Co-cultures of organoids and stromal cells

Abstract

The invention relates to an organoid co-culture and application thereof in disease research.

Description

[0001] All documents cited in this article are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to organoid co-cultures and their use in disease research. Background Technology

[0003] Organoids are promising tools for studying human physiology in vitro. They are self-organizing epithelial cell structures whose physiological characteristics resemble the tissue structure of cells in vivo. They have been widely used to mimic aspects of disease onset and progression. Therefore, stem cell-derived organoids provide sophisticated models for studying human development and disease.

[0004] Epithelial organoid models can be enhanced through the integration of non-epithelial cells, forming "co-cultures." However, designing protocols and conditions that allow highly diverse cell types to coexist in vitro is challenging, not to mention whether such coexistence would promote related cell-cell interactions.

[0005] Stromal cells are a type of non-epithelial cell. As components of the “matrix” associated with body organs, stromal cells provide structural support to the organs. Traditionally considered an “inert” (or purely structural) component, stromal cells have recently been identified as playing a crucial and active role in metabolism, signal transduction, and disease function—functions traditionally associated with non-stromal epithelial cell types.

[0006] For example, the matrix is ​​considered to play a crucial role in gastrointestinal diseases such as inflammatory bowel disease (IBD). Activation of the matrix compartment, including an overabundance of pro-inflammatory fibroblasts, has been observed in IBD patients. At the molecular level, intestinal cells interacting with these pro-inflammatory fibroblasts are thought to exhibit increased apoptosis, proliferation or hypertrophy, and remodeling of the intestinal stem cell microenvironment. These factors are thought to contribute to the chronicity of inflammatory bowel disease (IBD) and to treatment resistance. However, the etiology of IBD remains unknown, and previous in vitro models have failed to replicate in vivo physiology.

[0007] This invention provides co-cultures formed by combining at least one organoid and at least one stromal cell. As described in the examples, such co-cultures have been validated to exhibit reproducible in vivo microstructures, cell-cell interactions, and biochemical responses (such as inflammatory responses), and to sustain these responses over extended periods. Such co-cultures can be used in in vitro methods related to the treatment, diagnosis, and / or prognosis of diseases, particularly those involving inflammation and / or fibrosis.

[0008] For example, the inventors have reproduced in vivo inflammatory processes by exposing such co-cultures comprising at least one organoid and at least one stromal cell to at least one pro-inflammatory stimulant to induce a pro-inflammatory profile in the co-culture, and then exposing them to at least one inflammatory stimulant to induce an inflammatory response. Such co-cultures express the expected inflammatory biomarkers (such as IL-6 and CXCL2).

[0009] Such co-cultures can be used to test therapeutic agents, for example, by measuring a reduction in inflammatory biomarkers when a therapeutic agent is applied to the co-culture. A reduction in inflammatory biomarkers indicates, for example, that the therapeutic agent targets a specific molecular pathway that contributes to the production of inflammatory biomarkers, and therefore suggests that the therapeutic agent may be able to treat the disease in vivo.

[0010] Such cocultures can also be used for diagnosis and / or prognosis, for example, by determining whether a diagnostic agent known to induce inflammation via a specific molecular pathway can increase inflammation in a coculture containing at least one PDO. If such a diagnostic agent does not increase the biomarker of inflammation, this could indicate that the specific molecular pathway targeted by the diagnostic agent is saturated (i.e., cannot be further activated), and therefore the specific molecular pathway may function in the patient's disease. A similar inverse induction can be used when a known therapeutic agent is used as a diagnostic agent; if a known therapeutic agent can reduce the inflammatory biomarker in a coculture containing at least one PDO, then the molecular element targeted by the therapeutic agent may function in the patient's disease. Summary of the Invention

[0011] This invention provides a method for preparing a co-culture comprising at least one organoid and at least one stromal cell, the method comprising: At least one organoid and at least one stromal cell are combined in a co-culture medium to form a co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture 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, wherein the method comprises: Combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture; and Determine the presence or absence of at least one of the aforementioned changes in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture 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, wherein the method includes: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid; and Determine the presence or absence of at least one change in the organoids. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0014] The present invention further provides a method for testing at least one disease treatment agent, the method comprising: Combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture; Apply the at least one therapeutic agent to the co-culture; and Determine the presence or absence of at least one change in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0015] The present invention further provides a method for testing at least one disease treatment agent, the method comprising: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid. Apply the at least one therapeutic agent to the organoid; and Determine the presence or absence of at least one change in the organoids. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0016] The present invention further provides a method for determining the presence or absence of a disease diagnosis and / or prognosis 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; At least one diagnostic agent is applied to the co-culture; and Determine the presence or absence of at least one change in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0017] The present invention further provides a method for determining the presence or absence of a disease diagnosis and / or prognosis in a subject, the method comprising: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid. Apply at least one diagnostic agent to the at least one organoid; and Determine the presence or absence of at least one change in the at least one organoid. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0018] The present invention further provides co-cultures of the method described herein.

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

[0020] The present invention further provides a stromal cell culture medium for the method described herein. Attached Figure Description

[0021] The embodiments of the present invention will now be described with reference to the accompanying drawings, in which: Figure 1 A schematic diagram of the methods used to prepare co-cultures and induce pro-inflammatory profiles for analysis is shown, as described in the examples. Abbreviations: "FIB" for fibroblasts; "org" for organoids.

[0022] Figure 2Imaging results of patient-derived organoids (“PDO”) cultured with and without fibroblasts (“Fib”) and with and without pro-inflammatory stimulants (“OSM / IL-1b”) are shown, as described in Example 4. Figure 2 A shows a bright-field image. Figure 2 B and Figure 2 C shows the organoid area (µm) under various conditions. 2 ). Figure 2 D shows bright-field images of different types of PDO (“A”, “B”, and “C”) with and without fibroblasts, with or without pro-inflammatory stimuli (“+OSM / IL1b”) or without pro-inflammatory stimuli (“Control”). Figure 2 E and Figure 2 F shows the area (µm²) of organoids of different types of PDO with and without different types of immortalized fibroblasts (“IM-CoF”, “IM-SIF”) or primary fibroblasts (“Clone2”, “SIF”). 2 —See Table 1 below. Figure 2 G and Figure 2 H together show images and areas (µm) of organoids (“Org”) with or without a specific ratio (“F / O”), over a longer time period (up to day 3, “D3”, or up to day 6, “D6”), with and without pro-inflammatory stimulation (“OSM+IL1β”). 2 Measurement results.

[0023] Figure 3 Measurements of secretion results for organoids cultured with or without fibroblasts and with or without pro-inflammatory stimulants (“OSM+IL-1β”) are shown, as described in Example 5. The secretion of the following substances is shown: (A) IL-6; (B) CXCL2; (C) IL-6 for different fibroblast lines; and (D) IL-6 for different fibroblast lines; (E) CXCL2 for different primary organoids (“A”, “B”, and “C”) and different fibroblast lines; and (F) IL-6 and (G) CXCL2 for different fibroblast-organoid ratios (“F / O”) and different concentrations of IL-1β and OSM pro-inflammatory stimulants (0 ng / ml, 5 ng / ml, 15 ng / ml). See also Table 1.

[0024] Figure 4The measurements of (A) caspase activity, (B) caspase activity in different fibroblast lines, and (C) caspase activity in different organoids are shown as described in Example 6. “PDO” = patient-derived organoid; “Org” = organoid; “A,” “B,” and “C” = specific organoid types; “IM-CoF,” “IM-SIF” = specific immortalized fibroblast lines; “Clone2,” “SIF” = specific primary fibroblast lines. See also Table 1.

[0025] Figure 5 (A) organoid area measurements and (B) caspase activity measurements are shown, as well as how these are affected by pro-inflammatory stimuli and / or tofacitinib administration, as described in Example 7.

[0026] Figure 6 Imaging of organoids is shown with or without fibroblasts (“Fib”), fibroblast conditioned medium (“Fib-CM”), and pro-inflammatory stimulants (“OSM / IL1b”), and when cultured in expansion medium (“CNM”) or differentiation medium (“cCDM”), as described in Example 8.

[0027] Figure 7 Gene expression measurements of cocultures are shown with or without fibroblast conditioned medium (“Fib-CM”), pro-inflammatory stimulants (“OSM+IL1β”), and when cultured in expansion medium (“CNM”) or differentiation medium (“cCDM”), as described in Example 9. The expression of (A) ALPI and MUC2, (B) LGR5 and KI67, and (C) KRT20 and OLMF4 are shown.

[0028] Figure 8 Imaging results of organoids (“Org”) cultured in various culture media (A) without fibroblasts (“Fib”) and (B) with fibroblasts (“Fib”), and with (“INF”) and without (“Control”) pro-inflammatory stimulation with OSM (1 ng / ml) and IL-1β (1 ng / ml) are shown, as described in Example 10. “PD03” = PD0325901.

[0029] Figure 9Measurements of secretion results of organoids cultured in various media with and without fibroblasts (“Fib”), with (“INF”) or without (“Control”) pro-inflammatory stimulation with OSM (1 ng / ml) and IL-1β (1 ng / ml) are shown, as described in Example 10. The secretion of the following substances is shown: (A) IL-6; and (B) CXCL2. “PD03” = PD0325901.

[0030] Figure 10 The measurements of caspase activity in organoids cultured alone in various media, or with fibroblasts (“Fib”), with (“INF”) or without (“Control”) pro-inflammatory stimulation with OSM (1 ng / ml) and IL-1β (1 ng / ml), and with or without pro-inflammatory stimulation with TNF (15 ng / ml) and IFNγ (15 ng / ml), as described in Example 10. “PD03” = PD0325901. Detailed Implementation

[0031] definition

[0032] Unless otherwise stated, the practice of this invention will employ conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology, which are well explained in the literature. See, for example, references [1-7], etc.

[0033] The terms “approximately” or “about” as used herein are equivalent. Any numbers used herein, with or without the “approximately / about” modifier, are intended to cover any normal fluctuations as understood by those skilled in the art. As used herein, when applied to one or more intended values, the term “approximately” or “about” refers to a value similar to the reference value. In some embodiments, the term “approximately” or “about” refers to a range of values ​​falling within (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction, unless otherwise stated or clearly apparent from the context (except where such numbers would exceed 100% of the possible value). Values ​​can also be understood as precise values, and therefore the term “about” may be omitted. For example, the term “about 100” covers values ​​in the range of 90 to 110, and also includes 100.

[0034] "Co-culture" refers to two or more cell types maintained under conditions suitable for their mutual growth. In the context of this disclosure, "organoid co-culture" involves culturing a non-epithelial cell type (specifically a stromal cell type) with an epithelial organoid, as defined elsewhere. In some embodiments, the cell types in the co-culture are integrated because they can exhibit structural, biochemical, and / or phenomenological associations that they do not exhibit when isolated. In some embodiments, the cell types in the co-culture mimic the structural, biochemical, and / or phenomenological associations observed between cell types in vivo. In this application, the term "co-culture" can be used to refer to a normal (e.g., non-fibrotic, 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 has been derived from one or more epithelial cells or one or more non-epithelial cells with a disease phenotype, or in some embodiments, because the organoid has been exposed to one or more pro-inflammatory stimuli to induce the characteristics of a disease phenotype.

[0035] The word "comprising," and its variations are not intended to be restrictive, means that the item immediately following the word is considered included, but items not explicitly mentioned are not excluded. Additionally, if necessary, the verb "composed of" can be replaced with "substantially composed of," meaning that a product as defined herein may contain additional components besides the specifically identified components, which do not alter the distinctive features of the invention. Furthermore, a method as defined herein may include additional steps besides the specifically identified steps, which do not alter the distinctive features of the invention. Moreover, the use of the indefinite articles "a" or "an" to refer to an element does not preclude the possibility of more than one element unless the context explicitly requires one and only one element. Therefore, the indefinite articles "a" or "an" generally mean "at least one."

[0036] Fibroblasts are the main active cells of connective tissue, characterized by the secretion of collagen and other extracellular matrix proteins. They are one of the most abundant cell types in the matrix. Fibroblasts are a type of stromal cell.

[0037] "Fibrosis" or "fibrotic disease" refers to a condition or symptom of a condition characterized by excessive, uncontrolled, and / or inappropriate deposition of collagen and other extracellular matrix components. Examples of fibrotic diseases include intestinal fibrosis, solitary rectal ulcers, radiation enteropathy, and eosinophilic enteropathy.

[0038] The term "digestive system" encompasses the gastrointestinal tract and the liver, pancreas, and gallbladder.

[0039] The term "gastrointestinal tract" or "GI tract" encompasses the mouth, oral cavity, esophagus, stomach, intestines, rectum, and anus.

[0040] "Inflammation" refers to a collection of biochemical stimuli and cellular responses at the site of inflammation that result in physiological symptoms traditionally characterized by fever, pain, redness, and swelling. At the biochemical level, inflammation can be mediated by pro-inflammatory stimuli and inflammatory stimuli. "Inflammatory stimuli" are agents that trigger an "inflammatory response" in the cells and / or co-cultures they act on, thereby increasing caspase activity (and other types of cellular 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 cause the affected cells and / or cocultures to exhibit a "pro-inflammatory spectrum," characterized by altered gene expression (e.g., upregulated PDPN and / or TGF-β in stromal cells), altered secretomes (including increased secretion of IL-6 and / or CXCL2), increased organoid size and / or altered aggregation states, 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 typically signal through molecular pathways including STAT, NF-κB, MAPK, JUNK, mTOR, NFAT, and PI3K-AKT, but unlike inflammatory stimuli, they do not directly lead to increased apoptosis.

[0041] "Inflammatory condition" refers to a disease that presents with inflammation as a symptom or cause. Examples of inflammatory conditions include inflammatory bowel disease. In this article, the terms "disease," "symptom," and "condition" are used interchangeably.

[0042] Inflammatory bowel disease (“IBD”) is a condition characterized by chronic inflammation of the gastrointestinal tract (“GI”). Symptoms may include abdominal pain, diarrhea, weight loss, and gastrointestinal bleeding. Ulcerative colitis (“UC”) and Crohn’s disease (“CD”) are examples of IBD. UC may present as persistent inflammation localized to the colon. CD may present as discontinuous inflammation affecting the entire gastrointestinal tract.

[0043] The term "intestine" encompasses both the colon and the small intestine. The intestine is epithelial tissue and is part of the gastrointestinal tract and digestive system. Therefore, those skilled in the art will understand that the methods and uses described herein can be applied to other epithelial tissues, particularly those derived from the digestive system.

[0044] "Organoid" refers to a cellular structure obtained by expanding adult (post-embryonic) epithelial stem cells, preferably characterized by Lgr5 expression, and composed of tissue-specific cell types that are self-organized through cell sorting and spatially restricted lineage typing (e.g., as described in [8], particularly see the "Organoids Derived from Adult Stem Cells" section from page 1590 onwards). An organoid may be a "patient-derived organoid" (PDO). In this application, the term "organoid" may be used to refer to normal (e.g., non-fibrotic, 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, usually because the organoid has been derived from one or more epithelial stem cells with a disease phenotype, or in some embodiments, because the organoid has been genetically modified to exhibit specific characteristics of a disease phenotype.

[0045] "Safe" or "tolerable" means that the treatment for a disease has no side effects or only has side effects within a tolerable level according to standard clinical practice.

[0046] "Side effects" or "adverse effects" refer to physiological responses that are attributable to treatment rather than the desired effect.

[0047] The term "small intestine" encompasses the duodenum, jejunum, and ileum.

[0048] The "matrix" (sometimes called "mesenchyma" or "interstitium") refers to the supporting structure associated with epithelial organs, glands, or tumors, and it contains connective tissue. The matrix differs from the parenchyma, which refers to the portion of an organ that performs organ-specific functions. "Stromal cells" are cells found in or that produce the matrix, such as fibroblasts. The matrix (interstitium) can refer to the digestive interstitium (especially the gastrointestinal interstitium, such as the intestinal interstitium, or the colonic interstitium), the renal interstitium, or the pulmonary interstitium.

[0049] The terms "subject," "patient," or "individual" are used interchangeably herein and can refer to a human or any non-human animal (e.g., any mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In a preferred embodiment, the subject is a mammal, more preferably a human. "Human" can refer to a prenatal and / or postnatal form. The subject can be a person who does not have a disease. The subject can be a person who is seeking medical attention from a healthcare provider for a diagnosis or treatment of a disease. The subject may have or be susceptible to a disease or condition, but may or may not exhibit symptoms of said disease or condition.

[0050] "Having" means that the subject has been diagnosed with a disease, condition and / or symptom or has exhibited one or more symptoms of a disease, condition and / or symptom.

[0051] "Susceptibility" refers to a subject who has not yet been diagnosed with a disease, condition, and / or symptom. In some embodiments, a subject susceptible to a disease, condition, and / or symptom may not exhibit symptoms of the disease, condition, and / or symptom. In some embodiments, a subject susceptible to a disease, condition, symptom, or event may exhibit one or more of the following characteristics: (1) a gene mutation associated with the onset of the disease, condition, and / or symptom; (2) a genetic polymorphism associated with the onset of the disease, condition, and / or symptom; (3) increased and / or decreased expression and / or activity of a protein associated with the disease, condition, and / or symptom; (4) habits and / or lifestyles associated with the onset of the disease, condition, or symptom; and / or (5) having received, planning to receive, or needing to receive a transplant. In some embodiments, a subject susceptible to a disease, condition, and / or symptom will develop the disease, condition, and / or symptom. In some embodiments, a patient susceptible to a disease, condition, and / or symptom will not develop the disease, condition, and / or symptom.

[0052] "Therapeutic effective amount" means the amount of a therapeutic agent administered to a subject who has or is susceptible to a disease, condition, and / or symptom to treat, diagnose, prevent, and / or delay the onset of symptoms of such disease, condition, and / or symptom. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0053] "Treatment" means any method used to partially or completely reduce, improve, alleviate, suppress, prevent, or delay the onset of one or more symptoms or features of a particular disease, condition, and / or symptom; reduce the severity of one or more symptoms or features of a particular disease, condition, and / or symptom; and / or decrease the incidence of one or more symptoms or features of a particular disease, condition, and / or symptom. Treatment may be administered to a subject who has not yet developed symptoms of the disease or is only exhibiting early symptoms of the disease, in order to reduce the risk of developing a related pathological condition due to said disease. Any reference to a treatment method that includes administering a pharmaceutical agent to a subject also covers the pharmaceutical agent used in said treatment method, the use of said pharmaceutical agent in said treatment method, and the use of said pharmaceutical agent in the preparation of a medicament.

[0054] Overview

[0055] This 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 these (sometimes referred to as "co-culture"), and their use in in vitro methods, such as for testing therapeutic agents for diseases and / or methods for diagnosing and / or prognosticating diseases. Of particular interest are the uses of co-cultures in drug screening, toxicology screening, clinical studies, and drug development.

[0056] The present invention also provides co-cultures obtained by the method of the present invention.

[0057] Without wishing to be bound by any theory, the inventors believe that the co-culture of organoids and stromal cells as described herein encapsulates important in vivo interactions between the matrix and epithelium in both 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 altered aggregation).

