the culture medium optionally contains DAPT at about 10 μM. In some embodiments, the culture medium does not contain DAPT; for example, the culture medium does not contain a Notch pathway inhibitor.

[0166] In some embodiments, the culture medium contains an ERK inhibitor. In some embodiments, the culture medium optionally contains PD0325901 at approximately 100 nM. While we do not wish to be bound by any theory, we believe that organoids co-cultured in a medium containing an ERK inhibitor such as PD0325901 exhibit structures and lumen sizes that more strongly reflect interactions with stromal cells, and more clearly reveal the effects of inflammatory stimuli (such as TNF) on the co-culture. In some embodiments, the culture medium does not contain PD0325901; for example, the culture medium does not contain an ERK inhibitor.

[0167] In some embodiments, the culture medium contains a Wnt agonist, such as (a) Wnt-conditioned medium (optionally about 10% of the final volume) or NGS Wnt (optionally about 0.1 nM). Particularly preferred is a Wnt-conditioned medium such as Wnt3a-conditioned medium (optionally about 10% of the final volume). In some embodiments, the culture medium does not contain Wnt-conditioned medium or NGS Wnt; for example, the culture medium does not contain a Wnt agonist.

[0168] In some embodiments, the culture medium optionally contains serum, such as fetal bovine serum (FBS), at about 5% or 10% of the final volume, preferably at about 5% of the final volume. In some embodiments, the culture medium does not contain FBS; for example, the culture medium does not contain serum.

[0169] In some embodiments, the culture medium contains ECM. In some embodiments, the culture medium contains about 0.1% to about 40% ECM. In some embodiments, the culture medium contains about 1% to about 20% ECM. In some embodiments, the culture medium contains about 2% to about 10% ECM. In some embodiments, the culture medium contains about 5% ECM. In some embodiments, the ECM is Matrigel (optionally about 5%). In some embodiments, the culture medium does not contain Matrigel; for example, the culture medium does not contain ECM.

[0170] B27 and N-acetylcysteine ​​are optional components of the culture medium.

[0171] In some embodiments, the culture medium comprises N-Ac, a TGF-β inhibitor, B27, mitotic growth factor, gastrin, a BMP inhibitor, an antibiotic, R-spondin, a Notch pathway inhibitor, an ERK inhibitor, and a Wnt agonist. In some embodiments, the TGF inhibitor is A83-01, the mitotic growth factor is EGF, the R-spondin is Rspo3, the BMP inhibitor is Noggin-conditioned medium, the antibiotic is Primosin, the Notch pathway inhibitor is DAPT, the ERK inhibitor is PD0325901, and / or the Wnt agonist is Wnt-conditioned medium or NGS Wnt. In some embodiments, the culture medium further comprises FBS and ECM.

[0172] In some embodiments, the culture medium comprises N-Ac, A83-01, B27, EGF, gastrin, Noggin-conditioned medium, primosin, R-spongin, DAPT, PD0325901, and a Wnt agonist. In some embodiments, the culture medium further comprises FBS and ECM.

[0173] In some embodiments, the medium comprises 1.25 mM N-Ac, 500 nM A83-01, about 1 × B27 as indicated by the manufacturer, about 50 ng / mL EGF, about 5 nM gastrin, about 1–2% Noggin-conditioned medium, about 50 μg / mL Primosin, about 250 ng / mL Rspo3, about 10 μM DAPT, about 100 nM PD0325901, and about 10% of the final volume of Wnt3a-conditioned medium, or about 0.1 nM NGS Wnt. In some embodiments, the medium further comprises about 5% of the final volume of FBS and about 5% of the final volume of Matrigel.

[0174] Either the stromal cell medium or organoid medium described herein can be used as a co-culture medium. In some embodiments, organoid medium is used as a co-culture medium, for example, CNM is used as a co-culture medium.

[0175] The organoid media described herein can be used as a co-culture medium by selectively reducing the concentration of noggin by about 50% by reducing the concentration of a BMP inhibitor such as noggin, preferably by reducing the noggin-conditioned medium to about 1-2% of the final volume.

[0176] The organoid media described herein can be used as co-culture media by reducing the concentration of Wnt agonists, preferably by selectively reducing the concentration of Wnt agonists by about 80%, by reducing the Wnt-conditioned medium to about 10% of the final volume, or by reducing the concentration of NGS Wnt to about 0.1 nM. Particularly preferred is reducing the Wnt-conditioned medium, for example, by reducing the Wnt3a-conditioned medium (selectively to about 10% of the final volume).

[0177] The organoid media described herein can be used as co-culture media by adding a Notch pathway inhibitor such as DAPT to the medium, for example, by adding DAPT to a concentration of about 10 μM.

[0178] The organoid medium described herein can be used as a co-culture medium by adding an ERK inhibitor such as PD0325901 to the medium, for example, by adding PD0325901 to a concentration of about 100 nM.

[0179] The organoid media described herein can be used as co-culture media by adding serum such as FCS to the medium, for example, by adding FCS up to about 5% of the final volume.

[0180] The organoid media described herein can be used as a co-culture medium by adding an ECM such as Matrigel to the medium, for example, by adding Matrigel to a concentration of about 5% of the final volume.

[0181] More generally, those skilled in the art will recognize that some components of the differentiation medium (such as B27 and N-acetylcysteine) are optional and / or may be replaced with suitable alternative components. The differentiation medium may be any suitable differentiation medium for organoids and stromal cells, such as those described in WO2015 / 173425, WO2017 / 149025, and WO2017 / 220586.

[0182] In some embodiments, the differentiation medium suitable for co-culture may contain one or more of the following: a Wnt agonist, a BMP inhibitor, mitotic growth factor, and a TGF-beta inhibitor. For example, the differentiation medium may contain a Wnt agonist. The differentiation medium may further contain mitotic growth factor and / or a BMP inhibitor.

[0183] In preferred embodiments, the differentiation medium comprises a Wnt agonist, a BMP inhibitor, mitotic growth factor, and a TGF-beta inhibitor. The differentiation medium may further comprise one or more of the following: a p38 inhibitor, a cAMP agonist, a prostaglandin pathway activator, nicotinamide, gastrin, B27, and N-acetylcysteine.

[0184] In some embodiments, the differentiation medium comprises a basic medium for human or animal cells (e.g., DMEM / F12 containing B27 or Ad-DF+++ (Advanced Dulbecco's Modified Eagle / F12 medium supplemented with GultaMax, 1 M HEPES)), R-spongin family proteins, mitotic growth factor (EGF, etc.), BMP inhibitors (Noggin, etc.), TGF-beta inhibitors (A83-01, etc.), p38 inhibitors (SB202190, etc.), and optionally nicotinamide and N-acetylcysteine.

[0185] In some embodiments, the differentiation medium comprises advanced DMEM / F12 medium containing B27, nicotinamide, N-acetylcysteine, noggin, R-spongin 1-4, EGF, Wnt (either WNT-conditioned medium (50%, produced using stably transfected L cells) or NGS Wnt), TGF-β type I receptor inhibitor A83-01, and P38 inhibitor SB202190.

[0186] The co-culture medium may contain an extracellular matrix (ECM). Preferred ECMs include BME and Matrigel. These may constitute at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% (v / v) of the co-culture. Preferably, the ECM is Matrigel, constituting about 1% to about 10% (v / v) of the co-culture, for example, about 5% to about 10% (v / v), preferably about 5% (v / v) of the co-culture. In some embodiments, the co-culture medium does not contain an ECM.

[0187] In other embodiments, the co-culture medium may be described as “enterocyte-colon differentiation medium” (“eCDM”). Such a medium may contain a Wnt pathway inhibitor, which may be a porcupine (PORCN) inhibitor. Thus, such a medium may contain a PORCN inhibitor, for example, iWP-2, for example, 1.5 μM iWP-2.

[0188] eCDM may include a TGF-β inhibitor, mitotic growth factor, and BMP inhibitor. For example, the culture medium may include A-83-01, EGF, and noggin-conditioned medium, for example, 500 nM A-83-01, 50 ng / ml EGF, and 1-2% noggin-conditioned medium.

[0189] The culture medium may further contain R-spongin, such as Rspo3 (e.g., 250 ng / ml of Rspo3).

[0190] The culture medium may further contain antibiotics such as N-acetylcysteine ​​(e.g., 1.25 mM N-acetylcysteine), B27 supplement (e.g., 1 × B27 as directed by the manufacturer), gastrin (e.g., 5 nM gastrin), and / or primosine (e.g., 50 μg / ml primosine).

[0191] The culture medium may further contain serum such as FBS (e.g., 5% FBS) and / or ECM such as Matrigel (e.g., 5% Matrigel).

[0192] The culture medium may further contain serum such as FBS (e.g., 5% FBS) and / or ECM such as Matrigel (e.g., 5% Matrigel).

[0193] Therefore, in some embodiments, the co-culture medium comprises a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, R-spongin, serum, and ECM, and optionally one or more of N-acetylcysteine, B27 supplement, gastrin, and antibiotics.

[0194] In some embodiments, the co-culture medium comprises A-83-01, EGF, Noggin-conditioned medium, R-spongin, serum, and ECM, and optionally one or more of N-acetylcysteine, B27 supplement, gastrin, and primosine.

[0195] In some embodiments, the co-culture medium comprises 500 nM A-83-01, 50 ng / ml EGF, 1-2% Noggin-conditioned medium, 250 ng / ml Rspo3, 5% FBS, and 5% Matrigel, as well as optionally 1.25 mM N-acetylcysteine, 1× B27 supplement (as directed by the manufacturer), 5 nM gastrin, and 50 μg / ml primosine.

[0196] In some embodiments, the co-culture medium includes eCDM. eCDM may include a Wnt pathway inhibitor. In some embodiments, eCDM includes a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, and a Wnt pathway inhibitor. In some embodiments, eCDM includes a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, Wnt pathway inhibitor, and R-spondin. In some embodiments, the eCDM medium may include a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, Wnt pathway inhibitor, R-spondin, N-acetylcysteine, B27 supplement, gastrin, and an antibiotic.

[0197] In some embodiments, the co-culture medium includes eCDM. eCDM may include Wnt pathway inhibitors, such as PORCN inhibitors. In some embodiments, eCDM includes a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, and PORCN inhibitor. In some embodiments, eCDM includes a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, PORCN inhibitor, and R-spondin. In some embodiments, the eCDM medium may include a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, PORCN inhibitor, R-spondin, N-acetylcysteine, B27 supplement, gastrin, and antibiotics.

[0198] In some embodiments, the eCDM medium comprises A-83-01, EGF, Noggin-conditioned medium, and iWP2. In some embodiments, the eCDM medium may comprise A-83-01, EGF, Noggin-conditioned medium, iWP2, and Rspo3. In some embodiments, the eCDM medium may comprise A-83-01, EGF, Noggin-conditioned medium, iWP2, Rspo3, N-acetylcysteine, B27 supplement, gastrin, and primosine.

[0199] In some embodiments, the eCDM medium contains about 500 nM A-83-01, about 50 ng / ml EGF, about 1-2% noggin-conditioned medium, and about 1.5 μM iWP2. In some embodiments, the eCDM medium may contain about 500 nM A-83-01, about 50 ng / ml EGF, about 1-2% noggin-conditioned medium, about 1.5 μM iWP2, and about 250 ng / ml Rspo3. In some embodiments, the eCDM medium may contain about 500 nM A-83-01, about 50 ng / ml EGF, about 1-2% Nogging-conditioned medium, about 1.5 μM iWP2, about 250 ng / ml Rspo3, about 1.25 mM N-acetylcysteine, about 1 × B27 (based on manufacturer's instructions), about 5 nM gastrin, and about 50 μg / ml primosine.

[0200] Co-culture period In some embodiments, the co-culture of the present invention may be cultured or can be cultured for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, or at least about 144 hours. Preferably, the co-culture may be cultured or can be cultured for at least about 72 hours. More preferably, the co-culture may be cultured or can be cultured for at least about 144 hours.

[0201] In some embodiments, the co-culture of the present invention is cultured or can be cultured for at least about two months, for example, at least about ten weeks, at least about twelve weeks, at least about fourteen weeks, at least about sixteen weeks, at least about four months, at least about five months, at least about six months, at least about nine months, or at least about one year.

[0202] In some embodiments, the co-culture is cultured or can be cultured for at least about 5 passages, at least about 10 passages, at least about 15 passages, or at least about 20 passages, preferably at least about 10 passages.

[0203] In some embodiments, the cell count in the coculture increases exponentially over approximately 5 passages, 10 passages, 15 passages, or 20 passages, preferably over approximately 5 passages.

[0204] In preferred embodiments, the co-culture may be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months or longer. In some embodiments, the co-culture may be grown or maintained in culture for at least about 3 months, preferably at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 9 months, or at least about 12 months, or longer.

[0205] Single layer In some embodiments, the co-culture is prepared as a monolayer. Monolayer culture generally involves digesting or dissociating one or more organoids and stromal cells into a suspension of single cells and / or organoid fragments, seeding the suspension onto a semipermeable membrane, and culturing the cells and / or organoid fragments in the presence of differentiation medium until a monolayer is formed. Specific methods are disclosed in WO2023 / 281122, which is incorporated in whole by reference.

[0206] Components of organoid culture medium and co-culture medium Organoid media may contain one or more of the components listed in the following subsections. Co-culture media may contain one or more of the components listed in the following subsections. Therefore, the following subsections apply to both organoid media and co-culture media unless otherwise specified.

[0207] Basic culture medium Basic culture media for cell culture typically contain numerous components necessary to support the maintenance of cultured cells. Appropriate combinations of components can be readily formulated by those skilled in the art, given the following disclosures. The basic media for use in the present invention generally comprises a nutrient solution containing standard cell culture components such as amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers, as described in the literature and in more detail below. In some embodiments, the medium is further supplemented with one or more standard cell culture components selected from, for example, amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. Suitable basic media are known to those skilled in the art and are commercially available, and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Advanced-DMEM, Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Eagle Basic Medium (BME), DMEM / Ham F12, Advanced DMEM / Ham F12, Iskov Modified Dulbecco's Medium and Minimum Essential Medium (MEM), Ham F10, Ham F12, Medium 199, and RPMI 1640 Medium. For example, the basic medium may preferably be Advanced-DMEM supplemented with glutamax, penicillin / streptomycin, and HEPES.

[0208] Extracellular matrix (ECM) Epithelial stem cells typically grow in culture with an exogenous extracellular matrix (ECM) known to support cell growth (see, for example,

[10] ). Organoid media may contain ECM. The ECM may be exogenous ECM (meaning it is added to any extracellular matrix proteins spontaneously secreted by epithelial stem cells or populations of epithelial stem cells when in contact with the growth medium of the present invention). Any suitable ECM may be used. Cells are preferably cultured in a microenvironment, which at least partially mimics the cellular niche in which the cells naturally reside. The cellular niche is determined in part by the cells and by the ECM secreted by the cells in the niche. The cellular niche can be mimicked by culturing the cells in the presence of biomaterials or synthetic materials that enable interaction with cell membrane proteins such as integrins. Thus, the ECM described herein is any biomaterial or synthetic material or combination thereof that mimics the in vivo cellular niche by interacting with cell membrane proteins such as integrins.

[0209] In some embodiments, the ECM is in a suspension state, i.e., the cells are in contact with the ECM in a suspension system. In some embodiments, the ECM is in a suspension at a concentration of at least 1%, at least 2%, or at least 3%. In some embodiments, the ECM is in a suspension at a concentration of 1% to about 10% or 1% to about 5%. The suspension method may have advantages with respect to the upscaling method. In some embodiments, the ECM is in the form of one or more domes.

[0210] One type of ECM is secreted by epithelial cells, endothelial cells, parietal endoderm-like cells (e.g., Englebreth Holm Swarm parietal endoderm-like cells described in

[11] ), and connective tissue cells. This ECM consists of various polysaccharides, water, elastin, and glycoproteins, where the glycoproteins include collagen, entactin (nidogen), fibronectin, and laminin. Thus, in some embodiments, the ECM for use in the method of the present invention comprises one or more components selected from the list: polysaccharides, elastin, and glycoproteins, for example, the glycoproteins include collagen, entactin (nidogen), fibronectin, and / or laminin. For example, in some embodiments, collagen is used as the ECM. Various types of ECM are known, consisting of various compositions containing various types of glycoproteins and / or combinations of different glycoproteins.

[0211] ECM can be obtained, for example, by culturing ECM-producing cells such as epithelial cells, endothelial cells, parietal endoderm-like cells, or fibroblasts in a container, then removing these cells and adding isolated tissue fragments or isolated epithelial cells. Examples of extracellular matrix-producing cells include chondrocytes, which primarily produce collagen and proteoglycans; fibroblasts, which primarily produce type IV collagen, laminin, interstitial procollagen, and fibronectin; and colonic myofibroblasts, which primarily produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C. These are "naturally produced ECM." Naturally produced ECM can be commercially available. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.) or Matrigel® (BD Biosciences)).

[0212] In some embodiments, the extracellular matrix is ​​at least 50%, at least 60%, or at least 70% Matrigel, optionally 50–100%, 50–80% Matrigel, or optionally about 70% Matrigel. In preferred embodiments, the extracellular matrix is ​​at least 70% Matrigel.