[0058] Species

[0059] The cells (including epithelial cells and stromal cells), organoids, and / or co-cultures of this invention or applicable to the methods of this invention can be primarily derived from any multicellular organism. In some embodiments, the cells, organoids, and / or co-cultures of this invention are mammalian (meaning derived from mammals), such as mouse, primate, non-human primate, rat, dog, miniature pig, or human cells, organoids, and / or co-cultures. In a preferred embodiment, the cells, organoids, and / or co-cultures of this invention are human (meaning derived from humans).

[0060] sample

[0061] The co-culture of the present invention may comprise or consist of autologous cells, i.e., cells obtained from the same subject. For example, the co-culture may be obtained by preparing epithelial cells (e.g., colorectal cells) derived from tissue in the subject and stromal cells derived from the same subject (optionally from the same tissue in the same subject), which are then combined to form the 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 sample, such as an intestinal tissue biopsy sample. The sample may be a healthy sample (e.g., without inflammatory disease or fibrotic disease) or a disease sample (e.g., with inflammatory disease or fibrotic disease). In some embodiments, the epithelial cells and stromal cells may be obtained from different samples from the same subject.

[0062] Alternatively, the co-culture of the present invention may comprise or consist of non-autologous cells or, i.e., cells obtained from different subjects. For example, the co-culture may be obtained by culturing epithelial cells (e.g., colorectal cells) derived from tissue in a first subject and stromal cells derived from a second subject (optionally from the corresponding tissue of the second subject), which are then combined to form the co-culture. The sample may be a tissue biopsy sample, such as an intestinal tissue biopsy sample. The sample may be a healthy sample (e.g., without inflammatory disease or fibrotic disease) or a disease sample (e.g., with inflammatory disease or fibrotic disease). Different subjects may be antigenically matched, for example, different patients may be blood relatives, such as first-degree relatives.

[0063] Epithelial cells suitable for preparing organoids can, in principle, be derived from any epithelial tissue. For example, epithelial cells can be derived from the intestine, lung, kidney, pancreas, or liver. Epithelial cells derived from the digestive system, such as the gastrointestinal tract, are particularly preferred. Epithelial cells derived from the intestine are most preferred. In some embodiments, the epithelial cells are not derived from the lung, for example, not from the lung, kidney, pancreas, or liver. The tissue from which the epithelial cells are derived can be healthy (e.g., without inflammatory or fibrotic diseases) or diseased (e.g., with inflammatory or fibrotic diseases).

[0064] Stromal cells can, in principle, originate from any matrix. For example, stromal cells can originate from the lungs, kidneys, or intestines. Particularly preferred are stromal cells derived from the digestive system, such as those from the gastrointestinal tract. Most preferred are stromal cells derived from the intestines. In some embodiments, the stromal cells are not derived from the lungs, for example, not from the lungs, kidneys, pancreas, or liver. The tissue from which the stromal cells are derived can be healthy (e.g., non-inflammatory, non-fibrotic) or diseased (e.g., with inflammatory or fibrotic conditions).

[0065] Epithelial cells and / or stromal cells can be obtained during surgery from normal or diseased mucosa, such as from resected colon, rectum, small intestine and / or ileum of subjects with or without disease.

[0066] substrate culture

[0067] This invention provides at least one stromal cell that can be combined with at least one organoid to form a co-culture. This invention also provides at least one reference stromal cell that can be combined with at least one organoid to form a reference co-culture. This invention further provides stromal cells that can be cultured to prepare at least one stromal cell. All stromal cells such as these, and other stromal cells discussed herein, may be collectively referred to as “stromal cells of the invention” or “stromal cells,” and references to “stromal cells” may include any of these stromal cell types.

[0068] Cell types

[0069] This document describes features and methods relating to at least one stromal cell (in combination with at least one organoid to form a co-culture). These features and methods are generally applicable to stromal cells that can be cultured to prepare at least one stromal cell. Therefore, in this document, unless the context otherwise requires, the features, methods and other disclosures apply to both at least one stromal cell and stromal cells from which said at least one stromal cell is derived.

[0070] The at least one stromal cell (or the stromal cell from which the at least one stromal cell is derived) can be obtained from established cell lines available in the art (e.g., from ATCC or similar cell line libraries). Alternatively, the stromal cells can be purified from impure samples from the subject. Obtaining stromal cells from the same subject as the at least one organoid (or the epithelial cells from which the at least one organoid is derived) has associated advantages, as the resulting co-culture best represents the subject from whom the cells were derived (and is therefore the most realistic model). This is particularly useful in the context of personalized medicine.

[0071] stromal cells may be derived from the same tissue type, the same organ, and / or the same sample as the epithelial cells from which at least one organoid is derived.

[0072] 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 is not derived from lymphoid tissue. In some embodiments, at least one stromal cell is not part of any cell lineage derived from lymphoid tissue. In some embodiments, at least one stromal cell is not derived from hematopoietic tissue. In some embodiments, at least one stromal cell is not part of any cell lineage derived from hematopoietic tissue.

[0073] Preferred stromal cells are fibroblast cells (or "fibroblasts"). The fibroblasts may be intestinal fibroblasts (fibroblasts derived from the intestine), such as colonic fibroblasts or small intestinal fibroblasts.

[0074] The fibroblasts may be immortalized fibroblasts (derived from immortalized cell lines) or primary fibroblasts (e.g., fibroblasts derived from the patient).

[0075] Any of these types of stromal cells can be human stromal cells, such as human fibroblasts.

[0076] stromal cell culture medium

[0077] Matrix cell culture media can be used to prepare at least one matrix cell for co-culture, for example by promoting the growth and division (expansion) and / or differentiation of matrix cells to produce at least one matrix cell suitable for co-culture.

[0078] Any culture medium suitable for cultured matrix cells can be used, such as matrix cell culture medium containing fetal bovine serum suitable for culturing fibroblasts.

[0079] In some embodiments, the stromal cell culture medium comprises a basal medium, such as advanced DMEM / F-12 medium (“Ad-DF”), optionally about 500 ml AD-DF.

[0080] In some embodiments, the stromal cell culture medium contains an amino acid supplement, such as L-glutamine or an alternative to L-glutamine, preferably an alternative to L-glutamine, such as GlutaMax, optionally about 2 mM GlutaMax.

[0081] In some embodiments, the stromal cell culture medium contains a culture medium buffer, such as HEPES, optionally about 25 mM HEPES.

[0082] In some embodiments, the stromal cell culture medium contains one or more antibiotics, such as penicillin and / or streptomycin, optionally about 100 u / ml of penicillin and / or streptomycin.

[0083] In some implementations, the stromal cell culture medium contains serum, such as fetal bovine serum (FCS), for example, about 10% FCS.

[0084] In some embodiments, the stromal cell culture medium comprises basal medium, L-glutamine substitute, antibiotics, and optionally a culture medium buffer and / or serum.

[0085] In some embodiments, the stromal cell culture 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 culture medium comprises about 500 mL Ad-DF, about 2 mM GlutaMax, about 25 mM HEPES, about 100 u / ml penicillin and / or streptomycin, and about 10% FCS.

[0086] Preparation of stromal cells

[0087] In some embodiments, combining the at least one organoid and at least one stromal cell to form a co-culture includes preparing at least one stromal cell. This includes cultured the stromal cell in a stromal cell culture medium.

[0088] Cultured matrix cells may include thawed matrix cells and the addition of matrix cells to a matrix cell culture medium. Cultured matrix cells may also include incubation of matrix cells, for example, overnight.

[0089] The culture medium for matrix cells may include aspirating matrix cell culture medium and digesting the matrix cells with trypsin, followed by resuspending the matrix cells in matrix cell culture medium.

[0090] In some implementations, the cultured cell culture includes cultured cell culture in a container (such as a cell culture dish) coated with poly-L-lysine.

[0091] In some implementations, cultured matrix cells include passage of matrix cells before they reach a maximum threshold confluence and seeding matrix cells when they exceed a minimum threshold confluence. The maximum and minimum threshold confluence values ​​can be determined empirically.

[0092] single layer

[0093] In some embodiments, the at least one stromal cell is prepared as a monolayer. Monolayer culture typically involves a method comprising: digesting or dissociating at least one stromal cell into a single-cell suspension; seeding a semipermeable membrane with the suspension; and culturing the cells in the presence of an amplification medium until a monolayer is formed. Specific methods are disclosed in WO2023 / 281122, which is incorporated herein by reference in its entirety.

[0094] organoid culture

[0095] This invention provides at least one organoid that can be combined with at least one stromal cell to form a co-culture. This 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 “organoids of the invention” or “organoids”, and references to “organoid” may include at least one organoid that can be combined with at least one stromal cell to form a co-culture, as well as any reference organoid.

[0096] Preparation of organoids

[0097] As described herein, organoids can be prepared by culturing epithelial cells in organoid culture medium. The organoids of the present invention are characterized by Lgr5 expression.

[0098] Cultured epithelial cells can include obtaining epithelial cells from a sample, such as a tissue biopsy.

[0099] Culture of epithelial cells may include releasing crypts for organoid derivation, for example, by mechanically shearing the tissue and resuspending the crypts in organoid culture medium.

[0100] In some embodiments, the organoids of the present invention are (e.g., at a 1:1 ratio) destroyed (divided) into organoid fragments and then passaged. Dividation can be performed by mechanical force (referred to as "shearing") or enzymatically (e.g., by TrypLE).

[0101] organoid cell types

[0102] The organoids and / or organoid co-cultures of the present invention can be obtained from epithelial cells and can therefore be described as "epithelial organoids". Any epithelial cells from which organoids can be generated are suitable for the present invention. Preferred epithelial cells include intestinal cells, crypt cells, rectal cells, lung cells, hepatocytes, mammary gland cells, skin cells, pancreatic cells, endocrine cells, exocrine cells, ductal cells, kidney 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 be epithelial stem cells, preferably characterized by Lgr5 expression.

[0103] Suitable samples for obtaining epithelial cells include tissue biopsies, such as tissue samples obtained from a resected colon and / or rectum to obtain colorectal epithelial cells; or renal epithelial cells obtained from urine.

[0104] The organoids of the present invention can be gastrointestinal organoids, lung organoids, pancreatic organoids, tracheal organoids, skin organoids, or vaginal organoids—preferably gastrointestinal organoids. Further, the organoids of the present invention can 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.

[0105] Organoids and / or organoid co-cultures can be obtained from normal epithelial cells or from diseased epithelial cells (sometimes specifically referred to as "disease organoids" or "disease co-cultures"). In some embodiments, the epithelial cells are obtained from samples from subjects with inflammatory diseases. In one specific embodiment, diseased epithelial cells and normal epithelial cells are obtained from samples from the same subject, optionally from the same sample.

[0106] Organoids can be obtained by culturing epithelial stem cells. The epithelial stem cells can be obtained from adult tissues, i.e., the epithelial stem cells are adult epithelial stem cells. In this document, "adult" means mature tissue, including newborn infants or children, but excluding embryos or fetuses. Alternatively, the epithelial stem cells are not derived from embryonic stem cells or embryonic stem cell lines, such as embryonic stem cells or embryonic stem cell lines that have already differentiated in vitro. The adult tissue can be healthy adult tissue. In other embodiments, the adult tissue can be derived from a patient with a disease (such as an inflammatory disease).

[0107] The epithelial stem cells may be derived from the colorectal, small intestine, lung, stomach, pancreas, liver, breast, prostate, kidney, mouth, nasopharynx, larynx, hypopharynx, trachea, skin, fallopian tube, ovary, salivary gland, esophagus, hair follicle, and / or cochlear tissue. In some embodiments, the epithelial stem cells are colorectal cells. Methods for culturing epithelial stem cells from various epithelial tissues to obtain organoids have been previously described (e.g., in WO2009 / 022907, WO2010 / 090513, WO2012 / 014076, WO2012 / 168930, WO2015 / 173425, WO2016 / 083613 and WO2016 / 083612, WO2017 / 149025, WO2020 / 234250 and [8]). Cells taken directly from tissues, i.e., freshly isolated cells, are also referred to as “primary cells”. In some implementations, the epithelial stem cells are primary epithelial stem cells. In some implementations, the primary epithelial cells are epithelial cells derived from the patient.

[0108] In some implementations, the organoids contain only epithelial cells, meaning that no non-epithelial cells are present in the organoids. Even if other cell types are transiently present in the culture medium, such as in tissue fragments used as starting material, these cells are unlikely to survive and will instead be replaced by long-term expansion of stem cells that produce a pure epithelial cell population.

[0109] Organoids obtained using a suitable culture medium for expansion (“expansion medium”) are referred to as “expansion organoids”. Expanded organoids contain at least one epithelial stem cell that can divide and generate further epithelial stem cells or differentiated progeny. It should be understood that in preferred expanded organoids, the majority of cells are expanded cells (i.e., dividing cells) that retain an undifferentiated phenotype. While some spontaneous differentiation may occur, the cell population is typically an expanded cell population. The length of time that the organoid can sustain expansion while maintaining the core presence of epithelial stem cells and simultaneously maintaining the genotypic and phenotypic integrity of the cells is an important characteristic of the organoid. Organoids typically express Lgr5. The organoids also possess a unique structure that rapidly emerges with in vitro expansion and self-organization of the cells. These characteristics are described in detail herein.

[0110] In some embodiments, the organoids and / or co-cultures of the present invention (e.g., intestinal organoids) comprise one or more of the following cell types: Lgr5+ stem cells, intestinal epithelial cells, goblet cells, Paneth cells, and intestinal endocrine cells.

[0111] The organoids of the present invention may comprise or be derived from organoid fragments. Organoid fragments include any segment derived from an organoid, such as intestinal crypts derived from an organoid. In some embodiments, the organoid fragments are cell clumps, preferably composed of fewer than 10, fewer than 5, and preferably 2 to 4 cells. In a preferred embodiment, the one or more organoids are digested or dissociated into a suspension comprising single cells and cell clumps.

[0112] organoid structure

[0113] In some embodiments, the organoid is a three-dimensional cellular structure. In some embodiments, the organoid comprises a lumen surrounded by epithelial cells. In some embodiments, the epithelial cells surrounding the lumen are polarized. This polarization may be disrupted in diseased organoids. The epithelial cells used to obtain the organoid are preferably primary epithelial cells, such as those derived from a patient. Preferably, the adult epithelial stem cells are not derived from induced pluripotent stem cells (iPSCs).

[0114] In some embodiments, the epithelial cells in the organoid surround a lumen. In some embodiments, the organoid does not contain a lumen (in particular, disease organoids may not have a lumen). In some embodiments, the epithelial cells are polarized, meaning that proteins are differentially expressed at the apex or basal outer side of the epithelial cells. In some embodiments, the lumen is a sealed lumen (meaning a continuous cell barrier separates the contents of the lumen from the medium surrounding the organoid). In some embodiments, the organoid contains actively dividing stem cells, and these stem cells are preferably capable of differentiating into the same cell types as all major differentiated cell lineages present in the corresponding in vivo tissue, for example, when the organoid or cells are transferred to a differentiation culture medium. In some embodiments, the organoid contains basal cells on the outer side and more differentiated cells in the center.

[0115] In terms of the structure of intestinal epithelial cells, their apical surface faces the lumen and regulates their interaction with the lumen contents, including mediating nutrient absorption, detecting microbial products and secreting molecules that protect the epithelium from potentially harmful substances in the lumen. Their basolateral surface anchors the epithelial cells to the underlying basement membrane, transports nutrients from the lumen to the bloodstream, and communicates with nearby cells[9].

[0116] In some implementations, organoids include stratified epithelium. "Stratified" means the presence of multiple (more than one) cell layers. Such cells typically tend to have their nuclei located more centrally within the cell, i.e., they are non-polarized. Cells in a multilayered structure may self-organize, creating gaps or cavities between cells.

[0117] In some implementations, 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 greater.

[0118] In some implementations, the organoids have a diameter of about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 250 μm or larger at their widest point.

[0119] In some implementations, the organoids have a diameter at their widest point of up to about 50 μm, up to about 60 μm, up to about 70 μm, up to about 80 μm, up to about 90 μm, up to about 100 μm, up to about 125 μm, up to about 150 μm, up to about 175 μm, up to about 200 μm, up to about 250 μm, or greater.

[0120] In the context of this invention, tissue fragments are part of adult tissue, preferably human adult tissue. In contrast, organoids are formed through in vitro expansion to acquire their structural features and are thus distinguished from tissue fragments.

[0121] Organoid culture medium

[0122] Organoid culture media can be used to prepare organoids for co-culture, for example, by promoting the growth, division (expansion), structural tissue, or other development of epithelial cells to produce organoids suitable for co-culture. These organoid culture media are particularly suitable for tissues derived from the digestive system, such as gastrointestinal tissues like intestine or colonic rectum. Organoid culture media may also be referred to as “amplification medium,” “organoid amplification medium,” or “colonic normal medium” (“CNM”).

[0123] The culture medium may include a basal medium. The basal medium is any suitable basal medium for animal or human cells, subject to any limitations provided herein, such as complete advanced DMEM / F12 medium.

[0124] The culture medium may contain a Wnt agonist, such as (a) optionally about 50% of the final volume of WNT conditioned medium, or (b) about 0.5 nM NGS Wnt. Particularly preferred is a Wnt conditioned medium, such as Wnt3a conditioned medium (optionally about 10% of the final volume).

[0125] The culture medium may contain R-vertebral proteins, such as (a) R-vertebral protein-1 conditioned medium, optionally at about 20% of the final volume, or (b) about 250 ng / ml of Rspo3.

[0126] The culture medium may contain BMP inhibitors, such as head proteins, for example (a) head protein conditioned medium (“NCM”), optionally at about 1%-4% of the final volume, or (b) recombinant head proteins, optionally at a concentration of about 100 ng / ml. Head protein conditioned medium (“NCM”) is particularly preferred, optionally at about 1%-4% of the final volume.

[0127] The culture medium may contain B27, optionally about 1x B27 according to the manufacturer's instructions.

[0128] The culture medium may contain, for example, about 1.25 mM of N-acetylcysteine ​​(“N-Ac”).

[0129] The culture medium may contain nicotinamide, for example, about 10 mM.

[0130] The culture medium may contain mitotic growth factors, such as EGF, optionally at about 50 ng / mL.

[0131] The culture medium may contain gastrin, optionally at about 10 mM or about 5 mM.

[0132] The culture medium may contain a TGF-β inhibitor, such as A-83-01, optionally at about 500 nM.

[0133] The culture medium may contain a p38 MAPK inhibitor, such as SB202190, optionally at about 3 μM or about 10 μM.

[0134] The culture medium may contain a prostaglandin pathway activator, which may be a prostaglandin, such as prostaglandin E2, optionally at about 10 nM.

[0135] The culture medium may contain antibiotics, such as primocin, optionally at about 100 mg / mL or about 50 μg / mL.

[0136] Therefore, the culture medium may contain mitotic growth factor, BMP inhibitor, and R-vertebral protein. In some embodiments, the culture medium contains EGF, head protein, and R-vertebral protein. In some embodiments, the culture medium contains approximately 50 ng / mL EGF, approximately 1%-4% of the final volume of head protein conditioned medium, and approximately 20% of the final volume of R-vertebral protein-1 conditioned medium. Such culture media may include basal media.

[0137] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-vertebral protein. In some embodiments, the culture medium contains a Wnt agonist, EGF, head protein, and R-vertebral protein. In some embodiments, the culture medium contains approximately 50% of the final volume of WNT3A conditioned medium, approximately 50 ng / mL EGF, approximately 1%-4% of the final volume of head protein conditioned medium, and approximately 20% of the final volume of R-vertebral protein-1 conditioned medium. Such culture media may include basal media.

[0138] The culture medium may contain a Wnt agonist, mitotic growth factor, a BMP inhibitor, and R-vertebral protein. In some embodiments, the culture medium contains a Wnt agonist, EGF, head protein, and R-vertebral protein. In some embodiments, the culture medium contains approximately 50% of the final volume of WNT3A conditioned medium, approximately 50 ng / mL EGF, approximately 1%-4% of the final volume of head protein conditioned medium, and approximately 20% of the final volume of R-vertebral protein-1 conditioned medium. Such culture media may include basal media.

[0139] The culture medium may contain a Wnt agonist, mitotic growth factor, BMP inhibitor, R-vertebral protein, and TGF-β inhibitor. In some embodiments, the culture medium contains a Wnt agonist, EGF, cephalin, R-vertebral protein, and A-83-01. In some embodiments, the culture medium contains approximately 50% of the final volume of WNT3A conditioned medium, approximately 50 ng / mL EGF, approximately 1%-4% of the final volume of cephalin conditioned medium, approximately 20% of the final volume of R-vertebral protein-1 conditioned medium, and approximately 500 nM A-83-01. Such culture media may include basal media.

[0140] The culture medium may contain a Wnt agonist, mitotic growth factor, BMP inhibitor, R-vertebral protein, and p38 MAPK inhibitor. In some embodiments, the culture medium contains a Wnt agonist, EGF, cephalin, R-vertebral protein, and SB202190. In some embodiments, the culture medium contains approximately 50% of the final volume of WNT3A conditioned medium, approximately 50 ng / mLEGF, approximately 1%-4% of the final volume of cephalin conditioned medium, approximately 20% of the final volume of R-vertebral protein-1 conditioned medium, and approximately 3 μM SB202190. Such culture media may include basal media.

[0141] The culture medium may contain a Wnt agonist, mitotic growth factor, BMP inhibitor, R-vertebral protein, TGF-β inhibitor, and p38 MAPK inhibitor. In some embodiments, the culture medium contains a Wnt agonist, EGF, cephalin, R-vertebral protein, A-83-01, and SB202190. In some embodiments, the culture medium contains approximately 50% final volume of WNT3A conditioned medium, approximately 50 ng / mL EGF, approximately 1%-4% final volume of cephalin conditioned medium, approximately 20% final volume of R-vertebral protein-1 conditioned medium, approximately 500 nM A-83-01, and approximately 3 μM SB202190. Such culture media may include basal media.

[0142] B27 and N-acetylcysteine ​​are optional components of the culture medium. Therefore, the culture medium may contain basal medium, Wnt agonist, R-vertebral protein, head protein, B27, N-acetylcysteine, nicotinamide, mitotic growth factor, gastrin, TGF-β inhibitor, p38 MAPK inhibitor, prostaglandin pathway activator, and primary cell antibiotics. In some embodiments, the basal medium is a complete high-grade DMEM / F12 medium, the Wnt agonist is WNT3A conditioned medium, the R-vertebral protein is R-vertebral protein-1 conditioned medium, the head protein is head protein 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.

[0143] The culture medium may contain approximately 50% final volume of WNT3A conditioned medium, approximately 20% final volume of R-vertebral protein-1 conditioned medium, approximately 1%-4% final volume of head protein conditioned medium, approximately 1x B27 according to the manufacturer's instructions, approximately 1.25 mM of N-acetylcysteine, approximately 10 mM of nicotinamide, approximately 50 ng / mL of EGF, approximately 10 mM of gastrin, approximately 500 nM of A-83-01, approximately 3 μM of SB202190, approximately 10 nM of prostaglandin E2, and approximately 100 mg / mL of primary cell antibiotics.

[0144] More generally, those skilled in the art will recognize that some components of the organoid culture medium (such as B27 and N-acetylcysteine) are optional and / or can be replaced by suitable alternative components. Suitable organoid culture media for different tissues are known in the art (e.g., [8]). The culture medium can be any suitable expansion medium for epithelial stem cells or progenitor cells, preferably a suitable expansion medium for epithelial stem cells. Culture media suitable for culturing organoids are also described in the following references: 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 these culture media can be used in the context of this invention.

[0145] In some implementations, the culture medium contains a receptor tyrosine kinase ligand, a BMP inhibitor, and a Wnt agonist.

[0146] For example, in some embodiments, the culture medium comprises EGF, head protein, and Wnt conditioned medium. In some embodiments, the culture medium comprises EGF, head protein, R-vertebral protein, and Wnt substitutes.

[0147] 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-β inhibitor.

[0148] For example, preferred organoid culture media contain a Wnt agonist (e.g., any one of R-vertebral proteins 1-4), mitotic growth factors (e.g., selected from EGF, FGF, HGF, and BDNF), and a BMP inhibitor (e.g., head protein) (e.g., as described in WO2010 / 090513). In some embodiments, the organoid culture media further contain a TGF-β inhibitor (e.g., A83-01, Tocris) (e.g., as described in WO2012 / 168930). The addition of a TGF-β inhibitor is particularly suitable for culturing human cells. The TGF-β inhibitor preferably inhibits the ALK4 / 5 / 7 signaling pathway.

[0149] Preferred organoid culture media, particularly suitable for the culture of intestinal or colonic organoids, comprise one or more of the following: basal medium (e.g., advanced DMEM / F12 medium, Gibco), Wnt ligand (e.g., Wnt-3a), Wnt agonist (e.g., any one of R-vertebral proteins 1-4), BMP inhibitor (e.g., Noggin), EGF and TGF-β inhibitors (e.g., A83-01, Tocris), and optionally further comprise p38 MAPK inhibitors, gastrin, nicotinamide, prostaglandin E2, N-acetylcysteine, B27, and / or antimicrobial agents (e.g., primary cell antibiotics).

[0150] Specific organoid culture media

[0151] In a preferred embodiment, the organoid culture medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml); (ii) head protein (e.g., at a concentration of about 100 ng / ml); (iii) R-vertebral protein (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-β inhibitor (e.g., A83-01 at a concentration of about 500 nM); and (vii) nicotinamide (e.g., at a concentration of about 10 mM).

[0152] In another preferred embodiment, the organoid culture medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml); (ii) head protein (e.g., at a concentration of about 100 ng / ml); (iii) Wnt conditioned medium (e.g., about 50% final volume); (iv) p38 inhibitor (e.g., SB-203580 at a concentration of about 10 μM); (v) TGF-β inhibitor (e.g., A83-01 at a concentration of about 500 nM); and (vi) nicotinamide (e.g., at a concentration of about 10 mM).

[0153] In some embodiments, particularly in the case of lung-derived organoids, the organoid culture medium comprises one or more receptor tyrosine ligands, Wnt agonists, TGF-β inhibitors, and BMP inhibitors. In some embodiments, the organoid culture medium comprises FGF, R-vertebral protein, TGF-β inhibitors, BMP inhibitors, Rho-kinase inhibitors, and p38 inhibitors. In a preferred embodiment, the organoid culture 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) R-vertebral protein (e.g., R-vertebral protein-3 at a concentration of about 250 ng / mL); (iii) TGF-β inhibitor (e.g., A83-01 at a concentration of about 500 nM); (iv) BMP inhibitor (e.g., head protein-Fc fusion protein conditioned medium at a final volume of about 2%); (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).

[0154] In some embodiments, particularly in the case of organoids derived from kidneys, the organoid culture medium comprises one or more receptor tyrosine ligands, Wnt agonists, and TGF-β inhibitors. In some embodiments, the organoid culture medium comprises EGF, FGF, R-vertebral protein, TGF-β inhibitors, and Rho kinase inhibitors. In a preferred embodiment, the organoid culture 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) R-vertebral protein (e.g., Rspo 1-conditioned medium at about 10% final volume); (iv) TGF-β inhibitors (e.g., A83-01 at a concentration of about 500 nM); and (v) Rho kinase inhibitors (e.g., Y-27632 at a concentration of about 10 μM).

[0155] organoid culture duration

[0156] In some embodiments, epithelial cells are cultured in organoid culture 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, the epithelial cells are cultured in organoid culture medium for at least about 24 hours to prepare the at least one organoid.

[0157] In some embodiments, the at least one organoid of the present invention has been cultured in an organoid culture medium or is capable of being cultured for at least about 2 months, for example at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, or at least about 1 year.

[0158] In some embodiments, at least one organoid has been cultured or is capable of being 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.

[0159] In some implementations, the number of organoid cells increases exponentially at about 5 passages, about 10 passages, about 15 passages, or about 20 passages, preferably at about 5 passages.

[0160] In a preferred embodiment, the organoids used in the claimed method may be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or longer. In some embodiments, the organoids are expanded or maintained in the 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.

[0161] single layer

[0162] In some embodiments, the organoid is prepared as a single layer. In some embodiments, the organoid comprises a single-layer structure that is folded (or invaginated) to form two or more layers. Sometimes it is difficult to distinguish between folded (or invaginated) single-layer structures and multilayered cellular regions. In some embodiments, the organoid comprises both multilayered cellular regions and folded single-layered structural regions. In some embodiments, the organoid has segments formed by multiple layers and segments containing a single single-layered cell. In some embodiments, the organoid comprises or consists of a single single-layered cell. In some embodiments, the organoid does not comprise a single layer. The organoid may have a cell layer having at least one bud-like structure and a central lumen.

[0163] Organoid monolayer culture typically involves a method comprising the following steps: digesting or dissociating one or more organoids into a suspension of single cells and / or organoid fragments; seeding a semipermeable membrane with the suspension; 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 herein by reference in its entirety.

[0164] In particular, the following embodiments from WO2023 / 281122 are incorporated by reference and are especially relevant to intestinal organoids: Example 1 describes the preparation of an epithelial monolayer from a normal human intestinal organoid (e.g., the intestinal organoid of the present invention); Example 2 describes the establishment, differentiation, and characterization of a human gastrointestinal epithelial monolayer; Example 3 describes the process of constructing an in vitro biological system designed to mimic relevant components in the pathophysiological process of inflammatory bowel disease and to provide reliable readouts of barrier pathways for detection (e.g., for use in this invention and in the detection of inflammatory bowel disease). Example 4 describes the robustness verification of barrier function assays performed using a monolayer derived from intestinal organoids; Example 5 describes the establishment of a human gastrointestinal organoid epithelial monolayer; and Example 6 describes the polarization of a single layer of human gastrointestinal organoid epithelium.

[0165] co-culture

[0166] This invention provides co-cultures as described herein and uses co-cultures as described herein in methods of this invention.

[0167] combination

[0168] The method of the present invention may include combining the at least one organoid with the at least one stromal cell in a co-culture medium to form the co-culture.

[0169] The methods of the present invention, such as methods for determining the presence or absence of at least one variation in a co-culture (including methods for testing one or more therapeutic agents, and methods for determining the presence or absence of a diagnosis and / or prognosis), can be performed on already prepared co-cultures; that is, the step of combining the at least one organoid and at least one stromal cell is not a mandatory step in all methods of the present invention.

[0170] In a preferred embodiment, the organoid culture medium (optionally including any extracellular matrix, such as basement membrane matrix 'BME' or Matrigel) is removed from the at least one organoid before combining it with the at least one stromal cell. Removal can be performed using a protease such as a dispersant. The extracellular matrix can be disrupted using a commercially available kit such as CellRecovery Solution™ (Corning). Alternative matrices, such as collagen, can be used instead of the removed matrix.

[0171] In some implementations, a multi-drop dispenser is used to combine the at least one organoid and the at least one stromal cell.

[0172] In some embodiments, at least one stromal cell and the at least one organoid are combined in an equal ratio, i.e., one organoid is combined with one stromal cell (“1:1”). In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of about 0.5. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of about 2. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of about 2.5. A higher fibroblast-organoid ratio can lead to an enhanced pro-inflammatory spectrum (e.g., increased IL6 and CXCL2 secretion) and / or an enhanced inflammatory response.

[0173] In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of at least about 0.5. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of at least about 1:1. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of at least about 2. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of at least about 2.5.

[0174] In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of up to about 0.5. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of up to about 1:1. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of up to about 2. In some embodiments, at least one stromal cell is combined with the at least one organoid at a ratio of up to about 2.5.

[0175] In some embodiments, combining the at least one organoid with the at least one stromal cell includes 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.

[0176] In some embodiments, combining the at least one organoid with the at least one stromal cell includes a hanging drop method.

[0177] In some embodiments, combining the at least one organoid with the at least one stromal cell comprises a multi-well plate, optionally wherein the multi-well plate comprises an ultra-low adhesion plate.

[0178] In some implementations, at least one organoid can be prepared from epithelial cells while combining epithelial cells with the at least one stromal cell, such that co-culture and organoid formation occur simultaneously.

[0179] co-culture medium

[0180] This invention provides culture media for the co-culture of organoids and stromal cells (e.g., as described in the examples). The co-culture medium can also be described as a “differentiation medium” or a “combined colon differentiation medium” (“cCDM”). The co-culture medium can also be described as an “intestinal epithelial cell-colon differentiation medium” (“eCDM”).

[0181] The co-culture medium of the present invention advantageously allows for the co-culture of stromal cells and organoids. Such co-culture would be difficult or even impossible without the adaptability of the medium used in the co-culture medium of the present invention. The inventors have observed for the first time that stromal cell function is preserved in co-culture with organoids according to the present invention.

[0182] For example, and without wishing to be bound by any theory, a co-culture medium was chosen to identify one or more variations of organoid responses in co-culture of factors released by stromal cells. Stromal cells are known to secrete ligands that support the presence of stem cells and may therefore inhibit differentiation. The inventors theoretically believe that such responses can be determined based on the different morphologies of differentiated organoids (small lumens and thick epithelium) and undifferentiated organoids (large lumens and thin epithelium). The chosen co-culture medium also contains an ERK inhibitor (PD0325901), which the inventors believe will enhance the observability of the sensitivity of the co-culture to cytokine damage. The method of the present invention reveals active connectivity between organoids and the matrix. Surprisingly, it has been found that the inclusion of serum (e.g., fetal bovine serum, “FCS”, also known as fetal bovine serum, “FBS”) in the co-culture medium supports long-term culture of fibroblasts and organoids without disrupting organoid structure.

[0183] The co-culture medium is particularly suitable for tissues derived from the digestive system, such as gastrointestinal tissues like intestines or colonic tissues, as well as matrix tissues.

[0184] The at least one organoid and at least one stromal cell can be combined in cCDM to form a co-culture.

[0185] In some embodiments, the culture medium includes a basal medium. The basal medium is any suitable basal medium for animal or human cells, subject to no limitations provided herein, such as complete advanced DMEM / F12 medium.

[0186] In some embodiments, the culture medium contains N-acetylcysteine ​​(“N-Ac”), optionally at about 1.25 mM.

[0187] In some embodiments, the culture medium contains a TGF-β inhibitor, optionally A83-01, optionally about 500 nM.

[0188] In some implementations, the culture medium contains B27, optionally at about 1x according to the manufacturer's instructions.

[0189] In some embodiments, the culture medium contains a mitotic growth factor, such as EGF, optionally at about 50 ng / mL.

[0190] In some implementations, the culture medium contains gastrin, optionally at about 5 nM.

[0191] In some embodiments, the culture medium contains a BMP inhibitor, such as head protein, for example (a) head protein conditioned medium (“NCM”), optionally at about 1-2% of the final volume, or (b) recombinant head protein, optionally at a concentration of about 100 ng / ml. Head protein conditioned medium (“NCM”) is particularly preferred, optionally at about 1-2% of the final volume.

[0192] In some implementations, the culture medium contains, optionally, an antibiotic, such as a primary cell antibiotic, at a concentration of about 50 μg / mL.

[0193] In some embodiments, the culture medium contains, optionally, about 250 ng / mL of R-vertebral protein, such as Rspo3.

[0194] In some embodiments, the culture medium contains a Notch pathway inhibitor. In some embodiments, the culture medium contains DAPT, optionally 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.

[0195] In some embodiments, the culture medium contains an ERK inhibitor. In some embodiments, the culture medium contains PD0325901, optionally at about 100 nM. Without wishing to be bound by any theory, the inventors believe that organoids co-cultured in a medium containing an ERK inhibitor such as PD0325901 exhibit structures and lumen sizes that reflect interactions with stromal cells even more effectively, and more prominently 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.

[0196] 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 are Wnt conditioned media, 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.

[0197] In some embodiments, the culture medium contains serum, such as fetal bovine serum (FBS), optionally at about 5% or about 10% of the final volume, preferably 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.

[0198] 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.

[0199] B27 and N-acetylcysteine ​​are optional components of the culture medium.

[0200] In some embodiments, the culture medium comprises N-Ac, a TGF-β inhibitor, B27, mitotic growth factor, gastrin, a BMP inhibitor, an antibiotic, R-vertebral protein, a Notch pathway inhibitor, an ERK inhibitor, and a Wnt agonist. In some embodiments, the TGF-β inhibitor is A83-01, the mitotic growth factor is EGF, the R-vertebral protein is Rspo3, the BMP inhibitor is head protein conditioned medium, the antibiotic is a primary cell antibiotic, 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.

[0201] In some embodiments, the culture medium contains N-Ac, A83-01, B27, EGF, gastrin, head protein conditioned medium, primary cell antibiotics, R-vertebral protein, DAPT, PD0325901, and Wnt agonist. In some embodiments, the culture medium further contains FBS and ECM.

[0202] In some embodiments, the culture medium comprises 1.25 mM N-Ac, 500 nM A83-01, approximately 1x B27 according to the manufacturer's instructions, approximately 50 ng / mL EGF, approximately 5 nM gastrin, approximately 1-2% head protein conditioned medium, approximately 50 μg / mL primary cell antibiotic, approximately 250 ng / mL Rspo3, approximately 10 μM DAPT, approximately 100 nM PD0325901, and approximately 10% Wnt final volume or approximately 0.1 nM NGS Wnt Wnt Wnt conditioned medium. In some embodiments, the culture medium further comprises approximately 5% final volume FBS and approximately 5% final volume Matrigel.

[0203] Any of the stromal cell culture medium or the organoid culture medium described herein can be used as a co-culture medium. In some embodiments, organoid culture medium is used as a co-culture medium, for example, CNM is used as a co-culture medium.

[0204] The organoid culture medium described herein can be used as a co-culture medium by reducing the concentration of BMP inhibitors such as cephalin, optionally by reducing the concentration of cephalin by about 50%, and preferably by reducing the cephalin conditioned medium to about 1%-2% of the final volume.

[0205] The organoid culture medium described herein can be used as a co-culture medium by reducing the concentration of the Wnt agonist, optionally by reducing the concentration of the Wnt agonist by about 80%, preferably 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, reducing the Wnt3a conditioned medium (optionally to about 10% of the final volume).

[0206] The organoid culture medium described herein can be used as a co-culture medium by adding a Notch pathway inhibitor (e.g., DAPT) to the medium, for example by adding DAPT to a concentration of approximately 10 μM.

[0207] The organoid culture medium described herein can be used as a co-culture medium by adding an ERK inhibitor, such as PD0325901, for example by adding PD0325901 to a concentration of about 100 nM.