[0213] In some embodiments, the ECM is a three-dimensional matrix. In some embodiments, cells are embedded in the ECM. In some embodiments, cells are attached to the ECM. The culture medium of the present invention can be diffused into the three-dimensional ECM. In other embodiments, the ECM is in a suspension, i.e., cells are in contact with the ECM in a suspension system. In some embodiments, the ECM is in a suspension at a concentration of at least 1%, at least 2%, or at least 3%. In some embodiments, the ECM is in a suspension at a concentration of 1% to about 10% or 1% to about 5%.

[0214] In preferred methods of the present invention, cells are cultured in contact with the extracellular matrix (ECM). In some embodiments, methods of the present invention include culturing epithelial stem cells in contact with the extracellular matrix. "In contact" means physical, mechanical, or chemical contact, which means that force must be used to separate the resulting organoid or epithelial cell population from the matrix. The culture medium and / or cells may be placed on, embedded in, or mixed with the extracellular matrix or synthetic matrix.

[0215] In some embodiments, the organoid medium is placed on top of an extracellular matrix or synthetic matrix. The organoid medium can then be removed and replenished as needed. In some embodiments, the organoid medium is replenished every 1, 2, 3, 4, 5, 6, or 7 days. When components are “added” or “removed” from the medium, this may mean, in some embodiments, that the medium itself is removed from the extracellular matrix or synthetic matrix, and then a new medium containing the “added” components or excluding the “removed” components is placed on top of the extracellular matrix or synthetic matrix.

[0216] A three-dimensional matrix supports the culture of three-dimensional epithelial organoids. Therefore, in some embodiments, the extracellular matrix or synthetic matrix is ​​a three-dimensional matrix.

[0217] In some embodiments, the culture medium further comprises an integrin agonist (e.g., as described in WO2020 / 234250). Specific examples of integrin agonists include anti-integrin antibodies such as anti-b1 integrin antibodies (e.g., TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29, and 9EG7 antibodies). Integrin agonists may be used in place of or in addition to the extracellular matrix.

[0218] In some embodiments, the ECM is a laminin-containing ECM such as Matrigel® (BD Biosciences). In some embodiments, the ECM is Matrigel® (BD Biosciences) containing laminin, entactin, and type IV collagen. In some embodiments, the ECM contains laminin, entactin, type IV collagen, and heparin sulfate proteoglycan (e.g., Cultrex® Basement Membrane Extract Type 2 (Trevigen, Inc.)). In some embodiments, the ECM contains at least one glycoprotein such as collagen and / or laminin. If necessary, a mixture of naturally produced or synthetic ECM materials may be used. In some embodiments, the ECM is BME ("Basement Membrane Extract"), which is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumors (e.g., Cultrex® BME).

[0219] In another embodiment, the ECM may be a synthetic ECM. For example, a synthetic ECM such as ProNectin (Sigma Z378666) may be used. In further examples, the ECM may be a plastic (e.g., polyester or hydrogel). In some embodiments, the synthetic matrix may be coated with a biomaterial (e.g., one or more glycoproteins such as collagen or laminin).

[0220] Three-dimensional ECM supports the culture of three-dimensional epithelial organoids. The extracellular matrix material is typically a droplet on the bottom of a dish in which cells are suspended (this culture method can be described as the "hanging drop method"). Medium is added to the ECM and diffused, usually when the matrix solidifies at 37°C. Cells in the medium adhere to the ECM through interactions with the surface structure of the ECM, such as interactions with integrins.

[0221] Organoid media and / or cells may be placed on the extracellular matrix (ECM), embedded within it, or mixed with it.

[0222] In some embodiments, particularly when poly-L-lysine coating is used for culture, stromal cells are cultured without (exogenous) ECM. In other embodiments, stromal cells are cultured with (exogenous) ECM.

[0223] Wnt Agonist The culture medium may contain a Wnt agonist. The Wnt signaling pathway and small molecules that activate Wnt signaling are described in

[12] . A Wnt agonist is defined herein as an agent that activates or enhances TCF / LEF-mediated transcription in cells. Thus, Wnt agonists are selected from true Wnt agonists that bind to and activate the Wnt receptor complex, including all Wnt family proteins, inhibitors of intracellular β-catenin degradation, GSK inhibitors (such as CHIR9901), and TCF / LEF activators. One or more Wnt agonists in the culture medium may be selected from Wnt ligands derived from the Wnt family of secreted glycoproteins, inhibitors of intracellular β-catenin degradation, GSK-3 inhibitors, TCF / LEF activators, inhibitors of RNF43 or ZNRF3, and R-spondin family proteins. In some embodiments, the Wnt agonist in the culture medium comprises an R-spondin family protein and a GSK-3 inhibitor, and optionally further comprises a Wnt ligand derived from the Wnt family of secreted glycoproteins. One or more, for example, two, three, four or more Wnt agonists may be used in the culture medium.

[0224] The Wnt agonist in the culture medium is preferably any agonist capable of stimulating the Wnt pathway via the Lgr5 cell surface receptor; that is, in a preferred embodiment, the Wnt agonist in the culture medium is a Lgr5 agonist. Known Lgr5 agonists include Rspondin, its fragments and derivatives, and anti-Lgr5 antibodies (see, for example, WO2012 / 140274, particularly Figures 22-24, and

[13] ). A preferred Lgr5 agonist is Rspondin. Any suitable Rspondin may be used, for example, one or more of Rspondin 1, Rspondin 2, Rspondin 3, and Rspondin 4 or their derivatives may be selected. For example, any of Rspondin 1 (NU206, Nuvelo, San Carlos, CA), Rspondin 2 ((R&D systems), Rspondin 3, and Rspondin-4) may be used. Rspondin can be used at any appropriate concentration, for example, at least 100 ng / ml, more preferably at least 200 ng / ml, and more preferably about 250 ng / ml. An example of an agonistic anti-Lgr5 antibody is 1D9 (BDB562733, No: 562733, commercially available from BD Biosciences). Fragments of Rspondin may be used as Wnt agonists. For example, in some embodiments, the Wnt agonist is a fragment of Rspondin containing or consisting of a furin domain.

[0225] Therefore, in one embodiment, the culture medium comprises an Lgr5 agonist (e.g., Rspondin) and, in addition, a further Wnt agonist. In this regard, the further Wnt agonist may be selected from the group consisting of, for example, Wnt-3a, a GSK inhibitor (such as CHIR99021), Wnt-5, Wnt-6a Norrin, and NGS-Wnt. In one embodiment, the culture medium comprises Rspondin and, in addition, a soluble Wnt ligand such as Wnt3a or NGS-Wnt. The addition of a soluble Wnt ligand has been shown to be particularly advantageous for the proliferation of human epithelial stem cells (as described in WO2012 / 168930).

[0226] The R-spongin family proteins (also referred to herein as “R-spongins”) may be selected from R-spongin 1, R-spongin 2, R-spongin 3, R-spongin 4, and their analogs, fragments, variants, and derivatives. In this regard, fragments, variants, or derivatives can inhibit the action of the E3 ligase RNF43 / ZNRF3 on the Wnt receptor complex. R-spongin 1, R-spongin 2, R-spongin 3, and R-spongin 4 (also referred to herein as “R-spongins 1-4”) are all characterized by two amino-terminal furin-like repeats necessary and sufficient for Wnt signal enhancement and a thrombospongin domain located more towards the carboxyl-terminal member

[14] . Examples of R-spondin fragments, variants, 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 describes a furin domain fragment that can enhance Wnt signaling, incorporated herein by reference). Examples of R-spondin family protein analogs include, for example, antibodies that interact with RNF43 / ZnRF3 / Lgr. Agonist anti-Lgr5 antibodies that can enhance Wnt signaling are known in the art (see, for example, antibody 1D9 described in Example 3 of

[15] ).

[0227] Many GSK-3 inhibitors are known in the art (see, e.g.,

[16] and

[17] ) and commercially available (see, e.g., the list available here from Santa Cruz Biotechnology: https: / / www.scbt.com / scbt / browse / GSK-3-beta-Inhibitors / _ / N-x6oud All of these GSK-3 inhibitors are suitable for use in relation to the present invention, and those skilled in the art can determine suitable concentrations using the IC50 value.

[0228] CHIR-99021 (CAS: 252917-06-9; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride; 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, catalog 1126), CHIR98023, CHIR99021 (see above), and TWS119 (Tocris, catalog 3835). Therefore, in some embodiments, the GSK-3 inhibitor is an aminopyrimidine inhibitor selected optionally from CHIR98014, CHIR98023, CHIR99021, or TWS119. In some embodiments, the GSK-3 inhibitor is CHIR-99021.

[0229] Wnt ligands derived from the secreted glycoprotein Wnt family 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 (see

[18] ), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt-9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM1, and Wnt-16. An overview of human Wnt proteins is shown in

[19] . In some embodiments, the Wnt ligand is Wnt-3a, Wnt-5, or Wnt-6a, or optionally Wnt-3a. The addition of soluble Wnt ligands has been shown to be particularly advantageous for the enlargement of human organoids and organoid fragments (see, for example,

[20] ).

[0230] In some embodiments, the Wnt agonist in the culture medium is a Wnt substitute. The Wnt substitute is a water-soluble Wnt agonist manipulated by linking antagonistic Fzd and Lrp5 / 6 binding modules to a single polypeptide chain, and thus forces receptor heterodimerization while blocking endogenous Wnt binding. The Wnt substitute supports the growth of a wide range of cultures. Furthermore, the Wnt substitute is a non-lipidized Wnt agonist that can be produced in serum-free medium, maintained in a frozen state, and avoids differences in Wnt culture supernatant activity that may arise between laboratories

[21] . In some embodiments, the Wnt substitute is a next-generation substitute Wnt (NGS-Wnt), as described, for example,

[22] . NGS-Wnt may be supplied at concentrations of about 0.1 nM to about 0.5 nM. In some embodiments, the culture medium contains NGS-Wnt at a concentration of about 0.5 nM. In some embodiments, the culture medium contains NGS-Wnt at a concentration of about 0.1 nM.

[0231] In some embodiments, the Wnt agonist in the culture medium is a Wnt substitute. The Wnt substitute is a water-soluble Wnt agonist manipulated by linking antagonistic Fzd and Lrp5 / 6 binding modules to a single polypeptide chain, and thus forces receptor heterodimerization while blocking endogenous Wnt binding. The Wnt substitute supports the growth of a wide range of cultures. Furthermore, the Wnt substitute is a non-lipid-added Wnt agonist that can be produced in serum-free medium, maintained in a frozen state, and avoids differences in the activity of Wnt-conditioned media that may arise from different laboratories (

[23] ). In some embodiments, the Wnt substitute is a next-generation substitute Wnt (NGS-Wnt), such as described in

[24] . NGS-Wnt may be supplied at concentrations of about 0.1 nM to about 0.5 nM. In some embodiments, the culture medium contains NGS-Wnt at a concentration of about 0.5 nM. In some embodiments, the culture medium contains NGS-Wnt at a concentration of about 0.1 nM.

[0232] Soluble Wnt agonists such as Wnt-3a may be provided in the form of Wnt-conditioned medium. For example, Wnt-conditioned medium containing approximately 10% to 50% Wnt may be used.

[0233] Rspondin may be provided in the form of Rspo-acclimatized medium. For example, Rspo-acclimatized medium containing about 10% to about 30% (e.g., about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml) may be used.

[0234] Examples of Rspondin imitations suitable for use in the present invention are provided in WO2012 / 140274, which is incorporated herein by reference.

[0235] mitotic growth factor The culture medium may contain mitotic growth factors. Mitotic growth factors typically induce cell division via the signaling pathway of mitotic-activated protein kinases. Many receptor tyrosine kinase ligands are mitotic growth factors. In some embodiments, mitotic growth factors can bind to receptor tyrosine kinases. In some embodiments, mitotic growth factors can bind to one or more receptor tyrosine kinases. In some embodiments, one or more mitotic growth factors bind to receptor tyrosine kinases, e.g., EGFR, FGFR, or HGFR, and optionally, one or more mitotic growth factors are selected from EGF, FGF, and HGF.

[0236] In some embodiments, the mitotic growth factor binds to EGFR, HER1, HER2, HER3, or HER4. In some embodiments, the mitotic growth factor binds to EGFR. In some embodiments, in addition to the EGFR ligand, the HER2-4 ligand is included in the culture medium. For example, in some embodiments, in addition to EGF, neuregulin is included in the culture medium. Neuregulin has been shown to be favorable for culturing lung and breast tissue (see, for example,

[25] and

[26] ). In some embodiments, one or more mitotic growth factors in the culture medium are EGF. Any suitable EGF, e.g., EGF obtained from Peprotech, may be used.

[0237] FGFs stimulate cells by interacting with cell surface tyrosine kinase receptors (FGFRs). Four closely related receptors (FGFR1-FGFR4) have been identified. Therefore, in some embodiments, mitotic growth factors bind to members of the FGF receptor family. Members of the FGF receptor family 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 may be important in determining ligand specificity. Several FGFs exist that bind to members of the FGF receptor family, including (but are not limited to) FGF2, FGF4, FGF7, and FGF10. These are commercially available. Therefore, in some embodiments, mitotic growth factors are FGFs. In some embodiments, FGFs are selected from FGF2, FGF4, FGF7, and FGF10. In preferred embodiments, FGFs are FGF2 and / or FGF10. In the most preferred embodiment, the FGFs are FGF2 and FGF10.

[0238] Hepatocyte growth factor / scattering factor (HGF / SF) is a morphogenetic factor that regulates cell proliferation, cell motility, and morphogenesis by activating the tyrosine kinase signaling cascade after binding to pro-oncogenic HGFR. HGFR is also known as the c-Met receptor. HGF has been shown to be useful in the culture of epithelial stem cells. Therefore, in some embodiments, mitotic growth factor binds to HGFR. In some embodiments, mitotic growth factor is HGF. Any suitable HGF, for example, HGF obtained from Peprotech, may be used.

[0239] In some embodiments, the culture medium contains multiple mitotic growth factors, for example, two or three mitotic growth factors. For example, in some embodiments, one or more mitotic growth factors in the culture medium are EGF and FGF. In some embodiments, one or more mitotic growth factors in the culture medium are EGF, FGF2, and FGF10. In some embodiments, one or more mitotic 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.

[0240] In some embodiments, hepatocyte growth factor (HGF) is also present, with or without EGF and / or FGF.

[0241] BMP inhibitor The culture medium may contain a BMP inhibitor. A BMP inhibitor is defined as an agent that binds to a BMP molecule and forms a complex in which BMP activity is neutralized, for example, by preventing or inhibiting the binding of the BMP molecule to a 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 BMP from binding to the receptor. An example of the latter agent is an antibody that binds to the BMP receptor and prevents BMP from binding to an antibody-binding receptor.

[0242] BMP inhibitors can be added to the culture medium in amounts effective enough to inhibit BMP-dependent activity in 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%, and more preferably up to 0%, compared to the level of BMP activity in the absence of the inhibitor, when evaluated in the same cell type. As is known to those skilled in the art, BMP activity can be determined by measuring the transcriptional activity of BMPs, for example, as exemplified in

[27] .

[0243] Several classes of natural BMP-binding proteins are known, including noggin (Peprotech), cordins and cordin-like proteins containing a coding 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 alpha-2 macroglobulin (R&D systems).

[0244] Thus, in some embodiments, the BMP inhibitor is selected from noggin, DAN, and DAN-like proteins, including Cerberus and Gremlin (R&D systems). These secreted proteins can bind to BMP ligands with varying degrees of affinity and inhibit their access to signaling receptors. Addition of any of these BMP inhibitors to the basal medium prevents loss of stem cells. A preferred BMP inhibitor is noggin.

[0245] TGF-β inhibitor The medium can contain a TGF-beta (or "TGF-β") inhibitor. The presence of a TGF-beta inhibitor in the growth medium is particularly advantageous for increasing human organoid formation efficiency. A TGF-beta inhibitor is any agent that reduces the activity of the TGF-beta signaling pathway, also referred to herein as the ALK4, ALK5, or ALK7 signaling pathway. The TGF-beta inhibitors of the present invention can be proteins, peptides, small molecules, small interfering RNAs, antisense oligonucleotides, aptamers, or antibodies. The inhibitor can be natural or synthetic.

[0246] In some embodiments, the TGF-beta inhibitor is a small molecule inhibitor such as A83-01 (or "A-83-01"). A83-01 is a commercially available selective inhibitor of ALK4, ALK5, and ALK7 (Tocris catalog number 2939). The catalog describes it as a potent inhibitor (IC , , ,

[0248] , values are 12, 45, and 7.5 nM, respectively) that blocks phosphorylation of Smad2 and only slightly inhibits ALK-1, ALK-2, ALK-3, ALK-6, and MAPK activity. 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 bactobolin. The IC50 values of these inhibitors are known in the art, and one of ordinary skill in the art can select an appropriate inhibitor at an appropriate concentration based on the teachings provided in the examples of this application.