[0208] The organoid culture medium described herein can be used as a co-culture medium by adding serum, such as FCS, to the medium, for example by adding FCS to about 5% of the final volume.

[0209] The organoid culture medium described herein can be used as a co-culture medium by adding ECM to the medium, such as Matrigel, for example by adding Matrigel to a concentration of approximately 5% of the final volume.

[0210] 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 can be replaced by suitable alternative components. The differentiation medium can be any suitable differentiation medium for organoids and stromal cells, such as those described in WO2015 / 173425, WO2017 / 149025, and WO2017 / 220586.

[0211] In some embodiments, the differentiation medium suitable for co-culture may contain one or more of Wnt agonists, BMP inhibitors, mitotic growth factors, and TGF-β inhibitors. For example, the differentiation medium contains a Wnt agonist. The differentiation medium may further contain mitotic growth factors and / or BMP inhibitors.

[0212] In a preferred embodiment, the differentiation medium comprises a Wnt agonist, a BMP inhibitor, a mitotic growth factor, and a TGF-β inhibitor. The differentiation medium may further comprise one or more of a p38 inhibitor, a cAMP agonist, a prostaglandin pathway activator, nicotinamide, gastrin, B27, and N-acetylcysteine.

[0213] In some embodiments, the differentiation medium comprises a basal medium for human or animal cells (such as DMEM / F12, optionally including B27 or Ad-DF+++ (an advanced Dulbecco modified Eagle's / F12 medium supplemented with GultaMax, 1 M HEPES)), R-vertebral protein family proteins, mitotic growth factors (such as EGF), BMP inhibitors (such as head protein), TGF-β inhibitors (such as A83-01), p38 inhibitors (such as SB202190), and optionally nicotinamide and N-acetylcysteine.

[0214] In some implementations, the differentiation medium comprises advanced DMEM / F12 medium, which includes B27, nicotinamide, N-acetylcysteine, head protein, R-vertebral protein 1-4, EGF, Wnt (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.

[0215] The co-culture medium may contain 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 A co-culture of ( ). Preferably, the ECM is Matrigel and constitutes about 1% to about 10% ( ). v / v The co-culture, for example, about 5% to about 10% ( v / v The co-culture of the above, preferably about 5% ( v / v The co-culture of [the organism]. In some embodiments, the co-culture medium does not contain ECM.

[0216] In other embodiments, the co-culture medium may be described as "enterocellular colon differentiation medium" ("eCDM"). Such a medium may contain a Wnt pathway inhibitor, which may be a Porcupine (PORCN) inhibitor. Therefore, such a medium may contain a PORCN inhibitor, such as iWP-2, for example, 1.5 μM IWP-2.

[0217] eCDM may contain TGF-β inhibitors, mitotic growth factors and BMP inhibitors. For example, the culture medium may contain A-83-01, EGF and head protein conditioned medium, such as 500 nM A-83-01, 50 ng / ml EGF and 1-2% head protein conditioned medium.

[0218] The culture medium may further contain R-vertebral proteins, such as Rspo3 (e.g., 250 ng / ml Rspo3).

[0219] The culture medium may further contain N-acetylcysteine ​​(e.g., 1.25 mM N-acetylcysteine), B27 supplement (e.g., 1x B27 according to the manufacturer's instructions), gastrin (e.g., 5 nM gastrin), and / or antibiotics such as primary cell antibiotics (e.g., 50 μg / ml primary cell antibiotics).

[0220] The culture medium may further contain serum such as FBS (e.g., 5% FBS) and / or ECM such as Matrigel (e.g., 5% Matrigel).

[0221] The culture medium may further contain serum such as FBS (e.g., 5% FBS) and / or ECM such as Matrigel (e.g., 5% Matrigel).

[0222] Therefore, in some embodiments, the co-culture medium contains a TGF-β inhibitor, a mitotic growth factor, a BMP inhibitor, R-vertebral protein, serum and ECM, and optionally one or more of N-acetylcysteine, B27 supplement, gastrin and antibiotics.

[0223] In some embodiments, the co-culture medium comprises A-83-01, EGF, head protein conditioned medium, R-vertebral protein, serum and ECM, and optionally one or more of N-acetylcysteine, B27 supplement, gastrin and primary cell antibiotics.

[0224] In some embodiments, the co-culture medium comprises 500 nM A-83-01, 50 ng / ml EGF, 1-2% head protein conditioned medium, 250 ng / ml Rspo3, 5% FBS and 5% Matrigel, and optionally 1.25 mM N-acetylcysteine, 1x B27 supplement according to the manufacturer's instructions, 5 nM gastrin and 50 μg / ml primary cell antibiotic.

[0225] In some embodiments, the co-culture medium comprises eCDM, which may contain a Wnt pathway inhibitor. In some embodiments, the eCDM comprises a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, and Wnt pathway inhibitor. In some embodiments, the eCDM comprises a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, Wnt pathway inhibitor, and R-vertebral protein. In some embodiments, the eCDM medium may contain a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, Wnt pathway inhibitor, R-vertebral protein, N-acetylcysteine, B27 supplement, gastrin, and antibiotics.

[0226] In some embodiments, the co-culture medium comprises an eCDM, which may contain a Wnt pathway inhibitor, such as a PORCN inhibitor. In some embodiments, the eCDM comprises a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, and PORCN inhibitor. In some embodiments, the eCDM comprises a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, PORCN inhibitor, and R-vertebral protein. In some embodiments, the eCDM medium may contain a TGF-β inhibitor, mitotic growth factor, BMP inhibitor, PORCN inhibitor, R-vertebral protein, N-acetylcysteine, B27 supplement, gastrin, and antibiotics.

[0227] In some embodiments, the eCDM medium contains A-83-01, EGF, headprotein conditioned medium, and iWP2. In some embodiments, the eCDM medium may contain A-83-01, EGF, headprotein conditioned medium, iWP2, and Rspo3. In some embodiments, the eCDM medium may contain A-83-01, EGF, headprotein conditioned medium, iWP2, Rspo3, N-acetylcysteine, B27 supplement, gastrin, and primary cell antibiotics.

[0228] In some embodiments, the eCDM medium contains approximately 500 nM A-83-01, approximately 50 ng / ml EGF, approximately 1-2% headprotein conditioned medium, and approximately 1.5 μM iWP2. In some embodiments, the eCDM medium may contain approximately 500 nM A-83-01, approximately 50 ng / ml EGF, approximately 1-2% headprotein conditioned medium, approximately 1.5 μM iWP2, and approximately 250 ng / ml Rspo3. In some embodiments, the eCDM medium may contain approximately 500 nM A-83-01, approximately 50 ng / ml EGF, approximately 1-2% headprotein conditioned medium, approximately 1.5 μM iWP2, approximately 250 ng / ml Rspo3, approximately 1.25 mM N-acetylcysteine, approximately 1x B27 according to the manufacturer's instructions, approximately 5 nM gastrin, and approximately 50 μg / ml primary cell antibiotics.

[0229] Co-cultivation duration

[0230] In some embodiments, the co-culture of the present invention has been cultured or is capable of being 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 has been cultured or is capable of being cultured for at least about 72 hours. More preferably, the co-culture has been cultured or is capable of being cultured for at least about 144 hours.

[0231] In some embodiments, the co-cultures of the present invention have been or are able to be cultured for at least about 2 months, for example at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, or at least about 1 year.

[0232] In some implementations, the co-culture has been or is able to 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.

[0233] In some implementations, the number of cells in the co-culture increases exponentially with about 5 passages, about 10 passages, about 15 passages, or about 20 passages, preferably more than about 5 passages.

[0234] In a preferred embodiment, the co-culture can be cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or longer. In some embodiments, the co-culture is expanded or maintained in the 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.

[0235] single layer

[0236] In some embodiments, the co-culture is prepared as a monolayer. Monolayer culture typically involves a method comprising: digesting or dissociating one or more organoid and stromal cells into a suspension of single cells and / or organoid fragments; seeding the suspension with a semipermeable membrane; and culturing the cells and / or organoid fragments in the presence of a differentiation medium until a monolayer is formed. Specific methods are disclosed in WO2023 / 281122, which is incorporated herein by reference in its entirety.

[0237] Components of organoid culture medium and co-culture medium

[0238] Organoid culture media may contain one or more of the components listed in the following sections. Co-culture media may contain one or more of the components listed in the following sections. Therefore, unless otherwise stated, the following sections apply to both organoid culture media and co-culture media.

[0239] basal culture medium

[0240] The basal culture medium used for cell culture typically contains a large number of components essential for supporting the maintenance of cultured cells. Based on the following disclosure, those skilled in the art can readily formulate suitable combinations of components. The basal culture medium used in this invention typically 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 more detail in the literature and below. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components, such as those selected from amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. Suitable basal media are known to those skilled in the art and are commercially available. Non-limiting examples include Dulbecco modified Eagle medium (DMEM), advanced-DMEM, minimum essential medium (MEM), knockout-DMEM (KO-DMEM), Glasgow minimum essential medium (G-MEM), basal Eagle medium (BME), DMEM / Ham F12, advanced-DMEM / Ham F12, Iscove modified Dulbecco medium and minimum essential medium (MEM), Ham F-10, Ham F-12, medium 199, and RPMI 1640. For example, the basal medium may be advanced-DMEM, preferably supplemented with glutamax, penicillin / streptomycin, and HEPES.

[0241] Extracellular matrix (ECM)

[0242] Epithelial stem cells are typically grown in cultures containing a known exogenous extracellular matrix (ECM) that supports cell growth (see, for example,

[10] ). Organoid culture media may contain ECM. ECM may be exogenous ECM (meaning any extracellular matrix protein naturally secreted by epithelial stem cells or epithelial stem cell populations upon contact with the expanded culture medium of the present invention). Any suitable ECM may be used. Cells are preferably cultured in a cellular microenvironment that at least partially mimics the cell’s natural environment. The cellular microenvironment is determined in part by the cells and by the ECM secreted by the cells in that microenvironment. The cellular microenvironment can be mimicked by culturing the cells in the presence of biological or synthetic materials that interact with cell membrane proteins such as integrins. Thus, the ECM as described herein is any biological or synthetic material or combination thereof that mimics the in vivo cellular microenvironment, for example by interacting with cell membrane proteins such as integrins.

[0243] In some embodiments, the ECM is in a suspension, meaning the cells are in contact with the ECM in a suspension system. In some embodiments, the concentration of the ECM in the suspension is at least 1%, at least 2%, or at least 3%. In some embodiments, the concentration of the ECM in the suspension is from 1% to about 10% or from 1% to about 5%. This suspension method may be advantageous for scalable methods. In some embodiments, the ECM is in the form of one or more domes.

[0244] One type of ECM is secreted by epithelial cells, endothelial cells, wall endodermal-like cells (e.g., Englebreth Holm Swarm wall endodermal-like cells as described in

[11] ), and connective tissue cells. This ECM is composed of various polysaccharides, water, elastin, and glycoproteins, wherein the glycoproteins include collagen, entactin / nidogen, fibronectin, and laminin. Therefore, in some embodiments, the ECM used in the methods of the present invention comprises one or more components selected from the following list: polysaccharides, elastin, and glycoproteins, such as glycoproteins comprising collagen, entactin (nidogen), fibronectin, and / or laminin. For example, in some embodiments, collagen is used as the ECM. Different types of ECM are known, comprising different compositions including different types of glycoproteins and / or different combinations of glycoproteins.

[0245] The ECM can be provided by culturing ECM-producing cells, such as epithelial cells, endothelial cells, wall endodermal-like cells, or fibroblasts, in a container, then removing these cells and adding isolated tissue fragments or isolated epithelial cells. Examples of cells that produce the extracellular matrix include: chondrocytes (primarily producing collagen and proteoglycans), fibroblasts (primarily producing type IV collagen, laminin, mesenchymal procollagen, and fibronectin), and colonic myofibroblasts (primarily producing collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tendinogen-C). These are “naturally produced ECMs.” Naturally produced ECMs are commercially available. Examples of commercially available extracellular matrix include extracellular matrix proteins (Invitrogen) and mouse sarcoma cells from Engelbreth-Holm-Swarm (EHS) (e.g., Cultrex® basement membrane extract (Trevigen, Inc.) or Matrigel™ (BDBiosciences)).

[0246] In some embodiments, the extracellular matrix is ​​at least 50%, at least 60%, or at least 70% Matrigel, optionally 50-100%, 50-80% Matrigel, and optionally about 70% Matrigel. In a preferred embodiment, the extracellular matrix is ​​at least 70% Matrigel.

[0247] 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 diffuse 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 concentration of the ECM in the suspension is at least 1%, at least 2%, or at least 3%. In some embodiments, the concentration of the ECM in the suspension is from 1% to about 10% or from 1% to about 5%.

[0248] In a preferred method of the present invention, cells are cultured in contact with the extracellular matrix (ECM). In some embodiments, the method of the present invention includes culturing epithelial stem cells in contact with the extracellular matrix. “Contact” refers to physical, mechanical, or chemical contact, meaning that a certain force needs to be applied 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.

[0249] In some embodiments, the organoid culture medium is placed on an extracellular matrix or synthetic matrix. The organoid culture medium can then be removed and replenished as needed. In some embodiments, the organoid culture medium is replenished every 1, 2, 3, 4, 5, 6, or 7 days. If a component is “added” or “removed” from the culture medium, in some embodiments this may mean removing the culture medium itself from the extracellular matrix or synthetic matrix, and then placing a new culture medium containing the “added” component or excluding the “removed” component on the extracellular matrix or synthetic matrix.

[0250] 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.

[0251] 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). The integrin agonist may be used in place of or in conjunction with the extracellular matrix.

[0252] In some embodiments, the ECM is a laminin-containing ECM, such as Matrigel™ (BD Biosciences). In some embodiments, the ECM is Matrigel™ (BD Biosciences) which contains laminin, nestin, and collagen IV. In some embodiments, the ECM contains laminin, nestin, collagen IV, and heparan 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 desired, a mixture of naturally occurring or synthetic ECM materials may be used. In some embodiments, the ECM is BME (“Basement Membrane Extract”), which is a soluble basement membrane form purified from Engelbreth-Holm-Swarm (EHS) tumors (e.g., Cultrex® BME).

[0253] In another embodiment, the ECM may be a synthetic ECM. For example, a synthetic ECM such as pro-adhesion protein (Sigma Z378666) may be used. In further examples, the ECM may be a plastic, such as polyester, or a hydrogel. In some embodiments, the synthetic matrix may be coated with a biological material, such as one or more glycoproteins, such as collagen or laminin.

[0254] Three-dimensional ECM supports the culture of three-dimensional epithelial organoids. The extracellular matrix material is typically dropped onto the bottom of a culture dish, forming a capping layer that encapsulates the cells (this culture method can be described as the "hanging drop method"). Typically, once the matrix has solidified at 37°C, a culture medium is added and diffused into the ECM. The cells in the culture medium adhere to the ECM through interactions with their surface structures, such as integrins.

[0255] The organoid culture medium and / or cells can be placed on ECM, embedded in ECM, or mixed with ECM.

[0256] In some embodiments, stromal cells are cultured without (exogenous) ECM, particularly when poly-L-lysine coating is used in the culture. In other embodiments, stromal cells are cultured together with (exogenous) ECM.

[0257] Wnt agonist

[0258] Culture media may contain Wnt agonists. The Wnt signaling pathway and small molecules that activate Wnt signaling are described in

[12] . Wnt agonists are defined herein as agents that activate or enhance TCF / LEF-mediated transcription in cells. Thus, Wnt agonists are selected from true Wnt agonists that bind to and activate Wnt receptor complexes (including any and all Wnt family proteins), intracellular β-catenin degradation inhibitors, GSK inhibitors (such as CHIR9901), and TCF / LEF activators. One or more Wnt agonists in the culture medium may be selected from Wnt ligands of the Wnt family of secreted glycoproteins, intracellular β-catenin degradation inhibitors, GSK-3 inhibitors, TCF / LEF activators, RNF43 or ZNRF3, and inhibitors of R-vertebral family proteins. In some embodiments, the Wnt agonist in the culture medium comprises R-vertebral family proteins and GSK-3 inhibitors, and optionally further comprises Wnt ligands from the Wnt family of secreted glycoproteins. One or more, such as two, three, four or more, Wnt agonists may be used in the culture medium.

[0259] The Wnt agonist in the culture medium is preferably any agonist capable of stimulating the Wnt pathway via the Lgr5 cell surface receptor, i.e., in a preferred embodiment, the Wnt agonist in the culture medium is an Lgr5 agonist. Known Lgr5 agonists include R-vertebral proteins, fragments thereof, and derivatives thereof, as well as anti-Lgr5 antibodies (see, for example, WO2012 / 140274, particularly Figures 22-24, and

[13] ). The preferred Lgr5 agonist is R-vertebral proteins. Any suitable R-vertebral protein can be used, for example, it can be selected from one or more of R-vertebral protein 1, R-vertebral protein 2, R-vertebral protein 3, and R-vertebral protein 4, or derivatives thereof. For example, any R-vertebral protein 1 (NU206, Nuvelo, San Carlos, CA), R-vertebral protein 2 ((R&D systems), R-vertebral protein 3, and R-vertebral protein 4) can be used. Any suitable concentration of R-vertebral protein can be used, for example, at least 100 ng / ml, more preferably at least 200 ng / ml, and even more preferably about 250 ng / ml. An example of an agonistic anti-Lgr5 antibody is 1D9 (commercially available from BD Biosciences, BDB562733, part number: 562733). Fragments of R-vertebral protein can be used as Wnt agonists. For example, in some embodiments, the Wnt agonist is a fragment of R-vertebral protein containing or composed of a furin protease domain.

[0260] Therefore, in one embodiment, the culture medium contains an Lgr5 agonist, such as R-vertebral protein, and additionally contains a further Wnt agonist. In this case, the further Wnt agonist can be selected, for example, from the group consisting of: Wnt-3a, GSK inhibitors (such as CHIR99021), Wnt-5, Wnt-6a Norrin, and NGS-Wnt. In one embodiment, the culture medium contains R-vertebral protein and additionally contains 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 expansion of human epithelial stem cells (as described in WO2012 / 168930).

[0261] R-vertebral protein family proteins (also referred to herein as “R-vertebral proteins”) may be selected from R-vertebral protein 1, R-vertebral protein 2, R-vertebral protein 3, R-vertebral protein 4 and their analogues, fragments, variants and derivatives. In this case, the fragment, variant or derivative is able to block the action of the E3 ligase RNF43 / ZNRF3 on the Wnt receptor complex. R-vertebral protein 1, R-vertebral protein 2, R-vertebral protein 3 and R-vertebral protein 4 (also referred to herein as “R-vertebral protein 1-4”) are all characterized by two N-terminal furin-like repeats, which are necessary and sufficient for Wnt signal enhancement, and also have a platelet-reactive protein domain closer to the C-terminal member

[14] . Examples of R-vertebral protein fragments, variants and derivatives suitable for the present invention are known to those skilled in the art (e.g. see Example 2 of WO2012 / 140274, which describes a furin-like domain fragment capable of enhancing Wnt signaling, which is incorporated herein by reference). Examples of R-vertebral protein family analogues include, for example, antibodies that interact with RNF43 / ZNRF3 / Lgr. Agonistic anti-Lgr5 antibodies that can enhance Wnt signaling are known in the art (e.g., see antibody 1D9 in Example 3 of

[15] ).

[0262] Many GSK-3 inhibitors are known in the art (see, for example,

[16] ; and

[17] ) and commercially available (see, for example, a list available here from Santa Cruz Biotechnology: https: / / www.scbt.com / scbt / browse / GSK-3-beta-Inhibitors / _ / N-x6oud Any of these GSK-3 inhibitors is suitable for use in the context of this invention, and those skilled in the art will be able to determine appropriate concentrations using IC50 values.

[0263] CHIR-99021 (CAS: 252917-06-9; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxylonitrile; 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 number 1126), CHIR98023, CHIR99021 (see above), and TWS119 (Tocris, catalog number 3835). Therefore, in some embodiments, the GSK-3 inhibitor is an aminopyrimidine inhibitor, optionally selected from CHIR98014, CHIR98023, CHIR99021, or TWS119. In some embodiments, the GSK-3 inhibitor is CHIR-99021.

[0264] Wnt ligands from the Wnt family of secreted glycoproteins can be selected from Wnt-1 / 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 given 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 amplification of human organs and organoid fragments (e.g., as described in

[20] ).

[0265] In some embodiments, the Wnt agonist in the culture medium is a Wnt substitute. The Wnt substitute is a water-soluble Wnt agonist engineered by linking antagonistic Fzd and Lrp5 / 6 binding modules to a single polypeptide chain, thereby forcing receptor heterodimerization while blocking endogenous Wnt binding. The Wnt substitute supports the growth of a variety of cultures. Furthermore, the Wnt substitute is a non-lipidated Wnt agonist that can be produced in serum-free media, kept frozen, and circumvents activity differences in Wnt conditioned media produced in different laboratories

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

[22] . NGS-Wnt can be provided 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.

[0266] In some embodiments, the Wnt agonist in the culture medium is a Wnt substitute. The Wnt substitute is a water-soluble Wnt agonist engineered by linking antagonistic Fzd and Lrp5 / 6 binding modules to a single polypeptide chain, thereby forcing receptor heterodimerization while blocking endogenous Wnt binding. The Wnt substitute supports the growth of a variety of cultures. Furthermore, the Wnt substitute is a non-lipidated Wnt agonist that can be produced in serum-free media, kept frozen, and circumventing activity differences in Wnt conditioned media produced in different laboratories (

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

[24] . NGS-Wnt can be provided 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.

[0267] Soluble Wnt agonists such as Wnt-3a can be provided in the form of Wnt conditioned medium. For example, approximately 10% to approximately 50% Wnt conditioned medium can be used.

[0268] R-vertebral protein can be provided in the form of Rspo conditioned medium. For example, Rspo conditioned medium at about 10% to about 30%, such as about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml, can be used.

[0269] Examples of R-vertebral protein mimics suitable for use in this invention are provided in WO2012 / 140274, which is incorporated herein by reference.

[0270] Mitogenic growth factor

[0271] The culture medium may contain mitotic growth factors. Mitotic growth factors typically induce cell division via the mitogen-activated protein kinase signaling pathway. Many receptor tyrosine kinase ligands are mitotic growth factors. In some embodiments, mitotic growth factors may bind to receptor tyrosine kinases. In some embodiments, mitotic growth factors may bind to more than one receptor tyrosine kinase. In some embodiments, one or more mitotic growth factors bind to receptor tyrosine kinases such as EGFR, FGFR, or HGFR, optionally wherein said one or more mitotic growth factors are selected from EGF, FGF, and HGF.

[0272] In some embodiments, mitotic growth factor binds to EGFR, HER1, HER2, HER3, or HER4. In some embodiments, mitotic growth factor binds to EGFR. In some embodiments, the culture medium also includes HER2-4 ligands in addition to the EGFR ligand. For example, in some embodiments, the culture medium also includes a neuroregulatory protein in addition to EGF. Neuroregulatory proteins have been shown to be beneficial for the culture of lung and breast tissues (see, for example,

[25] and

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

[0273] FGF stimulates 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 factor binds to members of the FGF receptor family. Members of the FGF receptor family include (but are not limited to) FGFR1, FGFR2, FGFR3, or FGFR4. It has been shown that FGFR1-FGFR3 genes encode multiple isotypes, and these isotypes may be key in determining ligand specificity. Several FGFs bind to members of the FGF receptor family, including (but not limited to) FGF2, FGF4, FGF7, and FGF10. These are commercially available. Therefore, in some embodiments, the mitotic growth factor is FGF. In some embodiments, the FGF is selected from FGF2, FGF4, FGF7, and FGF10. In a preferred embodiment, the FGF is FGF2 and / or FGF10. In the most preferred embodiment, the FGF is FGF2 and FGF10.

[0274] Hepatocyte growth factor / scattering factor (HGF / SF) is a morphogenetic factor that regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogenic HGFR. The HGFR is also known as the c-Met receptor. HGF has been shown to be useful for epithelial stem cell culture. Therefore, in some embodiments, mitotic growth factor binds to HGFR. In some embodiments, the mitotic growth factor is HGF. Any suitable HGF can be used, such as HGF obtained from Peprotech.

[0275] In some embodiments, the culture medium includes more than one mitotic growth factor, such as 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 EGF, optionally at a final concentration of about 50 ng / ml; FGF2, optionally at a final concentration of about 5 ng / ml; and FGF10, optionally at a final concentration of about 10 ng / ml.

[0276] In some implementations, hepatocyte growth factor (HGF) is present, with or without EGF and / or FGF.

[0277] BMP inhibitors

[0278] The culture medium may contain a BMP inhibitor. A BMP inhibitor is defined as an agent that binds to a BMP molecule to form a complex, wherein BMP activity is neutralized, for example by preventing or inhibiting the binding of the BMP molecule to a BMP receptor. Alternatively, the inhibitor may be an agent that acts as an antagonist or inverse agonist. This type of inhibitor binds to a BMP receptor and prevents BMP from binding to that receptor. An example of the latter agent is an antibody that binds to a BMP receptor and prevents BMP from binding to the antibody-binding receptor.

[0279] A BMP inhibitor may be added to the culture medium in an amount sufficient to reduce BMP-related activity in the cells by up to 90%, more preferably up to 80%, more preferably up to 70%, more preferably up to 50%, more preferably up to 30%, more preferably up to 10%, and more preferably up to 0%, relative to the level of BMP activity without the inhibitor, as assessed 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 BMP, as illustrated, for example, in

[27] .

[0280] Several classes of natural BMP-binding proteins are known, including head protein (Peprotech), notochorin and notochorin-like proteins (R&D systems) containing a notochorin domain, follicle-suppressant and follicle-suppressant-related proteins (R&D systems) containing a follicle-suppressant domain, DAN and DAN-like proteins (R&D systems) containing a DAN cysteine ​​domain, sclerosing protein / SOST (R&D systems), core proteoglycan (R&D systems), and α-2 macroglobulin (R&D systems).

[0281] Therefore, in some embodiments, the BMP inhibitor is selected from head proteins, DANs, and DAN-like proteins, including Cerberus and Gremlin (R&D systems). These diffusible proteins are capable of binding BMP ligands with varying degrees of affinity and inhibiting their access to signal transduction receptors. Adding any of these BMP inhibitors to the basal culture medium can prevent stem cell loss. A preferred BMP inhibitor is a head protein.

[0282] TGF-β inhibitors

[0283] The culture medium may contain a TGF-β (or “TGF-β”) inhibitor. The presence of a TGF-β inhibitor in the amplification medium is particularly beneficial for improving human organogenesis efficiency. A TGF-β inhibitor is any agent that reduces the activity of the TGF-β signaling pathway (also referred to herein as the ALK4, ALK5, or ALK7 signaling pathway). The TGF-β inhibitor according to the invention may be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer, or antibody. The inhibitor may be naturally occurring or synthetic.

[0284] In some implementations, the TGF-β 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). It is described in the catalog as a TGF-β type I receptor ALK5 kinase, type I activin / nodule receptor ALK4, and type I nodule receptor ALK7 (IC). 50The values ​​(12, 45, and 7.5 nM, respectively) block the phosphorylation of Smad2 and only weakly inhibit the activity of ALK-1, -2, -3, -6, and MAPK. 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 Vactosertib. The IC50 values ​​of these inhibitors are known in the art, and those skilled in the art will be able to select appropriate concentrations of suitable inhibitors based on the teachings provided in the examples of this application.

[0285] Niacinamide

[0286] In some implementations, the culture 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 number 07154).

[0287] Prostaglandin pathway activators

[0288] In some embodiments, the culture medium further comprises a prostaglandin pathway activator. The prostaglandin pathway activator may be one or more of the following compounds: 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.

[0289] cAMP activator

[0290] In some embodiments, the culture medium contains a cAMP pathway activator. The cAMP pathway activator can be any suitable activator that increases cAMP levels in cells. In some embodiments, the cAMP pathway activator is an adenylate cyclase activator or a cAMP analog. Examples of suitable adenylate cyclase activators include trichodin, trichodin analogs, and cholera toxin. Examples of trichodin analogs are known in the art and include NKH477 (e.g., catalog number Tocris 1603). Examples of cAMP analogs are also known in the art and include, for example, 8-bromo-cAMP. 8-bromo-cAMP is a cell-permeable cAMP analog that is more resistant to phosphodiesterase hydrolysis than cAMP. In some embodiments, the cAMP pathway activator is therefore selected from trichodin, cholera toxin, NKH477, and 8-bromo-cAMP. In some embodiments, the cAMP pathway activator is trichodin. In some embodiments, the cAMP pathway activator is not cholera toxin.

[0291] Other components

[0292] One or more compounds selected from the group consisting of gastrin, B27, N-acetylcysteine ​​(or "N-Ac"), and N2 can be added to the culture medium. Therefore, in some embodiments, the culture medium further comprises one or more compounds selected from the group consisting of gastrin, B27, N2, and N-acetylcysteine. B27 (Invitrogen), N-acetylcysteine ​​(Sigma), N2 (Invitrogen), and gastrin (Sigma) are believed to control cell proliferation and contribute to DNA stability. In some embodiments, the culture medium further comprises B27 and N-acetylcysteine.

[0293] In some embodiments, the culture medium further comprises a ROCK inhibitor (Rho kinase inhibitor). ROCK inhibitors are particularly useful for cell attachment when establishing new cultures and / or when dividing (“passaging”) cells. Suitable ROCK inhibitors are known in the art and are commercially available (including, but not limited to, GSK 269962, GSK 429286, H 1152 dihydrochloride, glycyl-H 1152 dihydrochloride, SR 3677 dihydrochloride, SB 772077B dihydrochloride, and Y-27632 dihydrochloride, all 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. A particularly preferred ROCK inhibitor is Y-27632.

[0294] In some embodiments, preferably, the culture medium, particularly the amplification medium, does not contain undefined components (such as fetal bovine serum or fetal calf serum or feeder cells). Various serum substitutes are commercially available and known to those skilled in the art. When using a serum substitute, it can be used at approximately 1% to approximately 30% of the culture medium volume, according to conventional techniques. In some embodiments, the culture medium is serum-free and / or feeder-free.

[0295] The preferred culture medium is a defined synthetic culture medium buffered with carbonate-based buffer to a pH of about 7.4 (preferably pH of about 7.2 to about 7.6 or at least about 7.2 and not higher than about 7.6), while the cells are cultured in an atmosphere containing about 5% to about 10% CO2, or at least about 5% and not more than about 10% CO2, preferably about 5% CO2.

[0296] In vitro methods

[0297] Overview

[0298] The claimed method of this invention can be performed in vivo, in vitro, in situ, ex vivo, or any combination thereof. Preferably, the method is performed in vitro.

[0299] Traditionally, cell lines and, more recently, iPS cells have been used as ex vivo cell / organ and / or disease models (see, for example,

[28] ). However, these approaches suffer from numerous challenges and drawbacks. For instance, cell lines are not available from all patients (only certain biopsy samples yield cell lines successfully), thus limiting their use for personalized diagnosis and medical treatment. iPS cells typically require some degree of genetic manipulation to reprogram the cells to a specific cell fate. Alternatively, they are cultured under conditions that affect karyotype integrity, thus requiring the culture time to be kept to a minimum (as is the case with human embryonic stem cells). This means that iPS cells do not truly represent the in vivo situation but merely attempt to mimic the behavior of cells in vivo. Both cell lines and iPS cells also exhibit genetic instability.

[0300] Conversely, the organoids and co-cultures of the present invention provide a genetically stable platform that faithfully represents the in vivo condition. In some embodiments, the organoids and co-cultures include all differentiated cell types present in the corresponding cells in the corresponding in vivo condition. In other embodiments, the organoids and co-cultures can be further differentiated to provide all differentiated cell types present in vivo. Therefore, the organoids and co-cultures can be used to gain insights into the mechanisms of a variety of diseases and treatments for in vitro drug screening to evaluate potential therapeutic approaches, identify potential targets (e.g., proteins) for future novel (drug) therapy development, and / or explore gene repair in combination with cell replacement therapies.

[0301] Therefore, this invention also provides the use of organoids and co-cultures in the assay of epithelial viability, metabolic activity, permeability, barrier function integrity, and / or transporter activity. Methods for assessing the viability, permeability, and barrier function integrity of organoids and co-cultures, as well as the activity of transporters in organoids and co-cultures, are described herein.

[0302] The cocultures of the present invention can be used in in vitro methods, such as methods for studying (e.g., validating) cocultures, methods for testing therapeutic agents, and methods for diagnosing and / or prognosticating diseases. In some embodiments, organoids are used to mimic in vivo interactions. These methods can be used to test libraries of chemicals, antibodies, natural products (e.g., plant extracts or microbial compounds), etc., to assess their suitability as pharmaceuticals, diagnostics, cosmetics, and / or preventative medicines. For example, the cells are preferably exposed to multiple concentrations of a test agent for a period of time. At the end of the exposure period, the culture is evaluated (e.g., by determining the presence or absence of at least one change).

[0303] 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 predicting clinical outcomes via ex vivo methods.

[0304] This invention provides the use of organoids and co-cultures in histological and embryological studies, cell lineage and differentiation pathway studies; studies to identify chemical and / or neuronal signals that lead to the release of corresponding hormones; gene expression studies, including recombinant gene expression; studies of tissue damage and repair mechanisms; studies of inflammatory diseases; studies of fibrotic diseases; studies of pathogenesis; or studies of cell transformation mechanisms.

[0305] Determine at least one change

[0306] The method of the present invention may involve determining the presence or absence of at least one variation in a co-culture.

[0307] In principle, the presence or absence of any changes in the biochemical, genetic, phenotypic, or phenomenological properties of the co-culture can be determined. At least one change can be an increase or decrease in a property.

[0308] For example, at least one change may include changes in organoid morphology, organoid size (e.g., area), epithelial cell size (e.g., area), epithelial thickness, decreased cell viability, decreased cell proliferation, increased cell death, changes in secretory proteome profile, changes in cytokine secretion, increased apoptosis, increased caspase activity, and / or changes in the expression of one or more genes, optionally wherein the changes in the expression of one or more genes include changes in the expression of one or more disease biomarkers, one or more fibrosis biomarkers, and / or one or more inflammatory biomarkers.

[0309] At least one change may include changes in epithelial viability, metabolic activity, permeability, barrier integrity, and / or transporter activity. This article describes methods for assessing the viability, permeability, and barrier integrity of organoids and co-cultures, as well as the activity of transporters and co-cultures.

[0310] In some embodiments, at least one variation includes a change in organoid aggregation, such as an increase in organoid aggregation. An increase in organoid aggregation (e.g., compared to organoids before co-culturing) can be observed when the at least one organoid is co-cultured with at least one stromal cell in the absence of a pro-inflammatory stimulus. An increase in organoid aggregation (e.g., compared to organoids before co-culturing) can also be observed when the at least one organoid is co-cultured with at least one stromal cell in the presence of a pro-inflammatory stimulus. Aggregation in cocultures prepared with a pro-inflammatory stimulus may be less than that in cocultures prepared without a pro-inflammatory stimulus. Therefore, aggregation can be used as an indicator that at least one organoid has been successfully co-cultured with at least one stromal cell, and aggregation can also be used to determine whether the coculture exhibits a pro-inflammatory spectrum.

[0311] In some implementations, at least one variation includes a change in organoid surface adhesion, such as a reduction in organoid surface adhesion.

[0312] In some embodiments, at least one variation includes a change in organoid morphology, optionally wherein said variation in organoid morphology includes changes in organoid shape, organoid size (e.g., area), organoid lumen size (e.g., organoid lumen area), epithelial cell shape, or epithelial cell size (e.g., area). Preferably, at least one variation includes a change in organoid area. Without wishing to be bound by any theory, the inventors have found that organoid size, particularly cystic morphology, is a highly reproducible and specific readout for the functional presence of fibroblasts in co-cultures with a pro-inflammatory spectrum. Organoid size, preferably organoid area, can be measured by bright-field imaging.

[0313] In some embodiments, at least one variation includes an increase in the area of ​​at least one organoid, optionally said area is increased to at least about 10,000 μm. 2 At least approximately 15,000 µm 2 At least approximately 20,000 µm 2 Or at least about 25,000µm 2 .

[0314] In some embodiments, at least one variation includes an increase in the area of ​​the at least one organoid, wherein the increase 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%, or at least about 80%.

[0315] In some embodiments, at least one variation includes a variation in epithelial thickness, such as a reduction in epithelial thickness, optionally said 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%.

[0316] In some embodiments, at least one variation includes changes in the expression of one or more genes, preferably changes in the expression of one or more disease biomarkers. The term “expressed” is used to describe the presence of a biomarker within the cell. To be considered expressed, a biomarker must be present at a detectable level. “Detectable level” means that the biomarker can be detected using one of standard laboratory methods such as PCR, blot, or FACS analysis. If gene expression is reasonably detectable after 30 PCR cycles, and the expression level in the cell is at least about 100 copies per cell, then the gene is considered to be expressed by the organoid, stromal cell, or co-cultured cell of the present invention. The terms “express” and “expression” have corresponding meanings. Below this threshold expression level, the biomarker is considered not expressed. Comparison of the expression level of a biomarker in the cells of the present invention with the expression level of the same biomarker in another cell (e.g., embryonic stem cells) can preferably be made by comparing two cell types isolated from the same species. Preferably, this species is a mammal, more preferably a human. This comparison can be conveniently performed using reverse transcriptase polymerase chain reaction (RT-PCR) experiments.

[0317] Therefore, at least one change in the cellular composition of organoids can be determined by detecting the expression of one or more biomarker genes, for example, by detecting the presence or absence of at least one change in the expression of one or more biomarker genes. Lgr5 is Lgr5 + Stem cell biomarkers. Ki67 is a biomarker for proliferating cells, such as Lgr5.+ Stem cells. Goblet cells can be detected by mucus staining (e.g., by performing Alcian blue staining) or by detecting the expression of mucin-2 (Muc2), as described in this article. Intestinal alkaline phosphatase (ALPI or ALPI1) is a marker of intestinal epithelial cells. Lysozyme is a marker of Paneth cells. Chromogranin A is a marker of intestinal endocrine cells.