[0247] Nicotinamide In some embodiments, the medium contains nicotinamide. Nicotinamide is an amide derivative of vitamin B3, a poly(ADP-ribose) polymerase (PARP) inhibitor, and represents a primary precursor of NAD+. It is commercially available (e.g., from Stemcell Technologies catalog 07154).

[0248] Prostaglandin pathway activator In some embodiments, the culture medium further comprises a prostaglandin pathway activator. The prostaglandin pathway activator may be one or more compounds selected from the list comprising 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.

[0249] cAMP activator In some embodiments, the culture medium contains a cAMP pathway activator. The cAMP pathway activator can be any suitable activator that increases the level of cAMP in the cell. In some embodiments, the cAMP pathway activator is an adenylyl cyclase activator or a cAMP analog. Examples of suitable adenylyl cyclase activators include forskolin, forskolin analogs, and cholera toxin. An example of a forskolin analog is NKH477 (e.g., catalog number Tocris 1603), which is known in the art. An example of a cAMP analog is also known in the art, such as 8-bromo-cAMP. 8-bromo-cAMP is a cell-permeable cAMP analog that is more resistant to hydrolysis by phosphodiesterases than cAMP. Therefore, 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.

[0250] Additional ingredients The culture medium may be supplemented with one or more compounds selected from the group consisting of gastrin, B27, N-acetylcysteine ​​(or "N-Ac"), and N2. Therefore, in some embodiments, the culture medium further comprises one or more components selected from the group consisting of gastrin, B27, N2, and N-acetylcysteine. B27 (Invitrogen), N-acetylcysteine ​​(Sigma), N2 (Invitrogen), and gastrin (Sigma) are thought to control cell proliferation and support DNA stability. In some embodiments, the culture medium further comprises B27 and N-acetylcysteine.

[0251] In some embodiments, the culture medium further comprises a ROCK inhibitor (Rho-kinase inhibitor). ROCK inhibitors are particularly useful for cell adhesion when establishing new cultures and / or when dividing ("passaging") cells. Preferred ROCK inhibitors are known and commercially available in the art (not limited to GSK269962, GSK429286, H1152 dihydrochloride, glycyl-H1152 dihydrochloride, SR3677 dihydrochloride, SB772077B dihydrochloride, and Y-27632 dihydrochloride, all available from Tocris). In some embodiments, the culture medium is supplemented with about 5 μM to about 20 μM or about 8 μM to about 15 μM of ROCK inhibitor, optionally about 10 μM of ROCK inhibitor. A particularly preferred ROCK inhibitor is Y-27632.

[0252] In some embodiments, the culture medium, particularly the growth medium, is preferably free of undefined components (such as fetal bovine serum or feeder cells). Various different serum substitutes are commercially available and known to those skilled in the art. When serum substitutes are used, they may be used in an amount of about 1% to about 30% of the culture medium volume, according to the prior art. In some embodiments, the culture medium is serum-free and / or feeder-free.

[0253] A preferred medium is a defined synthetic medium buffered at a pH of about 7.4 (preferably about 7.2 to about 7.6, or at least about 7.2 and about 7.6) with a carbonate-based buffer while the cells are cultured in an atmosphere containing about 5% to about 10% CO2, or at least about 5% and less than or equal to about 10% CO2, preferably about 5% CO2.

[0254] In vitro method overview The claimed method of the present invention may be carried out in vivo, ex vivo, in vitro, in situ, exitu, or any combination thereof. Preferably, the method is carried out in vitro.

[0255] Traditionally, cell lines, and more recently, iPS cells, have been used as ex vivo cell / organ and / or disease models (see, e.g.,

[28] ). However, these methods have many challenges and drawbacks. For example, it is not possible to obtain cell lines from all patients (cell lines can only be obtained from specific biopsies), and therefore, cell lines cannot be used for personalized diagnosis and personalized medicine. iPS cells usually require some degree of genetic manipulation to reprogram the cells to a specific cell fate. Alternatively, iPS cells must be cultured for minimal time because they are subjected to culture conditions that affect karyotype integrity (this is also true for human embryonic stem cells). This means that iPS cells cannot accurately represent the in vivo state, but rather attempt to mimic the behavior of in vivo cells. Cell lines and iPS cells are also genetically unstable.

[0256] In contrast, the organoids and co-cultures of the present invention provide a genetically stable platform that faithfully reflects the in vivo situation. In some embodiments, the organoids and co-cultures of the present invention include all differentiated cell types present in the corresponding in vivo situation. In other embodiments, the organoids and co-cultures may further differentiate to provide all differentiated cell types present in vivo. Thus, the organoids and co-cultures may be used to gain mechanistic insights into various diseases and therapies, to conduct in vitro drug screening, to evaluate potential therapies, to identify candidate targets (e.g., proteins) for the development of future novel (drug) therapies, and / or to explore gene repair in conjunction with cell replacement therapy.

[0257] Accordingly, the present invention also provides the use of organoids and co-cultures in assays for evaluating the viability, metabolic activity, permeability, integrity of barrier function, and / or transport protein activity of epithelium. Methods for evaluating the viability, permeability, and integrity of barrier function of organoids and co-cultures, as well as the activity of transport proteins in organoids and co-cultures, are described herein.

[0258] The co-cultures of the present invention can be used in in vitro methods such as methods for investigating (e.g., verifying) co-cultures, methods for testing therapeutic agents, and methods for diagnosing and / or predicting the prognosis of diseases. In some embodiments, organoids are used to model in vivo interactions. These methods can be used to test libraries of chemicals, antibodies, natural products (e.g., plant extracts or microbial compounds) for suitability for use as drugs, diagnostic agents, cosmetics, and / or prophylactic agents. For example, cells are preferably exposed to multiple concentrations of test agents for a certain period of time. At the end of the exposure period, the cultures are evaluated (e.g., by determining the presence or absence of at least one change).

[0259] In some embodiments, the present invention provides the use of co-cultures in drug screening, target validation, target discovery, toxicology, toxicology screening, toxicity assays, or as ex vivo cell / organ models. In some embodiments, the present invention provides the use of organoids in ex vivo methods for predicting clinical outcomes.

[0260] The present invention provides for the use of organoids and co-cultures in the investigation of histology, cell lineage, and differentiation pathways; investigations to identify chemical and / or neuronal signals that lead to the release of various hormones; gene expression studies, including recombinant gene expression; investigations of mechanisms involved in tissue injury and repair; investigations of inflammatory diseases; investigations of fibrous diseases; studies of pathogenesis; or studies of mechanisms of cell transformation.

[0261] Decide on at least one change The method of the present invention may involve determining the presence or absence of at least one change in the co-culture.

[0262] In principle, it is possible to determine whether or not there has been any change in any biochemical, genetic, phenotypic, or phenomenological property of the co-culture. At least one change may be an increase or decrease in the property.

[0263] For example, at least one change may include a change in organoid morphology, a change in organoid size (e.g., area), a change in epithelial cell size (e.g., area), a change in epithelial thickness, a decrease in cell viability, a decrease in cell proliferation, an increase in cell death, a change in secretome profile, a change in cytokine secretion, an increase in cell apoptosis, an increase in caspase activity, and / or a change in the expression of one or more genes, and optionally, such change in the expression of one or more genes may include a change in the expression of one or more disease biomarkers, one or more fibrosis biomarkers, and / or one or more inflammatory biomarkers.

[0264] At least one change may include a change in epithelial viability, a change in metabolic activity, a change in permeability, a change in the integrity of the barrier function, and / or a change in the activity of a transport protein. Methods for assessing the viability, permeability, and integrity of the barrier function of organoids and co-cultures, as well as the activity of transport proteins in co-cultures, are described herein.

[0265] In some embodiments, at least one change includes a change in organoid aggregation, such as an increase in organoid aggregation. An increase in organoid aggregation can be observed without an inflammatory stimulus (e.g., compared to the organoids before co-culture) when at least one organoid is co-cultured with at least one stromal cell. An increase in organoid aggregation can be observed with an inflammatory stimulus (e.g., compared to the organoids before co-culture) when at least one organoid is co-cultured with at least one stromal cell. Aggregation in co-cultures prepared with an inflammatory stimulus may be less than aggregation in co-cultures prepared without an inflammatory stimulus. Thus, aggregation can be used as an indicator of the success of co-culturing at least one organoid with at least one stromal cell, and aggregation can also be used to determine whether the co-culture exhibits an inflammatory profile.

[0266] In some embodiments, at least one change includes a change in organoid surface attachment, such as a reduction in organoid surface attachment.

[0267] In some embodiments, at least one change includes a change in organoid morphology, and optionally, the change in organoid morphology includes changes in organoid shape, organoid size (e.g., area), organoid lumen size (e.g., area of ​​the organoid lumen), epithelial cell shape, or epithelial cell size (e.g., area). Preferably, at least one change includes a change in organoid area. While we do not wish to be bound by any theory, we have found that organoid size, particularly cystic morphology, is a highly reproducible and specific reading of the functional presence of fibroblasts in co-cultures having a pro-inflammatory profile. Organoid size, preferably organoid area, can be measured by bright-field imaging.

[0268] In some embodiments, at least one change includes an increase in the area of ​​at least one organoid, optionally, the area being at least about 10,000 μm². 2 , at least about 15,000 μm 2 , at least about 20,000 μm 2 , or at least about 25,000 μm 2 It increases to [a certain value].

[0269] In some embodiments, at least one change includes an increase in the area of ​​at least one organoid, the increase being at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%.

[0270] In some embodiments, at least one change includes a change in epithelial thickness, e.g., a reduction in epithelial thickness, optionally, the reduction being at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0271] In some embodiments, at least one change includes a change in the expression of one or more genes, preferably a change in the expression of one or more disease biomarkers. The term “expressed” is used to indicate the presence of a marker within a cell. For a marker to be considered expressed, it must be present at a detectable level. “Detectable level” means that the marker can be detected using one of the standard testing methods, such as PCR, blotting, or FACS analysis. A gene is considered expressed by the organoid cells, stromal cells, or co-culture of the present invention if moderate expression can be detected after 30 PCR cycles (this corresponds to an intracellular expression level of at least about 100 copies per cell). The terms “express” and “expression” are equivalent in meaning. Below this threshold, the marker is considered not expressed. Comparison of the expression level of a marker in the cells of the present invention with the expression level of the same marker in another cell (e.g., embryonic stem cells) can preferably be performed by comparing two cell types isolated from the same species. Preferably, this species is mammalian, and more preferably, this species is human. Such comparisons can be conveniently performed using reverse transcriptase polymerase chain reaction (RT-PCR) experiments.

[0272] Therefore, the presence or absence of at least one change in the cellular composition of an organoid can be determined by detecting the expression of one or more marker genes, for example, by detecting the presence or absence of at least one change in the expression of one or more marker genes. Lgr5 is Lgr5 + It is a stem cell marker. Ki67 is Lgr5 +These are markers for proliferative cells such as stem cells. Goblet cells can be detected by mucin staining, for example, Alcian blue staining, or by detecting the expression of mucin-2 (Muc2), as described herein. Enteric alkaline phosphatase (ALPI or ALPI1) is a marker for intestinal absorptive epithelial cells. Lysozyme is a marker for Paneth cells. Chromogranin A is a marker for enteroendocrine cells.

[0273] Therefore, in some embodiments, the presence or absence of a change in the expression of at least one of the following genes is determined: ALPI, MUC2, lysozyme, Ki67, and Lgr5. In some embodiments, the organoid and / or co-culture of the present invention expresses Lgr5 and Muc2, for example, the change is an increase in the expression of Lgr5 and Muc2. In some embodiments, the organoid and / or co-culture of the present invention does not express ALPI, for example, the change is a decrease in the expression of ALPI. In some embodiments, the organoid and / or co-culture of the present invention expresses Lgr5 and Muc2 but does not express ALPI, for example, the change is an increase in the expression of Lgr5 and Muc2, and a decrease in the expression of ALPI. In some embodiments, the organoid and / or co-culture of the present invention expresses lysozyme.

[0274] In some embodiments, at least one change includes upregulation of the expression of a stem cell marker, optionally the stem cell marker being OLFM4, and optionally the upregulation being at least about 10%, at least about 50%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1,000%.

[0275] In some embodiments, at least one change includes downregulation of the expression of an intestinal cell marker, optionally the intestinal cell marker being ALPI, and optionally the downregulation being at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0276] In some embodiments, at least one change includes downregulation of the expression of a growth marker, optionally the growth marker being KI67, and optionally the downregulation being at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0277] In some embodiments, at least one change includes a change in the secretome profile, optionally a change in cytokine secretion, and optionally a change in IL-6 and / or CXCL2 secretion. In some embodiments, at least one change is a change in IL-6 expression (preferably an increase in IL-6 expression), for example a change in IL-6 secretion (preferably an increase in IL-6 secretion). In some embodiments, at least one change is a change in CXCL2 expression (preferably an increase in CXCL2 expression), for example a change in CXCL2 secretion (preferably an increase in CXCL2 secretion).

[0278] In some embodiments, the changes include the expression of at least about 10,000 pg / ml of IL6, at least about 20,000 pg / ml of IL6, at least about 30,000 pg / ml of IL6, at least about 40,000 pg / ml of IL6, at least about 50,000 pg / ml of IL6, or at least about 100,000 pg / ml of IL6.

[0279] In some embodiments, the at least one change includes a change in RNA expression, which is optionally measured by RNA-seq. The change in expression may be an upregulation or downregulation of RNA. Preferably, the change in RNA expression is an upregulation of RNA expression (e.g., an increase in the normalized expression score, for example, the normalized expression score determined according to the median of ratios method): https: / / hbctraining.github.io / DGE_workshop / lessons / 02_DGE_count_normalization.html ) include. In some embodiments, the alteration of RNA expression includes upregulation of the expression of an RNA encoding any one of IL-6, CXCL2, MMP7, HIF3A, WNT7A, and IGFBP1.

[0280] In some embodiments, the at least one change includes a change in the expression of RNA encoding IL-6. In some embodiments, the change in the expression of RNA encoding IL-6 includes an upregulation, e.g., at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding IL-6 includes an upregulation, e.g., at least about 50x. In some embodiments, the at least one change includes a change in the expression of RNA encoding CXCL2. In some embodiments, the change in the expression of RNA encoding CXCL2 includes an upregulation, e.g., at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding CXCL2 includes an upregulation, e.g., at least about 25x. In some embodiments, the at least one change includes a change in the expression of RNA encoding IGFBP1. In some embodiments, the change in the expression of RNA encoding IGFBP1 includes upregulation, for example, upregulation of at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding IGFBP1 includes upregulation, for example, upregulation of at least about 10x. In some embodiments, the at least one change includes a change in the expression of RNA encoding WNT7A. In some embodiments, the change in the expression of RNA encoding WNT7A includes upregulation, for example, upregulation of at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding WNT7A includes upregulation, for example, upregulation of at least about 10x. In some embodiments, the at least one change includes a change in the expression of RNA encoding MMP7.In some embodiments, the change in the expression of RNA encoding MMP7 includes upregulation, e.g., upregulation of at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding MMP7 includes upregulation, e.g., upregulation of at least about 10x. In some embodiments, the at least one change includes a change in the expression of RNA encoding HIF3A. In some embodiments, the change in the expression of RNA encoding HIF3A includes upregulation, e.g., upregulation of at least about 2x, at least about 5x, at least about 10x, at least about 20x, at least about 25x, or at least about 50x. Preferably, the change in the expression of RNA encoding HIF3A includes upregulation, e.g., upregulation of at least about 25x.

[0281] In some embodiments, the alteration of RNA expression includes alteration of the expression of RNA encoding KRT20, MUC2, LYZ, ALPI, OLFM4, CCND1, LGR5, MKI67, CXCL2, IL6, THY1, or PDPN. In some embodiments, the alteration of RNA expression includes upregulation of the expression of RNA encoding KRT20, LYZ, ALPI, OLFM4, CCND1, CXCL2, IL6, or PDPN. In some embodiments, the alteration of RNA expression includes downregulation of the expression of RNA encoding MUC2, LGR5, MKI67, CXCL2, IL6, or THY1.

[0282] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 3. In some embodiments, the RNA is an upregulated RNA in Table 3. In some embodiments, the RNA is a downregulated RNA in Table 3. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 3 is a change in a co-culture of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus, as determined by comparison with a co-culture of the present invention in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus.

[0283] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 4. In some embodiments, the RNA is an upregulated RNA in Table 4. In some embodiments, the RNA is a downregulated RNA in Table 4. In some embodiments, the change in RNA expression encoding a marker listed in Table 4 is a change in organoids of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus, as determined by comparison with organoids of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus.

[0284] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 5. In some embodiments, the RNA is an upregulated RNA in Table 5. In some embodiments, the RNA is a downregulated RNA in Table 5. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 5 is a change in the organoid of the present invention cultured in an organoid medium of the present invention containing at least one pro-inflammatory stimulus, as determined by comparison with the organoid of the present invention cultured in an organoid medium of the present invention containing at least one pro-inflammatory stimulus.

[0285] In some embodiments, the change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 6. In some embodiments, the RNA includes RNA upregulated in Table 6. In some embodiments, the RNA is RNA downregulated in Table 6. In some embodiments, the change in RNA expression includes RNA encoding a marker listed in Table 6, and is the change in stromal cells of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus, as determined compared to stromal cells of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus.