[0318] Therefore, in some embodiments, the presence or absence of expression changes in at least one of the following genes is determined: ALPI, MUC2, lysozyme, Ki67, and Lgr5. In some embodiments, the organoids and / or co-cultures of the present invention express Lgr5 and Muc2, for example, the change is an increase in Lgr5 and Muc2 expression. In some embodiments, the organoids and / or co-cultures of the present invention do not express ALPI, for example, the change is a decrease in ALPI expression. In some embodiments, the organoids and / or co-cultures of the present invention express Lgr5 and Muc2 and do not express ALPI, for example, the change is an increase in Lgr5 and Muc2 expression and a decrease in ALPI expression. In some embodiments, the organoids and / or co-cultures of the present invention express lysozyme.

[0319] In some embodiments, at least one variation includes upregulation of the expression of a stem cell marker, optionally said stem cell marker is OLFM4, optionally said 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 1000%.

[0320] In some embodiments, at least one variation includes downregulation of the expression of an intestinal epithelial cell marker, optionally said intestinal epithelial cell marker being ALPI, optionally said 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%.

[0321] In some embodiments, at least one variation includes downregulation of the expression of a proliferation marker, optionally wherein the proliferation marker is KI67, optionally wherein 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%.

[0322] In some embodiments, at least one change includes a change in the secretome profile, optionally a change in cytokine secretion, optionally a change in IL6 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), such as 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), such as CXCL2 secretion (preferably an increase in CXCL2 secretion).

[0323] In some implementations, the variation includes expressing 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.

[0324] In some embodiments, at least one change includes a change in RNA expression, optionally wherein the change in expression is measured by RNA-seq. The change in expression can be an upregulation or downregulation of RNA. Preferably, the change in RNA expression includes an upregulation of RNA expression (e.g., an increased normalized expression score, such as a normalized expression score determined according to the median ratio method). https: / / hbctraining.github.io / DGE_workshop / lessons / 02_DGE_count_ normalization.html In some implementations, changes in RNA expression include upregulation of RNA encoding any one of IL-6, CXCL2, MMP7, HIF3A, WNT7A, and IGFBP1.

[0325] In some embodiments, at least one variation 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 upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in the expression of RNA encoding IL-6 includes upregulation, for example, at least about 50-fold upregulation. In some embodiments, at least one variation includes a change in the expression of RNA encoding CXCL2. In some embodiments, the change in the expression of RNA encoding CXCL2 includes upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in the expression of RNA encoding CXCL2 includes upregulation, for example, at least about 25-fold upregulation. In some embodiments, at least one variation includes a change in the expression of RNA encoding IGFBP1. In some embodiments, the change in expression of RNA encoding IGFBP1 includes upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in expression of RNA encoding IGFBP1 includes upregulation, for example, at least about 10-fold upregulation. In some embodiments, at least one change includes a change in expression of RNA encoding WNT7A. In some embodiments, the change in expression of RNA encoding WNT7A includes upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in expression of RNA encoding WNT7A includes upregulation, for example, at least about 10-fold upregulation. In some embodiments, at least one change includes a change in expression of RNA encoding MMP7. In some embodiments, the change in expression of the RNA encoding MMP7 includes upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in expression of the RNA encoding MMP7 includes upregulation, for example, at least about 10-fold upregulation. In some embodiments, at least one change includes a change in the expression of the RNA encoding HIF3A. In some embodiments, the change in expression of the RNA encoding HIF3A includes upregulation, for example, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold upregulation. Preferably, the change in expression of the RNA encoding HIF3A includes upregulation, for example, at least about 25-fold upregulation.

[0326] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding KRT20, MUC2, LYZ, ALPI, OLFM4, CCND1, LGR5, MKI67, CXCL2, IL6, THY1, or PDPN. In some embodiments, the change in RNA expression includes upregulation of the expression of RNA encoding KRT20, LYZ, ALPI, OLFM4, CCND1, CXCL2, IL6, or PDPN. In some embodiments, the change in RNA expression includes downregulation of the expression of RNA encoding MUC2, LGR5, MKI67, CXCL2, IL6, or THY1.

[0327] In some embodiments, at least one change in RNA expression includes a change in RNA expression encoding the markers listed in Table 3. In some embodiments, the RNA is an upregulated RNA in Table 3. In some embodiments, the RNA is an downregulated RNA in Table 3. In some embodiments, the change in RNA expression encoding the markers listed in Table 3 is a change that occurs when the co-culture of the present invention is cultured in the co-medium of the present invention containing at least one pro-inflammatory stimulant, and this change is determined relative to the co-culture of the present invention cultured in the co-medium of the present invention without said at least one pro-inflammatory stimulant.

[0328] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding the markers listed in Table 4 below. In some embodiments, the RNA is the upregulated RNA in Table 4. In some embodiments, the RNA is the downregulated RNA in Table 4. In some embodiments, the change in RNA expression encoding the markers listed in Table 4 is a change that occurs when the organoids of the present invention are cultured in the co-medium of the present invention containing at least one pro-inflammatory stimulant, and this change is determined relative to organoids of the present invention cultured in the co-medium of the present invention without said at least one pro-inflammatory stimulant.

[0329] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding the biomarkers listed in Table 5 below. In some embodiments, the RNA is an upregulated RNA in Table 5. In some embodiments, the RNA is an downregulated RNA in Table 5. In some embodiments, the change in RNA expression encoding the biomarkers listed in Table 5 is a change that occurs when the organoids of the present invention are cultured in the organoid culture medium of the present invention containing at least one pro-inflammatory stimulant, and this change is determined relative to organoids of the present invention cultured in the organoid culture medium of the present invention without said at least one pro-inflammatory stimulant.

[0330] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding the markers listed in Table 6 below. In some embodiments, the RNA comprises the upregulated RNAs in Table 6. In some embodiments, the RNA is the downregulated RNA in Table 6. In some embodiments, the change in RNA expression encoding the markers listed in Table 6 is a change that occurs when the stromal cells of the present invention are cultured in the present invention's co-medium containing at least one pro-inflammatory stimulant, and this change is determined relative to the stromal cells of the present invention cultured in the present invention's co-medium without said at least one pro-inflammatory stimulant.

[0331] In some embodiments, at least one change in RNA expression includes a change in RNA expression encoding the biomarkers listed in Table 7 below. In some embodiments, the RNA comprises the upregulated RNAs in Table 7. In some embodiments, the RNA is the downregulated RNA in Table 7. In some embodiments, the change in RNA expression encoding the biomarkers listed in Table 7 is a change that occurs when the organoids of the present invention are cultured in the present invention's co-culture medium containing at least one pro-inflammatory stimulant, and this change is determined relative to the co-cultures of the present invention cultured in the present invention's co-culture medium containing said at least one pro-inflammatory stimulant.

[0332] In some embodiments, at least one change in RNA expression includes a change in RNA expression encoding the biomarkers listed in Table 8 below. In some embodiments, the RNA comprises the upregulated RNAs in Table 8. In some embodiments, the RNA is the downregulated RNA in Table 8. In some embodiments, the change in RNA expression encoding the biomarkers listed in Table 8 is a change that occurs when the organoids of the present invention are cultured in the present invention's co-culture medium without at least one pro-inflammatory stimulant, and this change is determined relative to the co-cultures of the present invention cultured in the present invention's co-culture medium without said at least one pro-inflammatory stimulant.

[0333] In some embodiments, at least one change in RNA expression includes a change in RNA expression encoding the markers listed in Table 9 below. In some embodiments, the RNA comprises the upregulated RNAs in Table 9. In some embodiments, the RNA is the downregulated RNA in Table 9. In some embodiments, the change in RNA expression encoding the markers listed in Table 9 is a change that occurs when the organoids of the present invention are cultured in the present invention's co-medium without at least one pro-inflammatory stimulant, and this change is determined relative to organoids of the present invention cultured in a conditioned medium without at least one pro-inflammatory stimulant isolated from at least one stromal cell.

[0334] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding the markers listed in Table 10 below. In some embodiments, the RNA comprises the upregulated RNA in Table 10. In some embodiments, the RNA is the downregulated RNA in Table 10. In some embodiments, the change in RNA expression encoding the markers listed in Table 10 is a change that occurs when the organoids of the present invention are cultured in the present invention's co-medium containing at least one pro-inflammatory stimulant, and this change is determined relative to organoids of the present invention cultured in a conditioned medium containing at least one pro-inflammatory stimulant isolated from at least one stromal cell.

[0335] In some embodiments, at least one change in RNA expression includes a change in the expression of RNA encoding the markers listed in Table 11 below. In some embodiments, the RNA comprises the upregulated RNAs in Table 11. In some embodiments, the RNA is the downregulated RNA in Table 11. In some embodiments, the change in RNA expression encoding the markers listed in Table 11 is a change that occurs when the organoids of the present invention are cultured in the conditioned medium of the present invention isolated from at least one stromal cell without containing at least one pro-inflammatory stimuli, and this change is determined relative to the organoids of the present invention cultured in the conditioned medium of the present invention isolated from at least one stromal cell and without containing at least one pro-inflammatory stimuli.

[0336] In some embodiments, at least one variation includes a variation in caspase activity, optionally including a variation in caspase 3 / 7 activity, optionally wherein the caspase activity increases by at least about 500%, at least about 1000%, at least about 1500%, at least about 2000%, or at least about 2500%.

[0337] In some embodiments, a cytokine is added to the co-culture before determining the presence or absence of at least one change. Optionally, the cytokine is TNF and / or IFNγ, and the concentration of the cytokine is at most about 100 ng / ml, at most about 50 ng / ml, at most about 15 ng / ml, at most about 5 ng / ml, at most about 1 ng / ml, at most about 0.1 ng / ml, or at most about 0.01 ng / ml. Preferably, the cytokine is TNF at a concentration of at least about 1 ng / ml. Preferably, the cytokine is IFNγ at a concentration of at least about 15 ng / ml.

[0338] In some embodiments, a cytokine signaling inhibitor is added to the co-culture before determining whether at least one change is present or absent in the co-culture. Optionally, the cytokine signaling inhibitor reduces the activity of the JAK / STAT pathway. Optionally, the cytokine signaling inhibitor is tofacitinib, such as about 10 μM tofacitinib.

[0339] At least one change in a coculture containing at least one organoid can be determined relative to at least one organoid prior to the formation of the coculture. The at least one change in the coculture can be determined relative to a reference organoid (e.g., a reference organoid not incorporated into the coculture). At least one change in the coculture can be determined relative to a reference organoid (e.g., a reference organoid not containing stromal cells), optionally wherein the reference organoid does not contain non-epithelial cells. At least one change in the coculture can be determined after the application of at least one agent to the coculture, for example, relative to a coculture before the application of at least one agent. At least one change in the coculture can be determined after at least one agent has been applied to the coculture, for example, relative to a reference coculture without the application of at least one agent.

[0340] In some embodiments, the presence or absence of at least one change is determined in co-cultures that have been 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 co-cultures that have been cultured for at least about 72 hours.

[0341] In some embodiments, the presence or absence of the at least one change is determined at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days after the at least one organoid and the at least one stromal cell are combined to form the co-culture. Preferably, the presence or absence of the at least one change is determined at least seven days after the at least one organoid and the at least one stromal cell are combined to form the co-culture.

[0342] 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 cell death of the stromal cells in the co-culture.

[0343] 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.

[0344] 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.

[0345] 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 cell death of the non-epithelial cells in the co-culture.

[0346] Determining the presence or absence of at least one change may include determining whether the co-culture exhibits a pro-inflammatory spectrum, for example, after at least one agent has been applied to the co-culture.

[0347] technology

[0348] Any in vitro methods disclosed herein, including validation of co-cultures, testing of therapeutic agents and / or diagnosis and / or prognosis, and any methods involving the determination of at least one variation, may include standard laboratory techniques. For example, such in vitro methods may include any one of whole-genome sequencing, mRNA sequencing, peptidomimetics, and / or microscopy. One or more of these techniques may be used, in the form of information discovery and / or information validation, to ensure that the co-culture and / or organoids are consistent and / or as expected. For example, they may be used to determine mRNA transcriptional differences between organoids and co-cultures, and whether these differences in mRNA transcription are reflected in differences in protein expression. The presence of organoid-specific antigens may also be confirmed, and whether any neoantigens are generated only in the co-culture. The upregulation of inflammatory factors in the co-culture microenvironment may also be investigated.

[0349] In principle, any suitable laboratory method known to those skilled in the art can be used to determine the presence or absence of at least one change. In some embodiments, determination may include cell proliferation assays, viability assays, flow cytometry analysis, IFN-γ (interferon-γ) enzyme-linked immunosorbent assay (ELISA), gene expression analysis, and / or cell imaging. In some embodiments, determination may include cell proliferation assays, viability assays, flow cytometry analysis, CXCL2 ELISA, homogeneous time-resolved fluorescence assay (HTRF) of IL6 and CXCL2, IL6 HTRF, gene expression analysis, cell imaging, and / or RNA-seq.

[0350] Various assays are known in the art for measuring cell viability, including assays based on cell membrane integrity (e.g., using nucleic acid dyes such as propidium iodide, TO-PRO-3 iodide, or 7-AAD), cell function such as enzyme activity (e.g., using calcein or the CyQUANT cytotoxicity assay kit), or metabolic activity (e.g., using the alarmarBlue cell viability assay or the yQUANT MTT cell viability assay). In some embodiments, such as by a comet assay

[29] or yH2AX detection

[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 coculture is destroyed. Decreased cell viability can also be determined by the CellTiter Glo luminescent cell viability assay kit (Promega), intracellular flow cytometry staining (BD) of active caspase 3, or positive staining of dead cells. Staining agents that react positively to dead cells include non-cell membrane permeable DNA staining agents, such as NucRed Dead 647 ReadyProb.

[0351] Increased cell death can be detected using bright-field imaging.

[0352] To determine changes in gene expression, depending on the identity of the biomarker, as described herein, the expression of the biomarker can be assessed by RT-PCR, immunohistochemistry, or histological staining after approximately 3, 4, 5, 6, 7, 8, 9 days, or longer of culture. In some embodiments, as described herein, the expression of the biomarker is measured after approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days, or longer of culture, such as approximately 16 days.

[0353] In some implementations, at least one change includes changes in cytokine expression, such as changes in IL-6 and / or CXCL2 expression. These can be measured by HTRP or ELISA, respectively.

[0354] In some embodiments, at least one change includes changes in apoptosis, such as changes in caspase activity (e.g., caspase 3 / 7) or changes in cell viability. This can be measured by bright-field imaging with calcein staining (for cell death) and / or using a commercial caspase assay (e.g., caspase-Glo).

[0355] Image analysis can be used to assess the characteristics of cells in cultures, such as cell morphology; cell structure; evidence of apoptosis or cell lysis; and organoid composition and structure. Many types of imaging analysis are well-known in the art, such as electron microscopy, including scanning electron microscopy and transmission electron microscopy, confocal microscopy, stereomicroscopy, and fluorescence microscopy. Histological analysis can reveal basic structures and cell types.

[0356] The method of the present invention has high-throughput (HTP) capability. In some embodiments, the method of the present invention can be performed on a 96-well plate and / or a 384-well plate, preferably on a 96-well plate.

[0357] verify

[0358] The properties of the co-cultures of the present invention can be determined by studying them, such as whether the co-cultures have been successfully established and contain at least one organoid and at least one stromal cell under viable conditions. This is also known as "validation".

[0359] Verification may include determining the presence or absence of at least one change, as described herein. Therefore, 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 the co-culture), wherein the method includes determining the presence or absence of said at least one change in the co-culture.

[0360] For example, validation can be performed as a quality control step during the preparation of co-cultures, prior to other methods and uses described herein.

[0361] Validation may include determining whether the co-culture exhibits the expected interaction between epithelial cells and stromal cells as in vivo. Validation may include determining the presence or absence of at least one change when at least one stromal cell is combined with at least one organoid to form a co-culture, compared to the case of at least one stromal cell before being combined with at least one organoid to form the co-culture. Validation may include determining the presence or absence of at least one change when at least one organoid is combined with at least one stromal cell to form the co-culture, compared to the case of at least one organoid before being combined with at least one stromal cell to form the co-culture.

[0362] In a co-culture, at least one variation as described herein, relative to the presence or absence of the expected value (e.g., at least one reference organoid (the expected value) does not bind to at least one stromal cell to form a co-culture or forms at least one reference co-culture) can be determined as part of the validation.

[0363] inflammation

[0364] Pro-inflammatory stimuli

[0365] The cells, organoids, co-cultures, and methods of the present invention may involve pro-inflammatory stimuli, wherein the cells, organoids, or co-cultures are exposed to the pro-inflammatory stimuli. The pro-inflammatory stimuli cause the exposed cells to exhibit a pro-inflammatory spectrum and can be used to mimic healthy and disease states in vivo.

[0366] Therefore, the present invention provides cocultures exhibiting a pro-inflammatory spectrum, such as cocultures prepared in a culture medium containing at least one pro-inflammatory stimulant as described herein.

[0367] In some embodiments, the pro-inflammatory stimulant comprises cytokines. Cytokines that cause cells, organoids, or co-cultures to exhibit a pro-inflammatory spectrum are pro-inflammatory stimuli. These include IL-1β, oncokinase M (OSM), IL-6, IFNα, IFNβ, IFNλ, TGF-β, IL-23, IL-22, IL-4, IL-13, or IL-5. Pro-inflammatory stimuli of particular interest are IL-1β and / or OSM. In some embodiments, the pro-inflammatory stimulant comprises IL-1β. In some embodiments, the pro-inflammatory stimulant comprises OSM. In some embodiments, the pro-inflammatory stimulant comprises both IL-1β and OSM. In some embodiments, the pro-inflammatory stimulant is not IL-2. In some embodiments, the pro-inflammatory stimulant is not TNF or IFNγ. In some embodiments, the pro-inflammatory stimulant is not IL-2, IFNγ, or TNF. In some embodiments, the pro-inflammatory stimulant 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 stimulant does not contain IL-4, IL-13, or TNFα. In some embodiments, the pro-inflammatory stimulant does not contain IL-1β, IL-4, IL-13, or TNFα. In some embodiments, the pro-inflammatory stimulant does not contain OSM. In some embodiments, the pro-inflammatory stimulant does not contain IL-1β, IL-4, IL-13, TNFα, or OSM. Other pro-inflammatory stimulants that are not cytokines are considered, such as viral particles, single-stranded nucleic acids, double-stranded nucleic acids, RNA, DNA, and bacterial products (including lipopolysaccharide "LPS").

[0368] The step of contacting the co-culture with one or more pro-inflammatory stimuli can 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 be omitted before combining at least one organoid and at least one stromal cell to form the co-culture.

[0369] The inventors have discovered that co-cultures exposed to one or more pro-inflammatory stimuli do not require subsequent restimulation because they maintain the upregulation of pro-inflammatory genes, including PDPN and TGF-β. Therefore, in some embodiments, exposure to a pro-inflammatory stimuli results in the upregulation of PDPN and / or TGF-β. In some embodiments, the pro-inflammatory spectrum includes upregulated PDPN and / or TGF-β compared to the absence of pro-inflammatory stimulation.

[0370] The inventors did not observe a dose-dependent increase in pro-inflammatory gene expression after exposure to pro-inflammatory stimuli. Therefore, it is believed that the dosage described in the examples maximally induces the pro-inflammatory spectrum in the co-culture.

[0371] In some embodiments, at least one pro-inflammatory stimulant is applied to the co-culture such that the concentration of at least one pro-inflammatory stimulant reaches 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. A particularly preferred concentration is at least about 1 ng / ml, optionally at least about 15 ng / ml. These concentrations may refer individually to the concentration of each pro-inflammatory stimulant or collectively to the concentration of all pro-inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each pro-inflammatory stimulant. A particularly preferred pro-inflammatory stimulant comprises at least about 1 ng / ml IL-1β and at least about 1 ng / ml OSM.

[0372] In some embodiments, at least one pro-inflammatory stimulant is applied to the co-culture such that the concentration of at least one pro-inflammatory stimulant reaches 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. A particularly preferred concentration is about 1 ng / ml, optionally about 15 ng / ml. These concentrations may refer individually to the concentration of each pro-inflammatory stimulant or collectively to the concentration of all pro-inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each pro-inflammatory stimulant. A particularly preferred pro-inflammatory stimulant comprises about 1 ng / ml IL-1β and about 1 ng / ml OSM.

[0373] In some embodiments, at least one pro-inflammatory stimulant is applied to the co-culture such that the concentration of at least one pro-inflammatory stimulant reaches at most about 0.01 ng / ml, at most about 0.1 ng / ml, at most about 1 ng / ml, at most about 1.5 ng / ml, at most about 5 ng / ml, at most about 15 ng / ml, at most about 50 ng / ml, or at most about 100 ng / ml. A particularly preferred concentration is at most about 1 ng / ml, optionally at most about 15 ng / ml. These concentrations may refer individually to the concentration of each pro-inflammatory stimulant or collectively to the concentration of all pro-inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each pro-inflammatory stimulant. A particularly preferred pro-inflammatory stimulant comprises at most about 1 ng / ml IL-1β and at most about 1 ng / ml OSM.

[0374] At least one pro-inflammatory stimulant may be added to the co-culture medium at intervals of at least about 24 hours, at least about 48 hours, at least about 72 hours, or at least about 96 hours between two additions.

[0375] Therefore, in some embodiments, determining the presence or absence of at least one change includes applying at least one pro-inflammatory stimulant to the co-culture and determining the presence or absence of a pro-inflammatory spectrum.

[0376] In some implementations, pro-inflammatory stimulants are therapeutic agents.

[0377] In some implementations, pro-inflammatory stimulants are diagnostic agents.

[0378] In some implementations, determining the presence or absence of at least one change includes determining the presence or absence of a pro-inflammatory spectrum.

[0379] In some implementation schemes, pro-inflammatory stimulants are not therapeutic agents.

[0380] In some implementation schemes, pro-inflammatory stimulants are not diagnostic agents.

[0381] In some embodiments, determining the presence or absence of at least one change includes adding at least one pro-inflammatory stimulant to the co-culture medium, and optionally determining the presence or absence of a pro-inflammatory spectrum.

[0382] Inflammatory irritants

[0383] In some embodiments, at least one inflammatory stimulant 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 stimulant includes cytokines, such as damage-inducing cytokines. In some embodiments, the inflammatory cytokines include TNF. In some embodiments, the inflammatory cytokines include IFNγ. In some embodiments, the inflammatory stimulant does not contain alcohol. The response of cells, organoids, or co-cultures is influenced by whether the cells, organoids, or co-cultures have been exposed to a pro-inflammatory stimulant; therefore, cells, organoids, or co-cultures may be exposed to at least one pro-inflammatory stimulant and then to at least one inflammatory stimulant. Without wishing to be bound by any theory, the inventors consider that pro-inflammatory stimuli result in the presentation of a pro-inflammatory spectrum, which in turn leads to an inflammatory response (or an enhanced inflammatory response) upon exposure to an inflammatory stimulant. In some embodiments, neither the pro-inflammatory stimulant nor the inflammatory stimulant contains IL-1β, IL-4, IL-13, TNFα, alcohol, or OSM.

[0384] Therefore, the present invention provides co-cultures exhibiting an inflammatory response, such as co-cultures prepared in a culture medium containing at least one pro-inflammatory stimulant and / or at least one inflammatory stimulant, as described herein.

[0385] In some embodiments, at least one inflammatory stimulant is applied to the co-culture such that the concentration of at least one inflammatory stimulant reaches 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. A particularly preferred concentration is at least about 1.5 ng / ml, optionally at least about 15 ng / ml. These concentrations may refer individually to the concentration of each inflammatory stimulant or collectively to the concentration of all inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each inflammatory stimulant. A particularly preferred inflammatory stimulant comprises at least about 15 ng / ml TNF and at least about 15 ng / ml IFNγ.

[0386] In some embodiments, at least one inflammatory stimulant is applied to the co-culture such that the concentration of at least one inflammatory stimulant reaches 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. A particularly preferred concentration is about 1.5 ng / ml, optionally about 15 ng / ml. These concentrations may refer individually to the concentration of each inflammatory stimulant or collectively to the concentration of all inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each inflammatory stimulant. A particularly preferred inflammatory stimulant comprises about 15 ng / ml TNF and about 15 ng / ml IFNγ.

[0387] In some embodiments, at least one inflammatory stimulant is applied to the co-culture such that the concentration of at least one inflammatory stimulant reaches at most about 0.01 ng / ml, at most about 0.1 ng / ml, at most about 1 ng / ml, at most about 5 ng / ml, at most about 15 ng / ml, at most about 25 ng / mL, at most about 50 ng / mL, or at most about 100 ng / mL. A particularly preferred concentration is at most about 1.5 ng / ml, optionally at most about 15 ng / ml. These concentrations may refer individually to the concentration of each inflammatory stimulant or collectively to the concentration of all inflammatory stimulants. Preferably, these concentrations refer separately to the concentration of each inflammatory stimulant. A particularly preferred inflammatory stimulant comprises at most about 15 ng / ml TNF and at most about 15 ng / ml IFNγ.

[0388] In some implementations, at least one change is measured at least about 6 hours after the application of an inflammatory stimulant to the coculture, 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.

[0389] Therefore, in some embodiments, determining the presence or absence of at least one change includes applying at least one inflammatory stimulant to the co-culture and determining the presence or absence of an inflammatory response. In some embodiments, determining the presence or absence of at least one change includes applying at least one pro-inflammatory stimulant to the co-culture, applying at least one inflammatory stimulant to the co-culture, and determining the presence or absence of an inflammatory response.

[0390] Inflammatory responses may include increased caspase activity and / or apoptosis in co-cultures.

[0391] The presence or absence of an inflammatory response can be determined relative to a reference coculture, optionally wherein the reference coculture (a) does not accept at least one pro-inflammatory stimulant, (b) does not accept at least one inflammatory stimulant, and / or (c) is prepared by cultured stromal cells in a stromal cell culture medium and combining the stromal cells with at least one reference organoid.

[0392] In some implementations, determining the presence or absence of at least one change includes determining the presence or absence of an inflammatory response.

[0393] In some implementations, the inflammatory irritant is a therapeutic agent.

[0394] In some implementations, the inflammatory irritant is used as a diagnostic agent.

[0395] In some implementations, determining the presence or absence of at least one change includes determining the presence or absence of a pro-inflammatory spectrum.

[0396] In some implementations, inflammatory irritants are not considered therapeutic agents.

[0397] In some implementations, inflammatory irritants are not diagnostic agents.

[0398] In some embodiments, determining the presence or absence of at least one change includes adding at least one inflammatory stimulant to the co-culture medium and optionally determining the presence or absence of an inflammatory response.

[0399] In some embodiments, determining the presence or absence of at least one change includes (a) adding at least one pro-inflammatory stimulant to the co-culture medium and determining the presence or absence of a pro-inflammatory spectrum; and (b) adding at least one inflammatory stimulant 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.

[0400] disease

[0401] In some embodiments, at least one organoid is a disease organoid, such as an organoid having an inflammatory disease phenotype and / or a fibrotic disease phenotype. In some embodiments, the organoid is derived from a subject suffering from a disease (e.g., an inflammatory disease or a fibrotic disease).

[0402] Diseases involving stromal components (e.g., diseases involving stromal cells) can be studied using the co-cultures and methods of the present invention, for example, in the context of treatment, diagnosis, and / or prognosis. In principle, any condition affecting stromal cells can be studied. Preferred diseases include diseases of the digestive and respiratory systems, particularly intestinal and pulmonary diseases. Exemplary diseases include irritable bowel syndrome (IBD), ulcerative colitis (UC), celiac disease, leaky gut syndrome, chronic obstructive pulmonary disease (COPD), and asthma. Particularly preferred diseases include digestive system diseases, particularly intestinal diseases. Exemplary diseases include irritable bowel syndrome (IBD), ulcerative colitis (UC), celiac disease, and leaky gut syndrome.

[0403] In some implementations, at least one organoid may be co-cultured with at least one stromal cell from a subject with the disease to form a co-culture, and separately, at least one other organoid may be co-cultured with at least one stromal cell from a subject without the disease as a reference co-culture.

[0404] fibrosis

[0405] Conditions of particular interest include fibrotic conditions such as intestinal fibrosis, solitary rectal ulcers, radiation enteropathy, and eosinophilic enteropathy. Because of the presence of at least one stromal cell in the co-cultures of the present invention, these co-cultures are particularly suitable for investigating the applicability of therapeutic agents for treating fibrotic diseases. In some embodiments, at least one organoid and / or the at least one stromal cell is derived from fibrotic tissue.

[0406] inflammation

[0407] Conditions of particular interest include inflammatory conditions such as inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC). Because pro-inflammatory stimuli can be applied to the co-cultures of the present invention, these co-cultures are particularly suitable for investigating the suitability of therapeutic agents for treating inflammatory diseases. In some embodiments, at least one organoid and / or the at least one stromal cell is derived from inflamed tissue.

[0408] Therapeutics

[0409] This invention provides a method for testing therapeutic agents for treating diseases such as those disclosed elsewhere herein. Testing at least one therapeutic agent for a disease may include testing the tolerability and / or efficacy of an agent for treating a specific disease, testing the tolerability and / or efficacy of an agent for treating a broad class of diseases, or testing the tolerability and / or efficacy of an agent outside the context of any specific disease (e.g., by testing for activation of a specific molecular pathway).

[0410] Treatment agents for diseases can include anti-inflammatory agents, such as small molecule anti-inflammatory agents. Of particular interest are anti-inflammatory agents used to treat IBD, UC, and / or CD, including corticosteroids (e.g., prednisone), aminosalicylates (e.g., mesalamine, balsalazine, and oxalazine), immunosuppressants (e.g., azathioprine, mercaptopurine, and methotrexate), and anti-inflammatory agents (e.g., tofacitinib, utpatinib, and ozagide), biologics (e.g., infliximab, adalimumab, golimumab, cetuzumab, vedolizumab, ustekinumab, and ranitrazumab), and antibiotics (e.g., ciprofloxacin and metronidazole).

[0411] At least one therapeutic agent may contain at least one pro-inflammatory stimulant, such as at least one cytokine, such as IL-1β and / or OSM.

[0412] At least one therapeutic agent may contain at least one inflammatory stimulant, such as at least one cytokine, such as TNF and / or IFNγ.

[0413] Testing therapeutic agents may involve exposing cocultures to therapeutic levels of therapeutic agents with known or unknown efficacy and / or known or unknown tolerability.

[0414] Typically, the drug is dissolved in a solution to the (predicted) therapeutically effective concentration. The solution can be administered to the co-culture via injection (or other appropriate route of administration) into the container maintaining the co-culture.

[0415] In some embodiments, the agent is, for example, an approved or investigational drug for diseases or conditions of the digestive system, 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.

[0416] Testing at least one therapeutic agent may include testing the agent's efficacy in treating a disease. Testing at least one therapeutic agent may include testing the agent's tolerability in treating a disease.

[0417] 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 optionally using the therapeutic agent in a treatment method by administering the therapeutic agent to a subject.

[0418] In addition to testing therapeutic agents using the co-cultures of the present invention, the co-cultures of the present invention can also be used in methods for identifying at least one target of at least one therapeutic agent. For example, target genes in the at least one stromal cell and / or the at least one organoid can be genetically modified, and the presence or absence of at least one change in the co-culture can be determined according to the general principles disclosed elsewhere herein for determining the presence or absence of at least one change to identify whether the target gene is therapeutically significant (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 genes in the genome of the at least one stromal cell. Target genes may include gene 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 containing the genetic modification, relative to a reference co-culture that does not contain the genetic modification. In such embodiments, the at least one therapeutic agent does not need to be applied to the co-culture. In other embodiments, when the at least one therapeutic agent is applied to a co-culture containing the genetic modification, the presence or absence of at least one change can be determined by comparing it to a co-culture before the application of the at least one therapeutic agent, and / or to a reference co-culture that does not contain the genetic modification. Therefore, the methods for testing therapeutic agents described herein may include identifying therapeutic targets.

[0419] Personalized medicine

[0420] One approach to testing therapeutic agents can be described as a “personalized medicine” testing method. A personalized medicine approach may involve testing one or more therapeutic agents known to be suitable for treatment and determining whether one or more agents are suitable (e.g., effective and / or tolerable) for treating a subject’s condition (i.e., the specific subject testing one or more therapeutic agents). Personalized medicine approaches may use at least one organoid and / or at least one stromal cell derived from the same patient.

[0421] filter

[0422] Another way to test therapeutic agents can be described as a “screening” test. Screening methods may involve testing the efficacy or tolerability of one or more therapeutic agents in the treatment of a disease (e.g., in the treatment of a specific disease) and determining whether one or more agents are suitable (e.g., effective and / or tolerable) for treatment. Screening methods may use at least one organoid and / or at least one stromal cell from different patients. Screening methods may use at least one organoid and / or at least one stromal cell derived from an immortalized cell line. Screening methods may use more than one pair, such as more than two, three, four, five, ten, twenty, fifty, one hundred, or one thousand pairs of at least one organoid and / or at least one stromal cell from different patients.

[0423] diagnosis

[0424] Because the co-cultures and methods of the present invention can accurately mimic in vivo physiological states in an in vitro environment, they facilitate disease diagnosis or prognosis by determining the presence or absence of at least one change (as described elsewhere herein). Diagnosis and / or prognosis may involve applying diagnostic agents to the co-cultures, such as diagnostic agents that alter gene expression, signal transduction pathway activity, receptor function, ligand function, or other cellular processes, thereby allowing differential diagnosis or prognosis based on the presence or absence of at least one change after administration.

[0425] Diagnosis and / or prognosis may include determining the presence or absence of at least one change after applying at least one diagnostic agent to the 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 pathways. Particularly preferred are at least one agonist or antagonist of the JAK-STAT pathway. Also noteworthy are modulators of fluid transport and / or ion channel activity.

[0426] Diagnosis and / or prognosis 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 contains at least one cytokine, optionally including IL-1β or OSM.

[0427] At least one diagnostic agent may contain at least one pro-inflammatory stimulant, such as at least one cytokine, such as IL-1β and / or OSM.

[0428] At least one diagnostic agent may contain at least one inflammatory stimulant, such as at least one cytokine, such as TNF and / or IFNγ.

[0429] In some implementations, the diagnostic agent is a therapeutic agent.

[0430] Diagnosis and / or prognosis 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 therapeutic agent, particularly a known therapeutic agent. The presence or absence of at least one change (e.g., a response in the coculture indicating whether or not the molecular basis of disease symptoms has been treated) after the application of at least one therapeutic agent can aid in differential diagnosis by inferring the cause of the disease.

[0431] Diagnosis and / or prognosis 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 contain at least one organoid from a first patient with the disease and / or at least one stromal cell from the first patient, and the presence or absence of at least one change may be determined relative to a reference co-culture containing at least one organoid from a second patient without the disease and / or at least one stromal cell from the second patient.

[0432] Diagnostic and / or prognostic methods may include analyzing the presence or absence of at least one change, as well as identifying disease or clinical outcomes.

[0433] In some embodiments, the method includes selecting a therapeutic agent for treating a disease based on a diagnosis and / or prognosis determined in the method of the invention, and optionally using the therapeutic agent in a method of treating the disease by administering the therapeutic agent to a subject.

[0434] This invention provides methods for determining the presence or absence of a subject's disease diagnosis and / or prognosis, as described herein. These methods can be described as methods for determining the presence or absence of a subject's disease and / or susceptibility to the disease. These methods can be described as methods for diagnosing and / or prognosing a subject, wherein diagnosing a subject includes determining whether the subject has a disease, and / or wherein prognosing a subject includes determining whether the subject is susceptible to the disease.

[0435] Conditioned culture medium

[0436] The methods of the present invention, particularly methods for testing at least one therapeutic agent and for diagnosis and / or prognosis, 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 and at least one stromal cell to form a co-culture may alternatively involve combining at least one organoid with a conditioned medium obtained from at least one stromal cell (i.e., not involving the formation of a co-culture). This is because the inventors believe that the effect of stromal cells on organoids is at least partially attributable to soluble factors secreted by the stromal cells into the surrounding culture medium. The methods of the present invention may include the steps of isolating and collecting the conditioned medium from said co-culture and detecting the soluble factors in the conditioned medium.

[0437] These methods involving conditioned media may, but do not necessarily, involve the step of isolating the conditioned media from at least one stromal cell. Isolation of the conditioned media may be performed at least about 6 hours after culturing at least one stromal cell in a stromal cell culture medium, for example, at least about 12 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours.

[0438] Therefore, the present invention provides organoids exhibiting a pro-inflammatory spectrum, such as organoids prepared in a conditioned medium derived from stromal cells, wherein the stromal cells are prepared in a stromal cell culture medium containing at least one pro-inflammatory stimulant.

[0439] Other methods and products of the present invention

[0440] Reagent test kit

[0441] This invention provides a kit comprising any organoid, stromal cell, or co-culture of the present invention.

[0442] In some implementations, the kit includes one or more of the following: a syringe, an alcohol swab, a cotton ball, a gauze pad, and instructions for use for carrying out the method of the present invention.

[0443] Implementation Plan

[0444] The present invention further provides embodiments numbered as follows: 1. A method for preparing a co-culture comprising at least one organoid and at least one stromal cell, the method comprising: The at least one organoid is combined with the at least one stromal cell in a co-culture medium to form the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0445] 2. A method for determining the presence or absence of at least one variation in a co-culture comprising at least one organoid and at least one stromal cell, wherein the method comprises: Combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture; and Determine the presence or absence of at least one of the aforementioned changes in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0446] 3. A method for determining the presence or absence of at least one variation in at least one organoid, wherein the method comprises: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid; and Determine the presence or absence of at least one change in the organoids. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0447] 4. A method for testing at least one therapeutic agent for a disease, the method comprising: Combining at least one organoid with at least one stromal cell in a co-culture medium to form a co-culture; Apply the at least one therapeutic agent to the co-culture; and Determine the presence or absence of at least one change in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0448] 5. A method for testing at least one therapeutic agent for a disease, the method comprising: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid. Apply the at least one therapeutic agent to the organoid; and Determine the presence or absence of at least one change in the organoids. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0449] 6. The method as described in embodiment 4 or embodiment 5, wherein testing the at least one therapeutic agent includes testing the tolerability of the at least one therapeutic agent.