[0286] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 7. In some embodiments, the RNA includes RNA upregulated in Table 7. In some embodiments, the RNA is RNA downregulated in Table 7. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 7 is a change in organoids of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus, as determined by comparison with co-cultures of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus.

[0287] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 8. In some embodiments, the RNA includes RNA upregulated in Table 8. In some embodiments, the RNA is RNA downregulated in Table 8. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 8 is a change in organoids of the present invention cultured in a co-culture medium of the present invention that does not contain at least one pro-inflammatory stimulus, as determined by comparison with co-cultures of the present invention cultured in a co-culture medium of the present invention that does not contain at least one pro-inflammatory stimulus.

[0288] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 9. In some embodiments, the RNA includes RNA upregulated in Table 9. In some embodiments, the RNA is RNA downregulated in Table 9. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 9 is a change in organoids of the present invention cultured in a co-culture medium of the present invention that does not contain at least one pro-inflammatory stimulus, determined by comparing them with organoids of the present invention isolated from at least one stromal cell and cultured in a conditioned medium that does not contain at least one pro-inflammatory stimulus.

[0289] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 10. In some embodiments, the RNA includes RNA upregulated in Table 10. In some embodiments, the RNA is RNA downregulated in Table 10. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 10 is a change in organoids of the present invention cultured in a co-culture medium of the present invention containing at least one pro-inflammatory stimulus, determined by comparing them with organoids of the present invention cultured in a conditioned medium containing at least one pro-inflammatory stimulus, isolated from at least one stromal cell.

[0290] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding a marker listed in Table 11. In some embodiments, the RNA includes RNA upregulated in Table 11. In some embodiments, the RNA is RNA downregulated in Table 11. In some embodiments, the change in the expression of RNA encoding a marker listed in Table 11 is a change in organoids of the present invention isolated from at least one stromal cell and cultured in a conditioned medium free of at least one pro-inflammatory stimulus, as determined by comparison with organoids of the present invention isolated from at least one stromal cell and cultured in a conditioned medium free of at least one pro-inflammatory stimulus.

[0291] In some embodiments, at least one change includes a change in caspase activity, optionally including a change in caspase 3 / 7 activity, optionally increasing the caspase activity by at least about 500%, at least about 1,000%, at least about 1,500%, at least about 2,000%, or at least about 2,500%.

[0292] In some embodiments, a cytokine is added to the co-culture before determining whether at least one change is present in the co-culture, optionally the cytokine being TNF and / or IFNγ, and optionally the cytokine being at a maximum concentration of about 100 ng / ml, about 50 ng / ml, about 15 ng / ml, about 5 ng / ml, about 1 ng / ml, about 0.1 ng / ml, or about 0.01 ng / ml. Preferably, if the cytokine is TNF, the concentration is at least about 1 ng / ml. Preferably, if the cytokine is IFNγ, the concentration is at least about 15 ng / ml.

[0293] In some embodiments, a cytokine signaling inhibitor is added to the co-culture before determining whether at least one change is present in the co-culture, and optionally, the cytokine signaling inhibitor reduces the activity of the JAK / STAT pathway, and optionally, the cytokine signaling inhibitor is tofacitinib, for example, about 10 μM tofacitinib.

[0294] At least one change in a co-culture containing at least one organoid can be determined by comparing it to at least one organoid before the co-culture was formed. At least one change in a co-culture can be determined by comparing it to a reference organoid, such as a reference organoid not incorporated into the co-culture. At least one change in a co-culture can be determined by comparing it to a reference organoid, such as a reference organoid that does not contain stromal cells, and optionally the reference organoid does not contain non-epithelial cells. At least one change in a co-culture can be determined after at least one agent has been applied to the co-culture, for example, by comparing it to the co-culture before the application of the at least one agent. At least one change in a co-culture can be determined after at least one agent has been applied to the co-culture, for example, by comparing it to a reference co-culture in which the at least one agent has not been applied.

[0295] In some embodiments, the presence or absence of at least one change is determined in the co-culture after being cultured for at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, or at least about 120 hours. Preferably, the presence or absence of at least one change is determined in the co-culture after being cultured for at least about 72 hours.

[0296] In some embodiments, the presence or absence of at least one change is determined at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days after combining at least one organoid and at least one stromal cell to form a co-culture. Preferably, the presence or absence of at least one change is determined at least about 7 days after combining at least one organoid and at least one stromal cell to form a co-culture.

[0297] Determining the presence or absence of at least one change may include determining the presence or absence of stromal cells in the co-culture and, optionally, determining the amount of stromal cells in the co-culture. Determining the amount of stromal cells in the co-culture may include determining the degree of stromal cell death in the co-culture.

[0298] Determining the presence or absence of at least one change may include determining the presence or absence of organoid cells in the co-culture and, optionally, determining the amount of organoid cells in the co-culture. Determining the amount of organoid cells in the co-culture may include determining the degree of organoid cell death in the co-culture.

[0299] Determining the presence or absence of at least one change may include determining the presence or absence of epithelial cells in the co-culture and, optionally, determining the amount of epithelial cells in the co-culture. Determining the amount of epithelial cells in the co-culture may include determining the degree of epithelial cell death in the co-culture.

[0300] Determining the presence or absence of at least one change may include determining the presence or absence of non-epithelial cells in the co-culture and, optionally, determining the amount of non-epithelial cells in the co-culture. Determining the amount of non-epithelial cells in the co-culture may include determining the degree of non-epithelial cell death in the co-culture.

[0301] Determining the presence or absence of at least one change may include, for example, determining whether the co-culture exhibits a pro-inflammatory profile after at least one agent has been applied to the co-culture.

[0302] technique Any in vitro methods disclosed herein, including validation of co-cultures, testing of therapeutic agents, and / or diagnosis and / or prognosis prediction, and any methods involving the determination of at least one change, may include standard experimental techniques. For example, such in vitro methods may include any one of whole-genome sequencing, mRNA sequencing, peptidomed profiling, and / or microscopy. One or more of these techniques can be used, in the form of information discovery and / or information verification, to ensure that co-cultures and / or organoids are homogeneous and / or meet expectations. For example, they can be used to determine differences in mRNA transcription between organoids and co-cultures, and whether these differences in mRNA transcription are reflected in differences in protein expression. The presence of specific antigens on organoids and whether any new antigens arise only in co-cultures can also be confirmed. Upregulation of inflammatory factors in the co-culture microenvironment may also be investigated.

[0303] In principle, determining the presence or absence of at least one change can be carried out using any suitable experimental method known to those skilled in the art. In some embodiments, the determination may include cell proliferation assays, viability assays, flow cytometry analysis, ELISA for IFN-γ (interferon-gamma), gene expression analysis, and / or cell imaging. In some embodiments, the determination may include cell proliferation assays, viability assays, flow cytometry analysis, ELISA for CXCL2, IL6, HTRF for CXCL2, HTRF for IL6, gene expression analysis, cell imaging, and / or RNA-seq.

[0304] Various assays for measuring cell viability are known in the art, including assays that determine viability based on cell membrane integrity (e.g., using nucleic acid dyes, propidium iodide, TO-PRO-3 iodide, or 7-AAD), cellular function (e.g., using the Calcein or CyQUANT cytotoxicity assay kit), or metabolic activity (e.g., using the alarmarBlue cell viability reagent or the yQUANT MTT cell viability assay). In some embodiments, as measured by, for example, the Comet assay

[29] or the yH2AX assay

[30] , at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 20%, at least 30%, or at least 40% of the DNA in the organoid or co-culture fragment is damaged. Reduced cell viability can also be determined by the CellTiter Glo luminescence cell viability assay kit (Promega), intracellular flow cytometry staining (BD) for active caspase 3, or positive staining of dead cells. Positive strains of dead cells include non-cell membrane permeable DNA stains such as NucRed Dead 647 ReadyProb.

[0305] Increased cell death can be detected by bright-field imaging.

[0306] To determine changes in gene expression, depending on the uniqueness of the marker, the expression of the above markers may be evaluated by RT-PCR, immunohistochemistry, or tissue staining after culturing for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or longer, as described herein. In some embodiments, the expression of the markers is measured after culturing for about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, or longer, for example, about 16 days, as described herein.

[0307] In some embodiments, at least one change includes changes in cytokine expression, such as changes in the expression of IL-6 and / or CXCL2. These can be measured by HTRP or ELISA, respectively.

[0308] In some embodiments, at least one change includes apoptosis, e.g., a change in caspase activity (such as caspase 3 / 7), or a change in cell viability. This can be measured by bright-field imaging with calcein staining (for cell death) and / or using a commercially available caspase assay such as Caspase-Glo.

[0309] Image analysis can be used to evaluate the characteristics of cells in cultures, such as cell morphology; cell structure; evidence of apoptosis or cell lysis; and the composition and structure of organoids. Many types of image analysis are well known in the art, including electron microscopy (including scanning electron microscopy and transmission electron microscopy), confocal microscopy, stereomicroscopy, and fluorescence microscopy. Histological analysis can reveal the basic structure and cell type.

[0310] The method of the present invention has high throughput (HTP) capability. In some embodiments, the method of the present invention may be carried out on a 96-well plate and / or a 384-well plate, preferably a 96-well plate.

[0311] verification The co-cultures of the present invention can be investigated to determine whether their characteristics meet expectations, for example, whether the co-cultures have been successfully developed and contain at least one organoid and at least one stromal cell in a viable state. This is also called "verification."

[0312] Verification may include determining the presence or absence of at least one change, as described herein. Accordingly, the present invention provides a method for determining the presence or absence of at least one change in a co-culture comprising at least one organoid and at least one stromal cell (i.e., a method for verifying a co-culture), the method comprising determining the presence or absence of at least one change in the co-culture.

[0313] Verification can be used, for example, as a quality control step during the preparation of co-cultures before use by other methods described herein.

[0314] Validation may include determining whether the co-culture exhibits the expected epithelial-stromal cell interactions in vivo. Validation may include determining whether there is at least one change in at least one stromal cell when combined with at least one organoid to form a co-culture, compared to at least one stromal cell before combining with at least one organoid to form a co-culture. Validation may include determining whether there is at least one change in at least one organoid when combined with at least one stromal cell to form a co-culture, compared to at least one organoid before combining with at least one stromal cell to form a co-culture.

[0315] The presence or absence of at least one of the changes described herein in the co-culture, compared to an expected value (such as an expected value for at least one reference organoid that has not been combined with at least one stromal cell to form a co-culture, or for at least one reference co-culture), can be determined as part of the verification.

[0316] inflammation Pro-inflammatory stimulation The cells, organoids, co-cultures, and methods of the present invention may be accompanied by pro-inflammatory stimulation, where the cells, organoids, or co-cultures are exposed to the pro-inflammatory stimulation. The pro-inflammatory stimulation causes the exposed cells to adopt a pro-inflammatory profile, which is useful for modeling health and disease states in vivo.

[0317] Accordingly, the present invention provides a co-culture exhibiting a pro-inflammatory profile, for example, a co-culture prepared in a culture medium containing at least one pro-inflammatory stimulus as described herein.

[0318] In some embodiments, the pro-inflammatory stimulus includes cytokines. Cytokines that cause cells, organoids, or co-cultures to adopt a pro-inflammatory profile are pro-inflammatory stimuli. These include IL-1β, oncostatin M (OSM), IL-6, IFNα, IFNβ, IFNλ, TGF-β, IL-23, IL-22, IL-4, IL-13, or IL-5. Of particular interest as pro-inflammatory stimuli are IL-1β and / or OSM. In some embodiments, the pro-inflammatory stimulus includes IL-1β. In some embodiments, the pro-inflammatory stimulus includes OSM. In some embodiments, the pro-inflammatory stimulus includes IL-1β and OSM. In some embodiments, the pro-inflammatory stimulus is not IL-2. In some embodiments, the pro-inflammatory stimulus is not TNF or IFNγ. In some embodiments, the pro-inflammatory stimulus is not IL-2, IFNγ, or TNF. In some embodiments, the pro-inflammatory stimulus is selected from the group consisting of IL-1β, IL-6, IFNα, IFNβ, IFNλ, TGF-β, IL-23, IL-22, IL-4, IL-13, or IL-5. In some embodiments, the pro-inflammatory stimulus does not include IL-4, IL-13, or TNFα. In some embodiments, the pro-inflammatory stimulus does not include IL-1β, IL-4, IL-13, or TNFα. In some embodiments, the pro-inflammatory stimulus does not include OSM. In some embodiments, the pro-inflammatory stimulus does not include IL-1β, IL-4, IL-13, TNFα, or OSM. This specification intends to describe other pro-inflammatory stimuli that are not cytokines, such as viral particles, single-stranded nucleic acids, double-stranded nucleic acids, RNA, DNA, and bacterial products (including lipopolysaccharide "LPS").

[0319] The step of contacting the co-culture with one or more pro-inflammatory stimuli may be performed simultaneously with or after combining at least one organoid and at least one stromal cell to form the co-culture. The step of contacting the co-culture with one or more pro-inflammatory stimuli may not be performed before combining at least one organoid and at least one stromal cell to form the co-culture.

[0320] The inventors have found that co-cultures exposed to one or more pro-inflammatory stimuli do not require subsequent restimulation because they maintain upregulation of pro-inflammatory genes (including PDPN and TGF-β). Therefore, in some embodiments, exposure to pro-inflammatory stimuli leads to upregulation of PDPN and / or TGF-β. In some embodiments, the pro-inflammatory profile includes upregulated PDPN and / or TGF-β compared to the case without pro-inflammatory stimuli.

[0321] The inventors did not observe a dose-dependent increase in the expression of pro-inflammatory genes upon exposure to pro-inflammatory stimuli. Therefore, the doses described in the examples are considered to maximize the induction of a pro-inflammatory profile in the co-culture.

[0322] In some embodiments, at least one pro-inflammatory stimulus is applied to the coculture such that at least one pro-inflammatory stimulus reaches a concentration of at least about 0.01 ng / ml, at least about 0.1 ng / ml, at least about 1 ng / ml, at least about 1.5 ng / ml, at least about 5 ng / ml, at least about 15 ng / ml, at least about 50 ng / ml, or at least about 100 ng / ml. Particularly preferred concentrations are at least about 1 ng / ml, and optionally at least about 15 ng / ml. These concentrations may refer to each pro-inflammatory stimulus individually or to all pro-inflammatory stimuli collectively. Preferably, these concentrations refer to each pro-inflammatory stimulus individually. Particularly preferred is a pro-inflammatory stimulus containing at least about 1 ng / ml of IL-1β and at least about 1 ng / ml of OSM.

[0323] In some embodiments, at least one pro-inflammatory stimulus is applied to the coculture such that at least one pro-inflammatory stimulus reaches a concentration of about 0.01 ng / ml, about 0.1 ng / ml, about 1 ng / ml, about 1.5 ng / ml, about 5 ng / ml, about 15 ng / ml, about 50 ng / ml, or about 100 ng / ml. Particularly preferred concentrations are about 1 ng / ml, and optionally about 15 ng / ml. These concentrations may refer to each pro-inflammatory stimulus individually or to all pro-inflammatory stimuli collectively. Preferably, these concentrations refer to each pro-inflammatory stimulus individually. Particularly preferred is a pro-inflammatory stimulus containing about 1 ng / ml of IL-1β and about 1 ng / ml of OSM.

[0324] In some embodiments, at least one pro-inflammatory stimuli is applied to the coculture such that at least one pro-inflammatory stimuli reaches a maximum concentration of about 0.01 ng / ml, about 0.1 ng / ml, about 1 ng / ml, about 1.5 ng / ml, about 5 ng / ml, about 15 ng / ml, about 50 ng / ml, or about 100 ng / ml. Particularly preferred concentrations are up to about 1 ng / ml, and optionally up to about 15 ng / ml. These concentrations may refer to each pro-inflammatory stimuli individually or to all pro-inflammatory stimuli collectively. Preferably, these concentrations refer to each pro-inflammatory stimuli individually. Particularly preferred is a pro-inflammatory stimuli containing up to about 1 ng / ml of IL-1β and up to about 1 ng / ml of OSM.

[0325] At least one pro-inflammatory stimulus may be added to the co-culture medium at two intervals of at least approximately 24 hours, at least approximately 48 hours, at least approximately 72 hours, or at least approximately 96 hours apart.

[0326] Therefore, in some embodiments, determining the presence or absence of at least one change includes applying at least one pro-inflammatory stimulus to the co-culture and determining the presence or absence of a pro-inflammatory profile.

[0327] In some embodiments, the pro-inflammatory stimulus is a therapeutic agent.

[0328] In some embodiments, the pro-inflammatory stimulus is a diagnostic agent.

[0329] In some embodiments, determining the presence or absence of at least one change includes determining the presence or absence of a pro-inflammatory profile.

[0330] In some embodiments, pro-inflammatory stimuli are not therapeutic agents.

[0331] In some embodiments, pro-inflammatory stimuli are not diagnostic agents.

[0332] In some embodiments, determining the presence or absence of at least one change includes adding at least one pro-inflammatory stimulus to the co-culture medium and optionally determining the presence or absence of a pro-inflammatory profile.

[0333] inflammatory stimulus In some embodiments, at least one inflammatory stimulus is applied to cells, organoids, or co-cultures, and the presence or absence of at least one change indicates whether an inflammatory response is observed in the co-culture. In some embodiments, the at least one inflammatory stimulus includes cytokines, such as damage-inducing cytokines. In some embodiments, the inflammatory cytokine includes TNF. In some embodiments, the inflammatory cytokine includes IFNγ. In some embodiments, the inflammatory stimulus does not include alcohol. Since the response of cells, organoids, or co-cultures is influenced by whether the cells, organoids, or co-cultures are subjected to a pro-inflammatory stimulus, cells, organoids, or co-cultures may be exposed to at least one pro-inflammatory stimulus and subsequently exposed to at least one inflammatory stimulus. While we do not wish to be bound by any theory, we believe that a pro-inflammatory stimulus leads to the adoption of a pro-inflammatory profile, which then leads to an inflammatory response (or increased inflammatory response) when subsequently exposed to inflammatory stimuli. In some embodiments, neither the pro-inflammatory stimulus nor the inflammatory stimulus contains IL-1β, IL-4, IL-13, TNFα, alcohol, or OSM.

[0334] Accordingly, the present invention provides a co-culture exhibiting an inflammatory response, for example, a co-culture prepared in a culture medium containing at least one pro-inflammatory stimulus and / or at least one inflammatory stimulus as described herein.

[0335] In some embodiments, at least one inflammatory stimulus is applied to the coculture such that at least one inflammatory stimulus reaches a concentration of at least about 0.01 ng / ml, at least about 0.1 ng / ml, at least about 1 ng / ml, at least about 1.5 ng / ml, at least about 5 ng / ml, at least about 15 ng / ml, at least about 25 ng / mL, at least about 50 ng / mL, or at least about 100 ng / mL. Particularly preferred concentrations are at least about 1.5 ng / ml, and optionally at least about 15 ng / ml. These concentrations may refer to each inflammatory stimulus individually or to all inflammatory stimuli collectively. Preferably, these concentrations refer to each inflammatory stimulus individually. Particularly preferred is an inflammatory stimulus containing at least about 15 ng / ml of TNF and at least about 15 ng / ml of IFNγ.

[0336] In some embodiments, at least one inflammatory stimulus is applied to the coculture such that at least one inflammatory stimulus reaches a concentration of about 0.01 ng / ml, about 0.1 ng / ml, about 1 ng / ml, about 1.5 ng / ml, about 5 ng / ml, about 15 ng / ml, about 25 ng / ml, about 50 ng / ml, or about 100 ng / ml. Particularly preferred concentrations are about 1.5 ng / ml, and optionally about 15 ng / ml. These concentrations may refer to each inflammatory stimulus individually or to all inflammatory stimuli collectively. Preferably, these concentrations refer to each inflammatory stimulus individually. Particularly preferred is an inflammatory stimulus containing about 15 ng / ml of TNF and about 15 ng / ml of IFNγ.

[0337] In some embodiments, at least one inflammatory stimulus is applied to the coculture such that at least one inflammatory stimulus reaches a maximum concentration of about 0.01 ng / ml, about 0.1 ng / ml, about 1 ng / ml, about 5 ng / ml, about 15 ng / ml, about 25 ng / ml, about 50 ng / ml, or about 100 ng / ml. Particularly preferred concentrations are up to about 1.5 ng / ml, and optionally up to about 15 ng / ml. These concentrations may refer to each inflammatory stimulus individually or to all inflammatory stimuli collectively. Preferably, these concentrations refer to each inflammatory stimulus individually. Particularly preferred is an inflammatory stimulus containing up to about 15 ng / ml of TNF and up to about 15 ng / ml of IFNγ.

[0338] In some embodiments, at least one change is measured at least about 6 hours, optionally at least about 12 hours, at least about 24 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours after the inflammatory stimulus is applied to the coculture.

[0339] Therefore, in some embodiments, determining the presence or absence of at least one change includes applying at least one inflammatory stimulus to the co-culture and determining the presence or absence of an inflammatory response.

[0340] The inflammatory response may include increased caspase activity and / or apoptosis in the co-culture.

[0341] The presence or absence of an inflammatory response can be determined by comparison with a reference co-culture, which is optionally prepared by (a) being free from at least one pro-inflammatory stimulus, (b) being free from at least one inflammatory stimulus, and / or (c) culturing stromal cells in stromal cell medium and combining the stromal cells with at least one reference organoid.

[0342] In some embodiments, determining the presence or absence of at least one change includes determining the presence or absence of an inflammatory response.

[0343] In some embodiments, the inflammatory stimulus is a therapeutic agent.

[0344] In some embodiments, inflammatory stimuli are diagnostic agents.

[0345] In some embodiments, determining the presence or absence of at least one change includes determining the presence or absence of a pro-inflammatory profile.

[0346] In some embodiments, inflammatory stimuli are not therapeutic agents.

[0347] In some embodiments, inflammatory stimuli are not diagnostic agents.

[0348] In some embodiments, determining the presence or absence of at least one change includes adding at least one inflammatory stimulus to the co-culture medium and optionally determining the presence or absence of an inflammatory response.

[0349] In some embodiments, determining the presence or absence of at least one change includes (a) adding at least one pro-inflammatory stimulus to the co-culture medium and determining the presence or absence of a pro-inflammatory profile, and (b) adding at least one inflammatory stimulus to the co-culture medium and optionally determining the presence or absence of an inflammatory response. These steps may be performed in the order listed.

[0350] disease In some embodiments, at least one organoid is a disease organoid, such as an organoid having an inflammatory disease phenotype and / or a fibrous disease phenotype. In some embodiments, the organoid is derived from an object having a disease such as an inflammatory disease or a fibrous disease.

[0351] Diseases involving interstitial components (e.g., diseases involving interstitial cells) can be investigated using the co-cultures and methods of the present invention, for example, in relation to treatment, diagnosis, and / or prognosis prediction. In principle, any disorder affecting interstitial cells can be investigated. Preferred diseases include diseases of the digestive and respiratory systems, particularly the intestines and lungs. Exemplary diseases include irritable bowel disease (IBD), ulcerative colitis (UC), celiac disease, leaky gut syndrome, chronic obstructive pulmonary disease (COPD), and asthma. Particularly preferred diseases include diseases of the digestive system, particularly the intestines. Exemplary diseases include irritable bowel disease (IBD), ulcerative colitis (UC), celiac disease, and leaky gut syndrome.

[0352] In some embodiments, at least one organoid can be cultured with at least one stromal cell derived from a diseased subject to form a co-culture, and separately, at least one other organoid can be cultured with at least one stromal cell derived from a non-disease subject as a reference co-culture.

[0353] Fibrosis Particularly interesting disorders include fibrous disorders such as intestinal fibrosis, solitary rectal ulcers, radiation enteropathy, and eosinophilic enteropathy. Because at least one stromal cell is present in the co-culture of the present invention, this co-culture is particularly suitable for investigating the suitability of therapeutic agents for treating fibrous diseases. In some embodiments, at least one organoid and / or at least one stromal cell is derived from fibrous tissue.

[0354] inflammation Particularly interesting disorders include inflammatory disorders such as inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC). Because of its ability to accept pro-inflammatory stimuli, the co-culture of the present invention is particularly suitable for investigating the suitability of therapeutic agents for treating inflammatory diseases. In some embodiments, at least one organoid and / or at least one stromal cell are derived from inflammatory tissue.

[0355] Therapeutic drugs The present invention provides a method for testing therapeutic agents for treating diseases, such as those disclosed elsewhere in this specification. Testing of at least one therapeutic agent for a disease may include testing the tolerability and / or efficacy of the agent for treating a specific disease, testing the tolerability and / or efficacy of the agent for treating a broad group of diseases, or testing the tolerability and / or efficacy of the agent in a context other than any specific disease (e.g., by testing the activation of a specific molecular pathway).

[0356] Anti-inflammatory agents, such as small molecule anti-inflammatory drugs, can be used as treatments for these diseases. Of particular interest are anti-inflammatory agents for treating IBD, UC, and / or CD, including corticosteroids (such as prednisone), aminosalicylates (such as mesalamine, valsalazid, and orsalazine), immunosuppressants (such as azathioprine, mercaptopurine, and methotrexate), as well as anti-inflammatory agents (such as tofacitinib, upadacitinib, and ozanimod), biologics (such as infliximab, adalimumab, golimumab, certolizumab, vedolizumab, ustekinumab, and risankizumab), and antibiotics (such as ciprofloxacin and metronidazole).

[0357] At least one therapeutic agent may include at least one cytokine, for example, at least one pro-inflammatory stimulus such as IL-1β and / or OSM.

[0358] At least one therapeutic agent may include at least one cytokine, such as TNF and / or IFNγ, which are inflammatory stimuli.

[0359] Therapeutic testing may involve exposing co-cultures to therapeutic levels of the therapy with known or unknown efficacy and / or known or unknown tolerability.

[0360] Typically, the agent is dissolved in a solution to the (expected) therapeutically effective concentration. The solution can be administered to the co-culture by infusion into the container in which the co-culture is maintained (or by other appropriate administration).

[0361] In some embodiments, the agent is an approved or experimental agent for gastrointestinal diseases or disorders such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease, or leaky gut syndrome. In some embodiments, the agent is tofacitinib, preferably 10 μM tofacitinib.

[0362] Testing at least one therapeutic agent may include testing the effectiveness of the agent for treating a disease. Testing at least one therapeutic agent may include testing the tolerability of the agent for treating a disease.

[0363] In some embodiments, the method includes selecting a therapeutic agent based on the efficacy and / or tolerability determined by the method of the present invention, and using the agent in a treatment by selectively administering the therapeutic agent to a target.

[0364] In addition to testing therapeutic agents using the co-cultures of the present invention, the co-cultures of the present invention can be used in a method for identifying at least one target for at least one therapeutic agent. For example, a target gene can be genetically modified in at least one stromal cell and / or at least one organoid, and the presence or absence of at least one change in the co-culture can be determined according to the general principle of determining the presence or absence of at least one change as disclosed elsewhere herein, to determine whether the target gene has therapeutic significance (e.g., whether the target gene affects the tolerability and / or efficacy of at least one therapeutic agent). Genetic modification may include insertion, deletion, or mutation of a gene in the genome of at least one stromal cell. Target genes may include genes encoding cell surface receptors and cell signaling molecules such as cytokines. The presence or absence of at least one change can be determined in any co-culture of the present invention including genetic modification compared to a reference co-culture without genetic modification. In such embodiments, it is not necessary to apply at least one therapeutic agent to the co-culture. In other embodiments, the presence or absence of at least one change can be determined by comparing the co-culture containing the genetic modification with the co-culture before the application of at least one therapeutic agent, and / or with a reference co-culture without the genetic modification. Thus, the methods for testing therapeutic agents described herein may include identifying therapeutic targets.

[0365] Personalized medicine One approach to testing therapeutic agents can be described as a “personalized medicine” approach to testing. A personalized medicine approach may involve testing one or more therapeutic agents known to be suitable for treatment, and determining whether those agents are suitable (e.g., effective and / or tolerable) for treating a disorder in a subject (i.e., a specific subject for whom the therapeutic agents are being tested). A personalized medicine approach may utilize at least one organoid and / or at least one stromal cell derived from the same patient.

[0366] screening Another means of testing therapeutic agents can be described as a “screening” approach to testing. A screening approach may involve testing one or more therapeutic agents in terms of their efficacy or tolerability in treating a disease, for example, in treating a specific disease, and determining whether one or more agents are suitable for treatment (e.g., effective and / or tolerable). A screening approach may use at least one organoid and / or at least one stromal cell derived from different patients. A screening approach may use at least one organoid and / or at least one stromal cell derived from an immortalized cell line. A screening approach may use more than one pair, e.g., more than two, more than three, more than four, more than five, more than ten, more than 20, more than 50, more than 100, or more than 1,000 pairs, of at least one organoid and / or at least one stromal cell derived from different patients.

[0367] diagnosis The co-cultures and methods of the present invention enable faithful modeling of in vivo physiological functions in an in vitro environment, thereby facilitating disease diagnosis or prognosis prediction by determining the presence or absence of at least one change (as described elsewhere herein). Diagnosis and / or prognosis prediction may involve applying a diagnostic agent to the co-culture, e.g., a diagnostic agent that alters gene expression, signaling pathway activity, receptor function, ligand function, or other cellular processes, thereby enabling differential diagnosis or prognosis prediction based on the presence or absence of at least one change after administration.

[0368] Diagnosis and / or prognosis prediction may involve determining the presence or absence of at least one change after applying at least one diagnostic agent to a co-culture, wherein the at least one diagnostic agent comprises at least one agonist or antagonist of the NF-κB, MAPK, and / or JAK-STAT pathway. Particularly preferred are at least one agonist or antagonist of the JAK-STAT pathway. Regulators of fluid transport and / or ion channel activity are also of interest.

[0369] Diagnosis and / or prognosis prediction may include determining the presence or absence of at least one change after applying at least one diagnostic agent to a coculture, wherein the at least one diagnostic agent comprises at least one cytokine, and optionally, the at least one diagnostic agent comprises IL-1β or OSM.

[0370] At least one diagnostic agent may include at least one cytokine, for example, at least one pro-inflammatory stimulus such as IL-1β and / or OSM.

[0371] At least one diagnostic agent may include at least one cytokine, such as TNF and / or IFNγ, which are inflammatory stimuli.

[0372] In some embodiments, the diagnostic agent is a therapeutic agent.

[0373] Diagnosis and / or prognosis prediction may include determining the presence or absence of at least one change after applying at least one diagnostic agent to a co-culture, wherein the at least one diagnostic agent includes at least one therapeutic agent, in particular known therapeutic agents. The presence or absence of at least one change after administration of at least one therapeutic agent (such as a response in the co-culture indicating whether the molecular basis of the disease symptoms has been treated) may aid in differential diagnosis by inferring the cause of the disease.

[0374] Diagnosis and / or prognosis prediction may include determining the presence or absence of at least one change in the co-culture compared to a reference co-culture. For example, the co-culture may include at least one organoid and / or at least one stromal cell derived from a first patient with the disease, and the presence or absence of at least one change may be determined by comparison with a reference co-culture containing at least one organoid and / or at least one stromal cell derived from a second patient without the disease.

[0375] Methods for diagnosis and / or prognosis prediction may include analyzing the presence or absence of at least one change and identifying the disease or clinical outcome.

[0376] In some embodiments, the method includes selecting a therapeutic agent for treating a disease based on a diagnosis and / or prognosis determined by the method of the present invention, and optionally using the therapeutic agent in a treatment of the disease by administering the therapeutic agent to a target.

[0377] The present invention provides methods for determining the presence or absence of disease in a subject and / or predicting its prognosis, as described herein. These methods can be described as methods for determining the presence or absence of disease in a subject and / or susceptibility to disease. These methods can be described as methods for diagnosing and / or predicting the prognosis of a subject, where diagnosing a subject includes determining whether or not the subject has a disease, and / or predicting the prognosis of a subject includes determining whether or not the subject is susceptible to disease.

[0378] conditioned medium The methods of the present invention, in particular methods for testing at least one therapeutic agent and for diagnostic and / or prognostic prediction, may involve combining at least one organoid with a conditioned medium obtained from at least one stromal cell. Therefore, any method involving combining at least one organoid with at least one stromal cell to form a co-culture may instead involve combining at least one organoid with a conditioned medium obtained from at least one stromal cell (i.e., without forming a co-culture). While we do not wish to be bound by any theory, this is because we believe that the effect of stromal cells on organoids is at least partially due to soluble factors secreted by the stromal cells into the surrounding culture medium. The methods of the present invention may include the step of isolating and collecting the conditioned medium from the co-culture and detecting soluble factors in the conditioned medium.

[0379] These methods involving conditioned medium may, but are not necessarily, involve isolating the conditioned medium from at least one stromal cell. Isolation of the conditioned medium can be performed at least about 6 hours after culturing at least one stromal cell in stromal cell medium, for example, at least about 12 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.

[0380] Accordingly, the present invention provides organoids exhibiting a pro-inflammatory profile, for example, organoids prepared in a conditioned medium derived from stromal cells, the stromal cells being prepared in a stromal cell medium containing at least one pro-inflammatory stimulus.

[0381] Additional methods and products of the present invention kit The present invention provides a kit comprising any organoid, stromal cells, or co-culture of the present invention.

[0382] In some embodiments, the kit includes one or more of a syringe, an alcohol swab, a cotton ball, a gauze pad, and instructions for carrying out the method of the present invention.

[0383] Embodiment The present invention further provides the following numbered embodiments. 1. A method for preparing a co-culture comprising at least one organoid and at least one stromal cell, To form the aforementioned co-culture, the at least one organoid is combined with the at least one stromal cell in the co-culture medium. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 2. A method for determining the presence or absence of at least one change in a co-culture comprising at least one organoid and at least one stromal cell, To form the aforementioned co-culture, the at least one organoid is combined with the at least one stromal cell in the co-culture medium, To determine whether or not the aforementioned at least one change is present in the co-cultured product. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 3. A method for determining whether or not at least one change is present in at least one organoid, To isolate a conditioned medium from at least one stromal cell and combine the conditioned medium with at least one organoid, To determine whether or not at least one change has occurred in the organoid. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 4. A method for testing at least one therapeutic agent for a disease, To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, Applying the aforementioned at least one therapeutic agent to the co-culture, and determining whether or not there is at least one change in the co-culture. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 5. A method for testing at least one therapeutic agent for a disease, To isolate a conditioned medium from at least one stromal cell and combine the conditioned medium with at least one organoid, Applying the aforementioned at least one therapeutic agent to the organoid, To determine whether or not at least one change has occurred in the organoid. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 6. The method of Embodiment 4 or Embodiment 5, wherein testing the at least one therapeutic agent includes testing the tolerability of the at least one therapeutic agent. 7. The method according to any one of Embodiments 2 to 6, wherein the presence or absence of the at least one change is determined by comparison with a reference organoid, and optionally, the at least one therapeutic agent is not applied to the reference organoid and / or the reference organoid does not contain stromal cells. 8. The method according to any one of Embodiments 2 to 7, wherein the presence or absence of the at least one change is determined by comparison with a reference co-culture, and the at least one therapeutic agent is optionally not applied to the reference co-culture. 9. A method for determining the presence or absence of a disease in a subject and / or prognosis prediction, To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, Applying at least one diagnostic agent to the co-culture, To determine whether or not at least one change has occurred in the aforementioned co-culture product. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 10. A method for determining whether or not there is a diagnosis and / or prognosis prediction of a disease in a subject, To isolate a conditioned medium from at least one stromal cell and combine the conditioned medium with at least one organoid, Applying at least one diagnostic agent to the at least one organoid, To determine whether or not at least one change has occurred in the aforementioned at least one organoid. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in a stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in organoid medium, The aforementioned method. 11. The method according to Embodiment 9 or Embodiment 10, wherein the at least one diagnostic agent is an agonist or antagonist of the IL-1β and / or OSM pathway, optionally the at least one diagnostic agent is a cytokine, and optionally the at least one diagnostic agent comprises IL-1β and / or OSM. 12. The method according to any one of Embodiments 3, 5, or 10, wherein the isolation of the conditioned medium is performed at least about 6 hours after the preparation of the at least one stromal cell. 13. The method according to any one of Embodiments 2, 4, 6-9, or 11, wherein the presence or absence of the at least one change is determined by comparing it with the at least one organoid before the co-culture was formed. 14. The method according to any one of Embodiments 4 to 13, wherein the disease is an inflammatory disease or a fibrous disease. 15. The method according to Embodiment 14, wherein the disease is an inflammatory disease, optionally, the inflammatory disease is a gastrointestinal inflammatory disease, optionally, the inflammatory disease is inflammatory bowel disease (IBD), optionally, the IBD includes ulcerative colitis (UC) or Crohn's disease (CD). 16. The method according to any one of the prior embodiments, wherein the at least one organoid is derived from primary epithelial cells or immortalized epithelial cells. 17. The method according to any one of the prior embodiments, wherein at least one of the organoids is a patient-derived organoid. 18. The method according to any one of the prior embodiments, wherein the epithelial cells are primary epithelial cells or immortalized epithelial cells, and optionally the primary epithelial cells are patient-derived epithelial cells. 19. The method according to any one of the prior embodiments, wherein the at least one stromal cell comprises a fibroblast, optionally, the fibroblast being an intestinal fibroblast, for example, a colonic fibroblast or a small intestinal fibroblast, and optionally, the fibroblast being an immortalized human colonic fibroblast, a human colonic fibroblast, an immortalized human small intestinal fibroblast, or a human small intestinal fibroblast. 20. The co-culture, the at least one organoid, and / or the at least one stromal cell, (a) derived from the subject and / or (b) derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue, and / or (c) of fibrous tissue and / or (d) of inflammatory tissue and / or (e) Mammalian cells, optionally including or consisting of human cells, and / or (f) Derived from the intestine, and optionally from the colon, The method according to any one of Embodiments 4 to 19. 21. The method according to any one of the prior embodiments, wherein the at least one organoid does not contain epithelial cells derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue, and optionally, the at least one organoid does not contain epithelial cells or stromal cells derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue. 22. The epithelial cells and / or the stromal cells, (a) derived from the subject and / or (b) derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue, and / or (c) of fibrous tissue and / or (d) Mammalian cells, optionally human cells, and / or (e) of intestine, and optionally of colon, The method according to any one of Embodiments 4 to 21. 23. The method according to any one of the prior embodiments, wherein the at least one organoid and the at least one stromal cell originate from the same subject and optionally originate from (a) the same sample from the subject and / or (b) the same tissue within the same subject. 24. The method according to any one of the prior embodiments, wherein the epithelial cells and stromal cells originate from the same subject and are optionally derived from (a) the same sample from the subject and / or (b) the same tissue within the same subject. 25. The method according to Embodiment 23 or Embodiment 24, wherein the sample includes a tissue biopsy, and optionally, the sample includes an intestinal tissue biopsy. 26. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-25, wherein determining the presence or absence of at least one change in the co-culture includes determining the presence or absence of stromal cells in the co-culture and optionally determining the amount of stromal cells in the co-culture. 27. The method according to any one of the prior embodiments, wherein the at least one organoid is prepared by splitting a precursor organoid. 28. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-27, wherein combining the at least one organoid with the at least one stromal cell comprises seeding the at least one organoid and the at least one stromal cell simultaneously. 29. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-28, wherein the at least one organoid and the at least one stromal cell are combined in a ratio such that approximately 0.5 to approximately 2.5 stromal cells are seeded per organoid, and optionally the ratio is such that approximately 0.5 stromal cells are seeded per organoid or approximately 2.5 stromal cells are seeded per organoid. 30. The method according to any one of the prior embodiments, wherein the epithelial cells are cultured in the organoid medium for at least about 24 hours to prepare the at least one organoid. 31. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-30, wherein the co-culture is a three-dimensional co-culture, and optionally combining the at least one organoid with at least one stromal cell includes a hanging drop method. 32. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-31, wherein the at least one organoid and the at least one stromal cell are combined in a multiwell plate format, and optionally the multiwell plate includes an ultra-low adhesion plate. 33. The method according to any one of Embodiments 2, 4, 6-9, or 11-32, comprising isolating the conditioned medium from the co-culture and detecting soluble factors in the conditioned medium. 34. The method according to any one of the prior embodiments, wherein at least one stromal cell is modified to alter gene expression. 35. The method according to any one of the prior embodiments, wherein the epithelial cells are epithelial stem cells. 36. The method according to any one of the prior embodiments, wherein at least one organoid is characterized by Lgr5 expression. 37. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-36, wherein determining the presence or absence of at least one change comprises adding at least one inflammatory stimulus to the co-culture medium and determining the presence or absence of an inflammatory response. 38. The method according to Embodiment 37, wherein the at least one inflammatory stimulus comprises a cytokine, and optionally the cytokine comprises TNF and / or IFNγ. 39. The method according to Embodiment 37 or 38, wherein the inflammatory response includes an increase in caspase activity and / or apoptosis in the co-culture. 40. The method according to any one of Embodiments 37 to 39, wherein the presence or absence of an inflammatory response is determined by comparison with a reference co-culture, and optionally, the reference co-culture is prepared by (a) not receiving the at least one inflammatory stimulus and / or (b) culturing stromal cells in stromal cell proliferation medium and combining the stromal cells with at least one reference organoid. 41. The method according to any one of Embodiments 2 to 40, wherein the at least one change includes a change in organoid morphology, a change in organoid size or epithelial cell size, a decrease in cell viability, a decrease in cell proliferation, an increase in cell death, a change in secretome profile, a change in cytokine secretion, an increase in cell apoptosis, an increase in caspase activity, and / or a change in the expression of one or more genes, and optionally, the change in the expression of one or more genes includes a change in one or more disease biomarkers, one or more fibrosis biomarkers, and / or one or more inflammatory biomarkers. 42. The method according to any one of Embodiments 2 to 41, wherein determining the presence or absence of at least one change includes a cell proliferation assay, viability assay, flow cytometry analysis, ELISA, gene expression analysis, and / or cell imaging. 43. The method according to any one of Embodiments 2 to 42, wherein determining the presence or absence of at least one of the changes is used to determine the degree of organoid cell death. 44. The at least one change includes a change in the morphology of the organoid, and optionally the change in morphology is (a) Increase in organoid area, and / or (b) Increased organoid aggregation, and / or (c) Increase in lumen size, and / or (d) Increase in epithelial cell size, and / or (e) Reduction of adhesion to organoid surfaces The method according to any one of embodiments 2 to 43, including the method described above. 45. The at least one change includes an increase in the area of ​​the at least one organoid, optionally the area being at least about 10,000 μm² 2, at least about 15,000 μm 2 , at least about 20,000 μm 2 , or at least about 25,000 μm 2 The method according to any one of Embodiments 2 to 44, which increases to 46. The method according to Embodiment 44 or Embodiment 45, wherein the area of the at least one organoid increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 47. The method according to any one of Embodiments 2, 4, 6 to 9, 11, or 13 to 46, wherein the at least one change is determined at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days after combining at least one organoid and at least one stromal cell to form a co-culture. 48. The method according to any one of Embodiments 2 to 47, wherein the at least one change includes upregulation of the expression of a stem cell marker, optionally the stem cell marker is OLFM4, and optionally the upregulation is at least about 10%, at least about 50%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1,000%. 49. The method according to any one of Embodiments 2 to 48, wherein the at least one change includes downregulation of the expression of an intestinal cell marker, optionally the intestinal cell marker is ALPI, and optionally the downregulation is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. 50. The method according to any one of Embodiments 2 to 49, wherein the at least one change comprises downregulation of the expression of a growth marker, optionally the growth marker being KI67, and optionally the downregulation being at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. 51. The method according to any one of Embodiments 2 to 50, wherein the at least one change comprises a reduction in epithelial thickness, optionally wherein the reduction is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. 52. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-51, wherein the co-culture is cultured for at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, or at least about 120 hours before determining whether or not there is at least one change in the co-culture. 53. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-52, wherein the presence or absence of the at least one change is determined by comparing the state after the at least one organoid and at least one stromal cell have been combined to form the co-culture with the state before the at least one organoid and at least one stromal cell have been combined to form the co-culture. 54. The method according to any one of Embodiments 2 to 53, wherein the at least one change includes a change in the secretome profile, optionally a change in cytokine secretion, or optionally a change in IL-6 and / or CXCL2 secretion. 55. The method according to any one of Embodiments 2 to 54, wherein the at least one change includes the expression of at least about 10,000 pg / ml of IL-6, at least about 20,000 pg / ml of IL-6, at least about 30,000 pg / ml of IL-6, at least about 40,000 pg / ml of IL-6, at least about 50,000 pg / ml of IL-6, or at least about 100,000 pg / ml of IL-6. 56. The method according to any one of Embodiments 2 to 55, wherein the at least one change includes a change in caspase activity, optionally including a change in caspase 3 / 7 activity, optionally increasing the caspase activity by at least about 500%, at least about 1,000%, at least about 1,500%, at least about 2,000%, or at least about 2,500%. 57. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-56, wherein, before determining the presence or absence of at least one change in the co-culture, at least one inflammatory stimulus is added to the co-culture medium, optionally, the at least one inflammatory stimulus comprises a cytokine, optionally, the cytokine comprises TNF and / or IFNγ, and optionally, the cytokine is present in a concentration of up to about 100 ng / ml, up to about 50 ng / ml, up to about 15 ng / ml, up to about 5 ng / ml, up to about 1 ng / ml, up to about 0.1 ng / ml, or up to about 0.01 ng / ml. 58. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-57, wherein a cytokine signaling inhibitor is added to the co-culture before determining whether or not at least one change is present in the co-culture, optionally the cytokine signaling inhibitor reduces the activity of the JAK / STAT pathway, and optionally the cytokine signaling inhibitor is tofacitinib. 59. The method according to any one of the prior embodiments, wherein the organoid medium comprises mitotic growth factor, a BMP inhibitor, and R-spondin, and optionally, the organoid medium comprises a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-spondin. 60. The method according to any one of the prior embodiments, wherein the interstitial cell culture medium comprises a basic medium, an amino acid supplement, an antibiotic, and optionally serum. 61. The method according to any one of the prior embodiments, wherein the organoid medium and / or the stromal cell medium comprises an extracellular matrix, optionally the extracellular matrix is ​​Matrigel®, optionally the Matrigel® is 5% or 10% Matrigel®, and optionally the Matrigel® is 5% Matrigel®. 62. The co-culture medium is (A) mitotic growth factor, BMP inhibitor, and R-spongin (optionally, the co-culture medium comprises a Wnt agonist, mitotic growth factor, BMP inhibitor, and R-spongin; optionally, the co-culture medium comprises N-Ac, TGF-β inhibitor, mitotic growth factor, gastrin, BMP inhibitor, antibiotic, R-spongin, Notch pathway inhibitor, ERK inhibitor, and Wnt agonist), and / or (B) Serum (optional fetal bovine serum, optional 5% fetal bovine serum) The method according to any one of embodiments 2, 4, 6-9, 11, or 13-61, including the method described above. 63. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-62, wherein the organoid medium and / or co-culture medium does not contain an extracellular matrix. 64. The method according to any one of Embodiments 2, 4, 6-9, 11, or 13-63, wherein determining the presence or absence of the at least one change comprises adding at least one pro-inflammatory stimulus to the co-culture medium, and optionally, determining the presence or absence of the at least one change comprises (a) adding at least one pro-inflammatory stimulus to the co-culture medium and determining the presence or absence of a pro-inflammatory profile, and / or (b) adding at least one inflammatory stimulus to the co-culture medium and determining the presence or absence of an inflammatory response. 65. The method according to Embodiment 64, wherein the at least one pro-inflammatory stimulus is optionally added to the co-culture medium at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, or at least about 96 hours before adding the optional inflammatory stimulus to the co-culture medium. 66. The method according to any one of embodiments 37-40, 57, or 64-65, wherein at least one pro-inflammatory stimulus is added to the co-culture medium before the at least one inflammatory stimulus is added to the co-culture medium, and optionally, the at least one pro-inflammatory stimulus is added to the co-culture medium at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, or at least about 96 hours before the at least one inflammatory stimulus is added to the co-culture medium. 67. The method according to any one of embodiments 64 to 66, wherein the at least one pro-inflammatory stimulus comprises at least one cytokine. 68. The method according to Embodiment 67, wherein the at least one cytokine comprises IL-1β and / or oncostatin M (OSM). 69. The method according to any one of embodiments 64 to 68, wherein the at least one pro-inflammatory stimulus is added to the organoid co-culture medium at two intervals of at least about 24 hours, at least about 48 hours, at least about 72 hours, or at least about 96 hours apart. 70. The method according to any one of embodiments 64 to 69, wherein the at least one pro-inflammatory stimulus comprises IL-1β, optionally the concentration of IL-1β is at least about 20 ng / ml, optionally the concentration is at least about 5 ng / ml, at least about 1 ng / ml, at least about 0.1 ng / ml, or at least about 0.01 ng / ml, preferably the concentration is at least about 1 ng / ml. 71. The method according to any one of Embodiments 64 to 70, wherein the at least one pro-inflammatory stimulus comprises IL-1β, optionally the concentration of IL-1β is up to about 20 ng / ml, optionally the concentration is up to about 5 ng / ml, up to about 1 ng / ml, at least about 0.1 ng / ml, or at least about 0.01 ng / ml, preferably the concentration is up to about 1 ng / ml. 72. The method according to any one of Embodiments 64 to 71, wherein the at least one pro-inflammatory stimulus comprises IL-1β, optionally having a concentration of IL-1β of about 0.01 ng / ml to about 20 ng / ml, optionally having a concentration of about 0.1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 20 ng / ml, or about 1 ng / ml to about 5 ng / ml, optionally having a concentration of about 1 ng / ml. 73. The method according to any one of embodiments 64 to 69, wherein the at least one pro-inflammatory stimulus comprises OSM, optionally the concentration of OSM is at least about 20 ng / ml, optionally the concentration is at least about 5 ng / ml, at least about 1 ng / ml, at least about 0.1 ng / ml, or at least about 0.01 ng / ml, preferably the concentration is at least about 1 ng / ml. 74. The method according to any one of embodiments 64-69 or 73, wherein the at least one pro-inflammatory stimulus comprises OSM, optionally having a maximum concentration of OSM of about 20 ng / ml, optionally having a maximum concentration of about 5 ng / ml, a maximum of about 1 ng / ml, at least about 0.1 ng / ml, or at least about 0.01 ng / ml. 75. The method according to any one of Embodiments 64-69 or 73-74, wherein the at least one pro-inflammatory stimulus comprises OSM, optionally having a concentration of OSM of about 0.01 ng / ml to about 20 ng / ml, optionally having a concentration of about 0.1 ng / ml to about 20 ng / ml, about 1 ng / ml to about 20 ng / ml, or about 1 ng / ml to about 5 ng / ml, optionally having a concentration of about 1 ng / ml. 76. The method according to any one of Embodiments 64 to 75, wherein the at least one pro-inflammatory stimulus comprises IL-1β and OSM, and optionally, the at least one pro-inflammatory stimulus comprises at least about 1 ng / ml of IL-1β and at least about 1 ng / ml of OSM. 77. Co-culture according to any one of Embodiments 2, 4, 6-9, 11, or 13-76. 78. Co-culture medium according to any one of Embodiments 2, 4, 6-9, 11, or 13-76. 79. The stromal cell culture medium according to any one of the prior claims. 80. Co-cultures substantially as described herein. 81. Co-culture medium substantially as described herein. 82. Interstitial cell culture medium substantially as described herein. [Examples]

[0384] Other features, purposes, and advantages of the present invention will become apparent in the following examples. However, it should be understood that the examples illustrate embodiments of the present invention, but are presented only as examples and not as limitations. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the examples.

[0385] All patents and references cited herein are incorporated herein by reference in their entirety.

[0386] Culture medium The following culture media are used in the examples. The amount of reagent is indicated in the units listed, or as a percentage (or v / v%) of the final volume of the medium, or as a multiple (e.g., "1x") as directed by the manufacturer.

[0387] Ad-DF+++ TIFF2026525328000002.tif47165

[0388] Ad-DF+++ modified with additional HEPES TIFF2026525328000003.tif47165

[0389] Organoid growth medium (or "Colon Normal Medium", "CNM") TIFF2026525328000004.tif113165

[0390] Co-culture differentiation medium (“complex colonic differentiation medium”, “cCDM”) TIFF2026525328000005.tif113165

[0391] cCDM+serum TIFF2026525328000006.tif27165

[0392] Co-culture differentiation medium ("Intestinal Cell-Colon Differentiation Medium", "eCDM") TIFF2026525328000007.tif112165

[0393] If incubation is performed, it is overnight at 37°C and 5% CO2 unless otherwise instructed. Incubation may be performed for a longer period, with the culture medium being changed every 2-3 days.

[0394] Where indicated, the experiment involved the fibroblast cell lines shown in Table 1. TIFF2026525328000008.tif61165

[0395] Example 1: Preparation of fibroblasts and organoids This example provides an exemplary protocol used for preparing fibroblasts and organoids for subsequent use in Example 2.

[0396] Preparation of fibroblasts Fibroblast samples stored at -80°C were thawed and replaced with Ad-DF+++ medium containing 10% FBS and 25 mM HEPES. The fibroblasts were then transferred to a T75 flask coated with poly-L-lysine and incubated.

[0397] To treat the fibroblasts with trypsin, the culture medium was aspirated and the cells were washed with 10 mL of DPBS (Dulbeccio phosphate-buffered saline). The DPBS was then aspirated. 3 mL of TrypLE was added to the flask and shaken to ensure complete coating. The flask was then incubated at 37°C for 2 minutes.

[0398] Next, TrypLE containing fibroblasts was transferred to fresh Ad-DF+++ medium (5 ml of Ad-DF+++ per 1 ml of TrypLE) for neutralization, and then centrifuged at 1500 rpm (450 × g) for 5 minutes. Finally, the supernatant was aspirated, the cells were resuspended in 1 ml of Ad-DF+++ medium, and counted.

[0399] Preparation of organoids Biopsies derived from human colon tissue were collected in 50 mL canonical tubes containing 10–15 mL of ice-cold Advanced DMEM / F12 medium supplemented with penicillin / streptomycin (from a 100-fold stock solution of 10,000 U / mL penicillin and 10,000 μM / mL streptomycin), HEPES (from a 100-fold stock solution of 1 M), GlutaMAX (from a 100-fold stock solution; all Gibco®), and the Rho kinase inhibitor Y-27632 (Sigma-Aldrich). Biopsies were stored on ice and could be processed immediately or stored at 4°C for up to 24 hours until isolation was initiated.

[0400] The colonic mucosa is treated with EDTA to release the crypts in order to induce colonic organoids. The muscular layer and fat are removed using surgical scissors and forceps under a dissecting microscope. The washed tissue is cut into thin strips of approximately 1-2 mm. One strip is fixed in 4% formaldehyde (Sigma-Aldrich) for histological analysis, and the other strip is rapidly frozen (in dry ice or liquid nitrogen) and stored at -80°C for genetic and / or protein analysis.

[0401] The remaining strips were washed three times with fresh chelation solution (5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose, and 54.9 mM D-sorbitol dissolved in sterile water; all Sigma-Aldrich). After washing, the strips were incubated for 30 minutes on a rotating wheel (in a cold room) at 4°C in a chelation solution supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA; homemade) and 0.5 mM DL-dithiothreitol (DTT; Sigma-Aldrich).

[0402] The tube was vigorously shaken to release the colonic crypts from the mesenchymal tissue. If the crypts were not visible, incubation was repeated using freshly added chelate solution. The tissue fragments were allowed to settle for 1-2 minutes, and the supernatant containing the crypts was transferred to a new tube. 5-10 mL of fetal bovine serum (FCS; Sigma-Aldrich) was added, and the crypts were centrifuged at 300 × g for 5 minutes at 4°C.

[0403] The crypts were washed three times with supplemented advanced DMEM / F12. The crypts were resuspended in a mixture of Matrigel / BME and medium (70%), seeded in dome shapes at different densities, and placed in a humidified incubator at 37°C and 5% CO2 for 30 minutes. Once the Matrigel / BME had solidified, CNM supplemented with the Rho kinase inhibitor Y-27632 was added. Organoids formed from the crypts were subculturised every 7–10 days.

[0404] Subsequently, the organoid cultures can be subjected to primary analysis using whole-genome sequencing, mRNA sequencing, and peptidomedication profiling.

[0405] Organoid division Organoid cultures were fragmented ("divided") by pipetting a 1 mL micropipette (P1000 Gilson) up and down through the growth medium in Matrigel / BME droplets. The fragmented organoids were centrifuged at 500 × g for 5 minutes. The pelletized organoids were resuspended in an excess volume of fully advanced DMEM / F12 and centrifuged at 500 × g for 5 minutes. The organoid fragments were re-seed at the desired density on 70% Matrigel in CNM containing the Rho kinase inhibitor Y-27632 (1 / 1000) and placed in a humidified incubator at 37°C and 5% CO2.

[0406] The culture medium was changed every 3-4 days. The organoids were subcultured every 7-10 days.

[0407] Collection of organoids 20 μL of 100-fold dispase solution was added to each well of a 6-well plate (2 ml) containing the organoids prepared above, and the plate was incubated at 37°C and 5% CO2 for 30 minutes. Organoids were collected from all wells by pipetting through a 100 μm filter (pre-wetted twice with 10 mL of Ad-DF+++ medium) into a 50 mL plastic tube using a P1000 pipette. The 100 μm filter containing the organoids was then washed twice with 10 mL of Ad-DF+++ medium. The flow-through was filtered through a 20 μm filter (pre-wetted twice with 10 mL of Ad-DF+++ medium). The 20 μm filter (containing organoids) was inverted and washed with Ad-DF+++ medium (4 times the flow-through volume) supplemented with the Rho kinase inhibitor Y-27632. The flow-through was then centrifuged at 1500 rpm (450 x g) for 3 minutes at 8°C.

[0408] Finally, the supernatant was carefully aspirated, and the organoid pellet was resuspended in 1 mL of cCDM+ serum per 6-well plate before being counted.

[0409] Example 2: Formation of co-culture The organoid and fibroblast cultures prepared as described in Example 1 were placed on ice. The organoids and fibroblasts were mixed in cCDM containing 5% serum, 5% Matrigel, and the Rho kinase inhibitor Y-27632. The solution was kept at a low temperature and 100-200 μl was dispensed into each well of a 96-well plate.

[0410] The co-culture cells were released from BME using a cell recovery solution and fixed in 4% paraformaldehyde. The fixed whole mounts were stained with phalloidin to mark polymerized actin, and the nuclei were labeled with DAPI. The whole mounts were mounted on slides in ProLong Gold anti-fading mounting medium and imaged using a Leica SP8X confocal microscope (data not shown).

[0411] Example 3: Induction of pro-inflammatory profiles in co-cultures Co-cultures prepared as described in Examples 1 and 2 were induced to exhibit a pro-inflammatory profile by exposure to pro-inflammatory stimuli. Figure 1 shows a schematic diagram of the procedure in this example and subsequent examples.

[0412] Specifically, the stock solutions of OSM and IL-1β were diluted 1:1 in PBS with 0.6% Tween to a concentration of 1 ng / mL each. The OSM and IL-1β solutions were added together to the co-culture using a Tecan D300 and incubated at 37°C and 5% CO2 for 72 hours.

[0413] In Examples 4-11, fibroblast cultures, organoid cultures, and co-cultures of fibroblasts and organoids were prepared according to Examples 1-2 unless otherwise specified. OSM (1 ng / mL) and IL-1β (1 ng / mL) were added to the co-cultures according to Example 3, and "proinflammatory stimuli" or "proinflammatory stimulation" is defined in Examples 4-11. Subsequently, the co-cultures were incubated on plates at 37°C and 5% CO2 for 72 hours before cytokine or caspase analysis, or at 37°C and 5% CO2 for 96 hours before image analysis.

[0414] Example 4: Imaging shows an increase in organoid size when co-cultures are prepared using pro-inflammatory stimuli. This example uses imaging analysis to evaluate the morphology of co-cultured organoids. This demonstrates that organoids and fibroblasts, successfully co-cultured, exhibit normal organoid morphology (e.g., expected size, area, and appearance under bright-field imaging) consistent with organoids cultured in cCDM, and may show increased aggregation due to intercellular interactions mediated by fibroblasts.

[0415] This example also demonstrates that pro-inflammatory stimulation of organoids causes an increase in organoid area and affects aggregation (compared to co-cultures without pro-inflammatory stimulation), suggesting that organoid size is an indicator of the pro-inflammatory profile of the co-culture.

[0416] Organoids in the co-culture were first stained with Calcein AM. 48 hours after combining the organoids and fibroblasts in the co-culture, 50 μL of DMSO was added to the Calcein AM vial and mixed. 88 μL of the mixture was added to 4,312 μL of Ad-DF+++ medium. 10 μL of the final Calcein AM mixture was added to the co-culture and shaken for 5–10 seconds using a Tecan D300. The plates were then incubated at 37°C and 5% CO2 for 2 hours. Next, imaging was performed to evaluate the morphology of the organoids.

[0417] As shown in Figure 2A, co-cultures exposed to pro-inflammatory stimuli showed an increase in cystic morphology compared to organoids cultured alone or co-cultures not exposed to pro-inflammatory stimuli, which exhibited a smaller, more compact morphology. Aggregation was observed when organoids were co-cultured with fibroblasts compared to organoids not co-cultured with fibroblasts. Aggregation was less pronounced when co-cultures were prepared using pro-inflammatory stimuli.

[0418] As shown in Figure 2B, the area of ​​organoids is significantly increased in co-cultures with a pro-inflammatory profile.

[0419] As shown in Figure 2C, exposure to alternative stimuli such as IL-6 (20 ng / mL), LIGHT (20 ng / mL), or a combination of both did not cause changes in organoid size.

[0420] As shown in Figures 2D and 2E, similar results were obtained when alternative primary organoids "A," "B," and "C" (each derived from the small intestine) were used.

[0421] As shown in Figure 2F, similar results were obtained when the immortalized and primary fibroblast cell lines listed in Table 1 above were tested in the absence ("-") and in the presence ("+") of pro-inflammatory stimuli.

[0422] As shown in Figures 2G and 2H, similar results were obtained when the culture period was extended to day 3 ("D3") and day 6 ("D6") for different ratios of fibroblasts to organoids ("F / O").

[0423] Example 5: Expression measurements show increased secretion of markers indicating a pro-inflammatory profile when co-cultures are prepared using pro-inflammatory stimuli. The plate contents were centrifuged at 1500 rpm for 5 minutes, and the supernatant was collected. 100 μL was recovered, and the remaining supernatant was centrifuged again. Another 30 μL was recovered, for a total of 130 μL of supernatant. The supernatant container was sealed and stored at -80°C until use. IL-6 and CXCL2 expression were measured using HTRF (CisBio / Revvity, “Human IL-6 HTRF Kit”, https: / / uk.cisbio.eu / human-il6-kit-40419) and ELISA (Bio-Techne Ltd., “Human CXCL2 / GRO beta DuoSet ELISA”), respectively. https: / / www.rndsystems.com / products / human-cxcl2-gro-beta-duoset-elisa_dy276-05 The analysis was performed according to the supplier's protocol.

[0424] As shown in Figure 3A, in the absence of pro-inflammatory stimuli, no detectable levels of IL-6 expression were observed in organoids, fibroblasts, or co-cultures. Pro-inflammatory stimuli induced IL-6 expression in fibroblasts cultured alone and in co-cultures, but not in organoids cultured alone.

[0425] As shown in Figure 3B, pro-inflammatory stimuli separately promoted some expression of CXCL2 in fibroblasts and organoids (compared to fibroblasts and organoids cultured alone without pro-inflammatory stimuli), but co-culture with pro-inflammatory stimuli showed clear synergistic induction (compared to co-cultures without pro-inflammatory stimuli).

[0426] As shown in Figures 3C and 3D, similar results were obtained in the different fibroblast cell lines listed in Table 1 above, both in the absence ("-") and in the presence ("+") of pro-inflammatory stimuli.

[0427] As shown in Figure 3E, similar CXCL2 results were observed in three different primary organoids, "A," "B," and "C" (derived from the small intestine).

[0428] As shown in Figures 3F and 3G, a higher fibroblast-to-organoid ratio leads to increased secretion of IL-6 and CXCL2, regardless of the increase in the concentration of pro-inflammatory stimuli (from 0 ng / ml to 5 ng / ml and 15 ng / ml for IL-1β and OSM, respectively). Therefore, an increase in the presence of fibroblasts compared to organoids increases the pro-inflammatory profile in co-cultures prepared with pro-inflammatory stimuli.

[0429] Example 6: Caspase measurements show increased apoptosis when co-cultures are prepared using pro-inflammatory stimuli and exposed to inflammatory stimuli. After combining organoids and fibroblasts in co-culture, apoptosis in the co-culture was induced using inflammatory stimuli (in the form of damage-inducing cytokines). 24 hours later, apoptosis was measured in co-cultures prepared with pro-inflammatory stimuli and in co-cultures not prepared with pro-inflammatory stimuli.

[0430] TNF and IFNγ stock solutions were diluted 1:1 in PBS with 0.6% Tween and added to the co-culture at concentrations of 15 ng / mL and 15 ng / mL, respectively. The plates were then incubated for 24 hours.

[0431] To determine relative caspase 3 / 7 activity, apoptosis was evaluated using a Caspase-Glo assay (e.g., Promega). Briefly, 95 μL of caspase reagent at room temperature was added to each plate. The plates were then shaken for 1 minute and incubated in the dark at room temperature for 30 minutes. Subsequently, 160 μL of sample from each plate was transferred to a black-walled plate for analysis.

[0432] As shown in Figure 4A, in the absence of pro-inflammatory stimuli, fibroblasts in co-culture are protected against the increase in caspase activity induced by inflammatory stimuli. When co-cultures are prepared using pro-inflammatory stimuli, this protection is lost, and apoptosis increases compared to co-cultures not prepared with pro-inflammatory stimuli. Furthermore, pro-inflammatory stimuli increase baseline caspase activity (i.e., apoptosis in the presence of pro-inflammatory stimuli but not in the absence of inflammatory stimuli).

[0433] As shown in Figure 4B, similar results were obtained with the various fibroblast cell lines listed in Table 1 above.

[0434] As shown in Figure 4C, similar results were obtained using three alternative primary organoids "A," "B," and "C" (all derived from the small intestine).

[0435] Caspase activity was measured at different fibroblast-organoid ratios and TNF / IFNγ concentrations (0 ng / mL, 1 ng / mL, 5 ng / mL, 15 ng / mL, 50 ng / mL, and 100 ng / mL (data not shown)). As expected, co-cultures exposed to pro-inflammatory stimuli followed by inflammatory stimuli showed a greater increase in apoptosis than co-cultures exposed to inflammatory stimuli alone.

[0436] Example 7: Pro-inflammatory stimuli have a separable effect on apoptosis and organoid size; tofacitinib improves the increase in apoptosis in co-cultures using a pro-inflammatory profile upon exposure to inflammatory stimuli. Tofacitinib is a small molecule JAK inhibitor used to treat inflammatory conditions, including ulcerative colitis (UC). The effect of 10 μM tofacitinib on the parameters measured in Examples 4 and 6 was evaluated.

[0437] As shown in Figure 5A, tofacitinib treatment results in minor morphological changes (measured according to Example 4), but as shown in Figure 5B, tofacitinib significantly reduces caspase activity upon exposure to inflammatory cytokines (measured according to Example 6).

[0438] Similarly, as is evident from Figures 5A-5B, the pro-inflammatory stimulus IL-1β appears to have a dominant effect on morphological changes (Figure 5A), while OSM appears to have a dominant effect on apoptosis (Figure 5B). This suggests that different pro-inflammatory stimuli act via non-redundant pathways, leading to different phenotypic changes in co-cultures. This will have implications for the modeling, diagnosis, and treatment of inflammatory disorders.

[0439] Example 8: A conditioned medium from fibroblasts prepared with pro-inflammatory stimuli induces a pro-inflammatory profile in organoids cultured alone. The inventors hypothesized that the effect of pro-inflammatory stimuli on co-cultures can be at least partially mediated by soluble effectors secreted by fibroblasts into the surrounding culture medium ("conditioned medium"). Therefore, conditioned medium was isolated from fibroblasts cultured alone with pro-inflammatory stimuli, and this conditioned medium was combined with organoids to determine whether the organoid morphology measured in Example 4 could be replicated without combining the organoids with fibroblasts.

[0440] As shown in Figure 6, when organoids were exposed to a conditioned medium derived from fibroblasts cultured with pro-inflammatory stimuli, an increase in organoid area and aggregation was evident, comparable to that observed when organoids and fibroblasts were co-cultured with pro-inflammatory stimuli. This suggests that the inflammatory response in co-culture is at least partially mediated by soluble factors derived from fibroblasts.

[0441] Example 9: Gene expression analysis of co-cultured organoids The gene expression levels of several genotypes in organoids cultured in growth medium (CNM) or differentiation medium (cCDM) + serum, in or without conditioned medium derived from fibroblasts cultured with or without pro-inflammatory stimuli, were examined according to the method of Example 8.

[0442] As shown in Figure 7, the expression profiles of important inflammatory genes have changed.

[0443] Example 10: Effect of alternative media on co-culture measurements The inventors investigated alternative culture media for the methods of Examples 4-6 described above.

[0444] The methods of Examples 1-3 were repeated using cCDM + serum; cCDM; cCDM + serum (without PD0325901); cCDM + serum (without DAPT); CNM; or eCDM as the co-culture medium. Next, the methods of Examples 4-6 were applied as follows.

[0445] Imaging Imaging was performed on organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example, according to Example 4. Figure 8 shows the results for organoids alone (Figure 8A) and organoid-fibroblast co-cultures (Figure 8B).

[0446] As the imaging results show, the culture media showed an increase in organoid size and cystic morphology compared to the control when pro-inflammatory stimuli were applied (Figure 8B). No major morphological changes were observed in organoids that did not contain fibroblasts, regardless of the culture medium used (Figure 8A).

[0447] Measurement of expression IL-6 and CXCL2 expression were measured in organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example, according to Example 5. The results for IL-6 are shown in Figure 9A, and the results for CXCL2 are shown in Figure 9B.

[0448] As evidenced by the expression measurements, the culture medium allowed for the identification of changes in marker secretion associated with the presence of fibroblasts and pro-inflammatory stimuli. However, in the case of cnm, the induction of CXCL2 and IL-6 expression was much lower. cnm alone and cnm plus serum revealed increased induction of markers IL6 and CXCL2 in particular.

[0449] Caspase measurement Caspase measurements were performed on organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example, according to Example 6. The results are shown in Figure 10.

[0450] As evidenced by the caspase measurements, the culture medium enabled the identification of fibroblasts, pro-inflammatory stimuli, and the effects of inflammatory stimuli on caspase activity and apoptosis. In the absence of pro-inflammatory stimuli OSM and IL-1β, cCDM+ serum particularly highlighted the potential of fibroblasts to protect against inflammatory stimuli such as IFN and TNF.

[0451] Example 11: RNA-seq experiment for further markers indicating a pro-inflammatory profile To identify potential novel markers indicating pro-inflammatory profiles in co-cultures, RNA-seq was performed on organoids and organoid-fibroblast co-cultures prepared using the cCDM, CM, and CNM media described in Example 10, or the conditioned medium ("CM") described in Example 8, with or without the pro-inflammatory stimuli of OSM (1 ng / ml) and IL-1β (1 ng / ml) ("INF"). RNA extraction was performed using Qiagen QIASymphony SP, and library preparation was performed using the TruSeq® RNA Strand PolyA Kit, both following the manufacturer's standard protocols. All samples were collected 3 days after seeding. The mean normalized expression of various RNAs is shown in Table 2. Normalization was performed using the median ratio method (further information on the normalization method can be found here: https: / / hbctraining.github.io / DGE_workshop / lessons / 02_DGE_count_normalization.html).

[0452] As can be seen from the results in Table 2, the expression of IL-6, CXCL2, IGFBP1, WNT7A, MMP7, and HIF3A was particularly upregulated in co-culture of cCDM with pro-inflammatory stimuli, but not in culture of cCDM organoids alone with pro-inflammatory stimuli. This is in contrast to the expression profiles of certain other biomarkers, which were differentially upregulated in monoculture and co-culture, regardless of the presence or absence of pro-inflammatory stimuli.

[0453] (Table 2) Mean normalized expression scores of potential markers of the pro-inflammatory profile TIFF2026525328000009.tif232113

[0454] When additional markers were measured using the same RNA-seq protocol, significant downregulation or upregulation was observed among cultures prepared using various different methods. The results for the following culture types are shown in the table below. -Table 3: Fibroblast-organoid co-cultures in cCDM (with or without pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose significant changes in expression (upregulation or downregulation) are associated with pro-inflammatory stimulation of cCDM co-cultures. -Table 4: Organoid cultures in cCDM without fibroblasts (with or without pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose expression shows significant changes (upregulation or downregulation) in the absence of fibroblasts that are associated with pro-inflammatory stimulation of cCDM organoids. -Table 5: Organoid cultures in CNM without fibroblasts (with or without pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose significant changes in expression (upregulation or downregulation) in the absence of fibroblasts are associated with pro-inflammatory stimulation of CNM organoids. -Table 6: Fibroblast cultures in cCDM without organoids (with or without pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose expression shows significant changes (upregulation or downregulation) in the absence of organoids that are associated with pro-inflammatory stimulation of cCDM fibroblasts. -Table 7: Organoid cultures without fibroblasts in cCDM and organoid-fibroblast co-cultures in cCDM (both with pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers associated with organoid co-cultures containing fibroblasts that show significant changes in expression (upregulation or downregulation) compared to organoids in the absence of fibroblasts (both in cCDM and both with pro-inflammatory stimulation). -Table 8: Organoid cultures without fibroblasts in cCDM and organoid-fibroblast co-cultures in cCDM (neither containing pro-inflammatory stimuli). Therefore, the table lists markers associated with organoid co-cultures containing fibroblasts that show significant changes in expression (upregulation or downregulation) compared to organoids in the absence of fibroblasts (both in cCDM and neither containing pro-inflammatory stimuli). -Table 9: Organoid cultures without fibroblasts in cCDM and organoid cultures without fibroblasts in conditioned medium (CM) (neither containing pro-inflammatory stimuli). Therefore, the table lists markers whose significant changes in expression (upregulation or downregulation) in organoids in the absence of fibroblasts and without pro-inflammatory stimuli are associated with the change in culture medium from cCDM to CM. -Table 10: Organoid cultures without fibroblasts in cCDM and organoid cultures without fibroblasts in CM (both with pro-inflammatory stimulation using IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose significant changes in expression (upregulation or downregulation) in organoids present with pro-inflammatory stimulation in the absence of fibroblasts are associated with the change in culture medium from cCDM to CM. -Table 11: Organoid cultures in CM that do not contain fibroblasts (with or without pro-inflammatory stimulation by IL-1β (1 ng / ml) and OSM (1 ng / ml)). Therefore, the table lists markers whose significant changes in expression (upregulation or downregulation) in the absence of fibroblasts are associated with pro-inflammatory stimulation of organoids in CM.

[0455] Various markers showed significant downregulation or upregulation in co-cultures (Tables 3, 7, and 8), fibroblast-only cultures (Table 6), and cultures in fibroblast-conditioned medium (Tables 9, 10, and 11), while these markers were less abundant in organoid-only cultures (Tables 4 and 5). This suggests that fibroblasts play a crucial role in the response to pro-inflammatory stimuli.

[0456] (Table 3) Changes in expression of co-culture (cCDM) in response to pro-inflammatory stimulation TIFF2026525328000010.tif150165

[0457] (Table 4) Changes in expression of fibroblast-free organoids (cCDM) induced by pro-inflammatory stimulation TIFF2026525328000011.tif93165

[0458] (Table 5) Changes in expression of organoids without fibroblasts (CNM) in response to pro-inflammatory stimulation TIFF2026525328000012.tif59165

[0459] (Table 6) Changes in expression of organoid-free fibroblasts (cCDM) induced by pro-inflammatory stimulation TIFF2026525328000013.tif150165

[0460] (Table 7) Changes in expression in co-cultured organoids (cCDM) induced by pro-inflammatory stimulation compared to organoids (cCDM) without fibroblasts (cCDM). TIFF2026525328000014.tif150165

[0461] (Table 8) Changes in expression in organoids without fibroblasts (cCDM) compared to co-cultured organisms (cCDM) (neither without pro-inflammatory stimulation) TIFF2026525328000015.tif150165

[0462] (Table 9) Changes in expression in fibroblast-free organoids in cCDM compared with fibroblast-free organoids in conditioned medium (CM) (both without pro-inflammatory stimulation) TIFF2026525328000016.tif150165

[0463] (Table 10) Changes in expression in organoids without fibroblasts in cCDM after pro-inflammatory stimulation, compared to organoids without fibroblasts in CM after pro-inflammatory stimulation. TIFF2026525328000017.tif149165

[0464] (Table 11) Changes in expression in organoids without fibroblasts in CM without pro-inflammatory stimulation, compared to organoids without fibroblasts in CM with pro-inflammatory stimulation. TIFF2026525328000018.tif150165

[0465] References [1] Gennaro (2000) Remington: The Science and Practice of Pharmacy.20th edition, ISBN:0683306472. [2]Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press). [3]Methods In Enzymology(S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [4] Handbook of Experimental Immunology, Vols. I IV (DMWeir and CC Blackwell, eds, 1986, Blackwell Scientific Publications) [5] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press). [6]Ausubel et al.(eds)(2002)Short protocols in molecular biology,5th edition(Current Protocols). [7]PCR(Introduction to Biotechniques Series),2nd ed.(Newton & Graham eds.,1997,Springer Verlag). [8]Clevers,Cell.2016 Jun 16;165(7):1586-1597 [9]Co et al.(2019)Cell Reports 26,2509-2520

[10] M van de Wetering et al.(2015)Cell.;161(4):933-45

[11] Hayashi et al.(2004)Matrix Biology 23:47 62

[12] Nusse and Clevers(2017)Cell 169(6):985-999

[13] De Lau,W.et al.Nature,2011 Jul 4;476(7360):293-7

[14] Lau et al.Genome Biol.2012;13(3):242(2012

[15] WO2012 / 140274

[16] 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

[17] 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

[18] Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-805

[19] ”THE WNT FAMILY OF SECRETED PROTEINS”,R&D Systems Catalog,2004

[20] WO2012 / 168930

[21] Janda CY,et al.(2017)Nature;545(7653):234-237

[22] Miao,Y.et al.(2020)Cell Stem Cell.27(5),840-851

[23] Janda CY,et al.Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling.Nature.2017 May 11;545(7653):234-237

[24] Miao,Y.et al.(Next-generation surrogate Wnts support organoid growth and deconvolute Frizzled pleiotropy in vivo.Cell Stem Cell.27(5),840-851(2020)

[25] WO2016 / 083613

[26] WO2016 / 083612

[27] Zilberberg et al.,2007.BMC Cell Biol.8:41

[28] Robinton et al.Nature 481,295,2012

[29] Olive and Banath(2006)Nature Protocols volume 1,pages 23-29

[30] Ivashkevich et al(2012)Cancer Lett.327(1-2):123-133.doi:10.1016 / j.canlet.2011.12.025.

Claims

1. A method for determining the presence or absence of at least one change in a co-culture comprising at least one organoid and at least one stromal cell, To form the aforementioned co-culture, the at least one organoid is combined with the at least one stromal cell in the co-culture medium, To determine whether or not the aforementioned at least one change is present in the co-cultured product. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in an organoid medium, The aforementioned method.

2. A method for testing at least one therapeutic agent for a disease, To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, Applying the aforementioned at least one therapeutic agent to the co-culture, To determine whether or not at least one change has occurred in the co-cultured product. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in an organoid medium, The aforementioned method.

3. A method for determining whether or not there is a diagnosis and / or prognosis prediction of a disease in the subject, To form a co-culture, at least one organoid is combined with at least one stromal cell in the co-culture medium, Applying at least one diagnostic agent to the co-culture, To determine whether or not at least one change has occurred in the co-cultured product. Includes, Optionally, (a) preparing at least one stromal cell by culturing stromal cells in stromal cell medium, and / or (b) Preparing at least one organoid by culturing epithelial cells in an organoid medium, The aforementioned method.

4. Determining whether or not at least one change occurs in the co-culture is (a) Determining the presence or absence of stromal cells in the co-culture, and optionally determining the amount of stromal cells in the co-culture, and / or (b) Adding at least one inflammatory stimulus to the co-culture medium and determining whether or not an inflammatory response is present, optionally, i. The at least one inflammatory stimulus comprises a cytokine, optionally comprising TNF and / or IFNγ, and / or ii. The inflammatory response includes an increase in caspase activity and / or apoptosis in the co-culture. To make the aforementioned determination, and / or (c) Adding at least one pro-inflammatory stimulus to the co-culture medium, optionally, i. At least one pro-inflammatory stimulus is added to the co-culture medium before any inflammatory stimulus is added to the co-culture medium, and / or ii. The at least one pro-inflammatory stimulus comprises at least one cytokine, optionally comprising IL-1β and / or oncostatin M (OSM), Adding the above The method according to any one of the prior claims, including the method described in any one of the prior claims.

5. The method according to any one of the prior claims, wherein determining the presence or absence of the at least one change includes (a) adding at least one pro-inflammatory stimulus to the co-culture medium and determining the presence or absence of a pro-inflammatory profile, and optionally (b) adding at least one inflammatory stimulus to the co-culture medium and determining the presence or absence of an inflammatory response.

6. The method according to any one of claims 2 to 5, wherein the disease is an inflammatory disease or a fibrous disease, optionally, the disease is an inflammatory disease, optionally, the inflammatory disease is a gastrointestinal inflammatory disease, optionally, the inflammatory disease is an inflammatory bowel disease (IBD), and optionally, the IBD includes ulcerative colitis (UC) or Crohn's disease (CD).

7. The method according to any one of the prior claims, wherein the at least one change includes a change in organoid morphology, a change in organoid size or epithelial cell size, a decrease in cell viability, a decrease in cell proliferation, an increase in cell death, a change in secretome profile, a change in cytokine secretion, an increase in cell apoptosis, an increase in caspase activity, and / or a change in the expression of one or more genes, and optionally, the change in the expression of one or more genes includes a change in one or more disease biomarkers, one or more fibrosis biomarkers, and / or one or more inflammatory biomarkers.

8. The method according to any one of the prior claims, wherein the at least one change includes a change in the secretome profile, optionally a change in cytokine secretion, and optionally a change in IL-6 and / or CXCL2 secretion.

9. The method according to any one of claims 1 to 7, wherein the at least one change includes a change in the expression of IGFBP1, WNT7A, MMP7, and / or HIF3A.

10. The method according to any one of the prior claims, wherein the presence or absence of at least one of the changes is determined by comparison with a reference organoid, and optionally the reference organoid does not contain stromal cells.

11. The method according to any one of the prior claims, wherein the at least one organoid is a patient-derived organoid.

12. The method according to any one of the prior claims, wherein the at least one stromal cell comprises a fibroblast, optionally, the fibroblast is an intestinal fibroblast, for example, a colonic fibroblast or a small intestinal fibroblast, optionally, the fibroblast is an immortalized human colonic fibroblast, a human colonic fibroblast, an immortalized human small intestinal fibroblast, or a human small intestinal fibroblast.

13. The co-culture, the at least one organoid, and / or the at least one stromal cell, (a) derived from the subject and / or (b) derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue, and / or (c) of fibrous tissue and / or (d) of inflammatory tissue and / or (e) Mammalian cells, optionally including or consisting of human cells, and / or (f) Derived from the intestines, and optionally from the colon, The method according to any one of the prior claims.

14. The method according to any one of the prior claims, wherein the at least one organoid and the at least one stromal cell originate from the same subject and optionally originate from (a) the same sample from the subject and / or (b) the same tissue within the same subject.

15. The method according to any one of the prior claims, wherein the co-culture medium comprises mitotic growth factor, a BMP inhibitor, and R-spondin, and optionally comprises a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-spondin, and optionally comprises N-Ac, a TGF-β inhibitor, mitotic growth factor, gastrin, a BMP inhibitor, an antibiotic, R-spondin, a Notch pathway inhibitor, an ERK inhibitor, and a Wnt agonist.

16. The method according to any one of the prior claims, wherein the co-culture medium comprises serum, optionally fetal bovine serum, or optionally 5% fetal bovine serum.

Images