[0450] 7. The method of any one of embodiments 2-6, wherein the presence or absence of the at least one change is determined relative to a reference organoid, optionally wherein the at least one therapeutic agent is not applied to the reference organoid and / or wherein the reference organoid does not contain stromal cells.

[0451] 8. The method of any one of embodiments 2-7, wherein the presence or absence of said at least one change is determined relative to a reference co-culture, and optionally wherein said at least one therapeutic agent is not applied to said reference co-culture.

[0452] 9. A method for determining the presence or absence of a disease diagnosis and / or prognosis 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; At least one diagnostic agent is applied to the co-culture; and Determine the presence or absence of at least one change in the co-culture. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0453] 10. A method for determining the presence or absence of a diagnosis and / or prognosis of a disease in a subject, the method comprising: The conditioned medium is separated from at least one stromal cell, and the conditioned medium is combined with at least one organoid. Apply at least one diagnostic agent to the at least one organoid; and Determine the presence or absence of at least one change in the at least one organoid. Optional, wherein: (a) The method comprises preparing the at least one stromal cell by cultured stromal cells in a stromal cell culture medium; and / or (b) The method includes preparing the at least one organoid by culturing epithelial cells in an organoid culture medium.

[0454] 11. The method of 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 wherein the at least one diagnostic agent is a cytokine, and optionally wherein the at least one diagnostic agent comprises IL-1β and / or OSM.

[0455] 12. The method as described in 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 matrix cell.

[0456] 13. The method of any one of embodiments 2, 4, 6-9 or 11, wherein the presence or absence of the at least one change is determined relative to the at least one organoid prior to the formation of the co-culture.

[0457] 14. The method as described in any one of embodiments 4-13, wherein the disease is an inflammatory disease or a fibrotic disease.

[0458] 15. The method of embodiment 14, wherein the disease is an inflammatory disease, optionally wherein the inflammatory disease is a gastrointestinal inflammatory disease, optionally wherein the inflammatory disease is inflammatory bowel disease (IBD), optionally wherein the IBD includes ulcerative colitis (UC) or Crohn's disease (CD).

[0459] 16. The method according to any of the foregoing embodiments, wherein the at least one organoid is derived from primary epithelial cells or immortalized epithelial cells.

[0460] 17. The method according to any of the foregoing embodiments, wherein the at least one organoid is an organoid derived from a patient.

[0461] 18. The method according to any of the foregoing embodiments, wherein the epithelial cells are primary epithelial cells or immortalized epithelial cells, optionally wherein the primary epithelial cells are epithelial cells derived from the patient.

[0462] 19. The method according to any of the foregoing embodiments, wherein the at least one stromal cell comprises a fibroblast, optionally wherein the fibroblast is an intestinal fibroblast, such as a colonic fibroblast or a small intestinal fibroblast, optionally wherein the fibroblast is an immortalized human colonic fibroblast, a human colonic fibroblast, an immortalized human small intestinal fibroblast, or a human small intestinal fibroblast.

[0463] 20. The method of any one of embodiments 4-19, wherein the co-culture, the at least one organoid, and / or the at least one stromal cell: (a) derived from the said subject; and / or (b) Derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue; and / or (c) Originating from fibrotic tissue; and / or (d) Originating from inflamed tissue; and / or (e) Contains or consists of: mammalian cells, optionally human cells; and / or (f) Derived from the intestine, optionally from the colon.

[0464] 21. The method according to any of the foregoing 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 wherein the at least one organoid does not contain epithelial cells or stromal cells derived from lung tissue, kidney tissue, pancreatic tissue or liver tissue.

[0465] 22. The method according to any one of embodiments 4-21, wherein the epithelial cells and / or the stromal cells are: (a) derived from the said subject; and / or (b) Derived from lung tissue, kidney tissue, pancreatic tissue, or liver tissue; and / or (c) Originating from fibrotic tissue; and / or (d) Mammalian cells, optionally human cells; and / or (e) Derived from the intestine, optionally from the colon.

[0466] 23. The method according to any of the foregoing embodiments, wherein the at least one organoid and the at least one stromal cell are derived from the same subject, optionally from (a) the same sample from the subject and / or (b) the same tissue from the same subject.

[0467] 24. The method according to any of the foregoing embodiments, wherein the epithelial cells and the stromal cells are derived from the same subject, optionally from (a) the same sample from the subject and / or (b) the same tissue from the same subject.

[0468] 25. The method of embodiment 23 or embodiment 24, wherein the sample comprises a tissue biopsy, and optionally the sample comprises an intestinal tissue biopsy.

[0469] 26. The method as described in any one of embodiments 2, 4, 6-9, 11 or 13-25, wherein determining the presence or absence of at least one variation in the co-culture includes determining the presence or absence of matrix cells in the co-culture, optionally including determining the amount of matrix cells in the co-culture.

[0470] 27. The method according to any of the foregoing embodiments, wherein the at least one organoid is prepared by splitting a precursor organoid.

[0471] 28. The method as described in 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 simultaneously seeding the at least one organoid and the at least one stromal cell.

[0472] 29. The method of 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 at a ratio of about 0.5 to about 2.5 stromal cells / organoid, optionally wherein the ratio is about 0.5 stromal cells / organoid or about 2.5 stromal cells / organoid.

[0473] 30. The method according to any of the foregoing embodiments, wherein the epithelial cells are cultured in the organoid culture medium for at least about 24 hours to prepare the at least one organoid.

[0474] 31. The method of any one of embodiments 2, 4, 6-9, 11 or 13-30, wherein the co-culture is a three-dimensional co-culture, optionally wherein combining the at least one organoid and at least one stromal cell includes the hanging drop method.

[0475] 32. The method of 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 the form of a porous plate, optionally wherein the porous plate comprises an ultra-low adhesion plate.

[0476] 33. The method of any one of embodiments 2, 4, 6-9 or 11-32, comprising separating the conditioned medium from the co-culture and detecting soluble factors in the conditioned medium.

[0477] 34. The method according to any of the foregoing embodiments, wherein the at least one stromal cell is modified to alter gene expression.

[0478] 35. The method according to any of the foregoing embodiments, wherein the epithelial cells are epithelial stem cells.

[0479] 36. The method according to any of the foregoing embodiments, wherein the at least one organoid is characterized by Lgr5 expression.

[0480] 37. The method of 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 stimulant to the co-culture medium and determining the presence or absence of an inflammatory response.

[0481] 38. The method of embodiment 37, wherein the at least one inflammatory stimulant comprises a cytokine, optionally wherein the cytokine comprises TNF and / or IFNγ.

[0482] 39. The method as described in embodiment 37 or 38, wherein the inflammatory response includes increased caspase activity and / or apoptosis in the co-culture.

[0483] 40. The method of any one of embodiments 37-39, wherein the presence or absence of an inflammatory response is determined relative to a reference co-culture, optionally wherein the reference co-culture (a) does not accept the at least one inflammatory stimulant and / or (b) is prepared by cultured stromal cells in a stromal cell expansion medium and combining the stromal cells with at least one reference organoid.

[0484] 41. The method of any one of embodiments 2-40, wherein the at least one change includes changes in organoid morphology, changes in organoid size or epithelial cell size, decrease in cell viability, decrease in cell proliferation, increase in cell death, changes in secretome profile, changes in cytokine secretion, increase in apoptosis, increase in caspase activity and / or changes in the expression of one or more genes, optionally, wherein the changes in the expression of one or more genes include changes in one or more disease biomarkers, one or more fibrosis biomarkers and / or one or more inflammatory biomarkers.

[0485] 42. The method of any one of embodiments 2-41, wherein determining the presence or absence of at least one change includes cell proliferation assay, viability assay, flow cytometry analysis, ELISA, gene expression analysis and / or cell imaging.

[0486] 43. The method of any one of embodiments 2-42, wherein determining the presence or absence of the at least one change includes determining the degree of organoid cell death.

[0487] 44. The method of any one of embodiments 2-43, wherein the at least one variation includes a variation in organoid morphology, optionally wherein the variation in morphology includes: (a) Increased organoid area; and / or (b) Increased organoid aggregation; and / or (c) Increased lumen size; and / or (d) Increased epithelial cell size; and / or (e) Reduced organoid surface adhesion.

[0488] 45. The method of any one of embodiments 2-44, wherein the at least one variation comprises an increase in the area of ​​the at least one organoid, optionally wherein the area is increased to at least about 10,000 μm. 2 At least approximately 15,000 µm 2 At least approximately 20,000 µm 2 Or at least approximately 25,000 µm 2 .

[0489] 46. ​​The method of embodiment 44 or embodiment 45, wherein the area of ​​the at least one organoid is increased 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%.

[0490] 47. The method as described in any one of embodiments 2, 4, 6-9, 11 or 13-46, wherein the at least one change is determined at least one day, at least two days, at least three days, at least four days, at least five days, at least six days or at least seven days after the at least one organoid and the at least one stromal cell are combined to form the co-culture.

[0491] 48. The method of any one of embodiments 2-47, wherein the at least one variation includes upregulation of the expression of a stem cell marker, optionally wherein the stem cell marker is OLFM4, and optionally wherein 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 1000%.

[0492] 49. The method of any one of embodiments 2-48, wherein the at least one variation comprises downregulation of the expression of an intestinal epithelial cell marker, optionally wherein the intestinal epithelial cell marker is ALPI, and optionally wherein 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%.

[0493] 50. The method of any one of embodiments 2-49, wherein the at least one variation comprises downregulation of the expression of a proliferation marker, optionally wherein the proliferation marker is KI67, and optionally wherein 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%.

[0494] 51. The method of any one of embodiments 2-50, wherein the at least one change includes 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%.

[0495] 52. The method as described in 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 at least one change is present or absent in the co-culture.

[0496] 53. The method as described in any one of embodiments 2, 4, 6-9, 11 or 13-52, wherein the presence or absence of the at least one variation is determined after combining the at least one organoid and at least one stromal cell to form the co-culture, relative to before combining the at least one organoid and at least one stromal cell to form the co-culture.

[0497] 54. The method of any one of embodiments 2-53, wherein the at least one change includes a change in the secretome profile, optionally a change in cytokine secretion, optionally a change in IL-6 and / or CXCL2 secretion.

[0498] 55. The method of any one of embodiments 2-54, wherein the at least one variation comprises the expression of at least about 10,000 pg / ml IL-6, at least about 20,000 pg / ml IL-6, at least about 30,000 pg / ml IL-6, at least about 40,000 pg / ml IL-6, at least about 50,000 pg / ml IL-6, or at least about 100,000 pg / ml IL-6.

[0499] 56. The method of any one of embodiments 2-55, wherein the at least one change includes a change in caspase activity, optionally including a change in caspase 3 / 7 activity, wherein the caspase activity is optionally increased by at least about 500%, at least about 1000%, at least about 1500%, at least about 2000%, or at least about 2500%.

[0500] 57. The method of any one of embodiments 2, 4, 6-9, 11 or 13-56, wherein at least one inflammatory stimulant is added to the co-culture medium before determining the presence or absence of at least one change in the co-culture, optionally wherein the at least one inflammatory stimulant comprises a cytokine, optionally wherein the cytokine comprises TNF and / or IFNγ, optionally wherein the concentration of the cytokine is at most about 100 ng / ml, at most about 50 ng / ml, at most about 15 ng / ml, at most about 5 ng / ml, at most about 1 ng / ml, at most about 0.1 ng / ml or at most about 0.01 ng / ml.

[0501] 58. The method of 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 at least one change is present or absent in the co-culture, optionally wherein the cytokine signaling inhibitor reduces the activity of the JAK / STAT pathway, optionally wherein the cytokine signaling inhibitor is tofacitinib.

[0502] 59. The method of any of the foregoing embodiments, wherein the organoid culture medium comprises mitotic growth factor, BMP inhibitor and R-vertebral protein, optionally wherein the organoid culture medium comprises Wnt agonist, mitotic growth factor, BMP inhibitor and R-vertebral protein.

[0503] 60. The method of any of the foregoing embodiments, wherein the matrix cell culture medium comprises a basal culture medium, an amino acid supplement, an antibiotic, and optionally serum.

[0504] 61. The method of any of the foregoing embodiments, wherein the organoid culture medium and / or stromal cell culture medium comprises an extracellular matrix, optionally wherein the extracellular matrix is ​​Matrigel®, optionally wherein the Matrigel® is 5% or 10% Matrigel®, optionally wherein the Matrigel® is 5% Matrigel.

[0505] 62. The method according to any one of embodiments 2, 4, 6-9, 11 or 13-61, wherein the co-culture medium comprises: (A) Mitogenic growth factor, BMP inhibitor, and R-vertebral protein, optionally wherein the co-medium contains a Wnt agonist, mitogenic growth factor, BMP inhibitor, and R-vertebral protein, optionally wherein the co-medium contains N-Ac, TGF-β inhibitor, mitogenic growth factor, gastrin, BMP inhibitor, antibiotic, R-vertebral protein, Notch pathway inhibitor, ERK inhibitor, and Wnt agonist; and / or (B) Serum, optionally fetal bovine serum, optionally 5% fetal bovine serum.

[0506] 63. The method of any one of embodiments 2, 4, 6-9, 11 or 13-62, wherein the organoid culture medium and / or co-culture medium does not contain extracellular matrix.

[0507] 64. The method of any one of embodiments 2, 4, 6-9, 11 or 13-63, wherein determining the presence or absence of at least one change comprises adding at least one pro-inflammatory stimulant to the co-culture medium, optionally wherein determining the presence or absence of at least one change comprises (a) adding at least one pro-inflammatory stimulant to the co-culture medium, determining the presence or absence of a pro-inflammatory spectrum, and / or (b) adding at least one pro-inflammatory stimulant to the co-culture medium and determining the presence or absence of an inflammatory response.

[0508] 65. The method of embodiment 64, wherein the at least one pro-inflammatory stimulant is added to the co-culture medium, optionally 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 any inflammatory stimulant to the co-culture medium.

[0509] 66. The method of any one of embodiments 37-40, 57 or 64-65, wherein at least one pro-inflammatory stimulant is added to the co-culture medium before the at least one inflammatory stimulant is added to the co-culture medium, optionally wherein the at least one pro-inflammatory stimulant 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 stimulant is added to the co-culture medium.

[0510] 67. The method of any one of embodiments 64-66, wherein the at least one pro-inflammatory stimulant comprises at least one cytokine.

[0511] 68. The method of embodiment 67, wherein the at least one cytokine comprises IL-1β and / or oncokinetic M (OSM).

[0512] 69. The method of any one of embodiments 64-68, wherein the at least one pro-inflammatory stimulant is added to the organoid co-culture medium at intervals of at least about 24 hours, at least about 48 hours, at least about 72 hours, or at least about 96 hours between two additions.

[0513] 70. The method of any one of embodiments 64-69, wherein the at least one pro-inflammatory stimulant comprises IL-1β, optionally wherein the concentration of IL-1β is at least about 20 ng / ml, optionally wherein 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 wherein the concentration is at least about 1 ng / ml.

[0514] 71. The method of any one of embodiments 64-70, wherein the at least one pro-inflammatory stimulant comprises IL-1β, optionally wherein the concentration of said IL-1β is at most about 20 ng / ml, optionally wherein said concentration is at most about 5 ng / ml, at most about 1 ng / ml, at least about 0.1 ng / ml or at least about 0.01 ng / ml, preferably wherein said concentration is at most about 1 ng / ml.

[0515] 72. The method of any one of embodiments 64-71, wherein the at least one pro-inflammatory stimulant comprises IL-1β, optionally wherein the concentration of IL-1β is from about 0.01 ng / ml to about 20 ng / ml, optionally wherein the concentration is from about 0.1 ng / ml to about 20 ng / ml, from about 1 ng / ml to about 20 ng / ml, or from about 1 ng / ml to about 5 ng / ml, optionally wherein the concentration is about 1 ng / ml.

[0516] 73. The method of any one of embodiments 64-69, wherein the at least one pro-inflammatory stimulant comprises OSM, optionally wherein the concentration of the OSM is at least about 20 ng / ml, optionally wherein 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 wherein the concentration is at least about 1 ng / ml.

[0517] 74. The method of any one of embodiments 64-69 or 73, wherein the at least one pro-inflammatory stimulant comprises OSM, optionally wherein the concentration of the OSM is at most about 20 ng / ml, optionally wherein the concentration is at most about 5 ng / ml, at most about 1 ng / ml, at least about 0.1 ng / ml, or at least about 0.01 ng / ml.

[0518] 75. The method of any one of embodiments 64-69 or 73-74, wherein at least one pro-inflammatory stimulant comprises OSM, optionally wherein the concentration of OSM is from about 0.01 ng / ml to about 20 ng / ml, optionally wherein the concentration is from about 0.1 ng / ml to about 20 ng / ml, from about 1 ng / ml to about 20 ng / ml, or from about 1 ng / ml to about 5 ng / ml, optionally wherein the concentration is about 1 ng / ml.

[0519] 76. The method of any one of embodiments 64-75, wherein the at least one pro-inflammatory stimulant comprises IL-1β and OSM, optionally wherein the at least one pro-inflammatory stimulant comprises at least about 1 ng / ml IL-1β and at least about 1 ng / ml OSM.

[0520] 77. A co-culture as described in any one of embodiments 2, 4, 6-9, 11 or 13-76.

[0521] 78. A co-culture medium as described in any one of embodiments 2, 4, 6-9, 11 or 13-76.

[0522] 79. A stromal cell culture medium, as described in any of the preceding claims.

[0523] 80. A co-culture, which is essentially as described herein.

[0524] 81. A co-culture medium, which is essentially as described herein.

[0525] 82. A stromal cell culture medium, which is essentially as described herein.

[0526] Example

[0527] Other features, objects, and advantages of the invention will become apparent from the following embodiments. However, it should be understood that while these embodiments illustrate implementations of the invention, they are given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art based on the embodiments.

[0528] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0529] culture medium

[0530] The following culture media were used in the examples. The amounts of reagents are given in described units, or as a percentage (or %) of the final volume of the culture medium. v / v (or as indicated in the manufacturer's instructions for use, such as "1x")

[0531] Ad-DF+++

[0532] Ad-DF+++ modified with additional HEPES

[0533] Organoid amplification medium (or "normal colonic medium", "CNM")

[0534] Co-culture differentiation medium ("combined colon differentiation medium", "cCDM")

[0535] cCDM + serum

[0536] Co-culture differentiation medium ("intestinal epithelial cell-colon differentiation medium", "eCDM")

[0537] Incubate overnight at 37°C and 5% CO2, unless otherwise specified. Longer incubation periods are possible, with the medium replaced every 2-3 days.

[0538] If indicated, the experiments involved fibroblast cell lines, as shown in Table 1:

[0539] Example 1: Preparation of fibroblasts and organoids

[0540] This embodiment provides an exemplary scheme for preparing fibroblasts and organoids, which will be used subsequently in Embodiment 2.

[0541] Fibroblast preparation

[0542] The fibroblast samples stored at -80°C were thawed and refreshed using Ad-DF+++ medium containing 10% FBS and 25 mM HEPES. The fibroblasts were then transferred to T75 flasks coated with poly-L-lysine and incubated.

[0543] To digest the fibroblasts with trypsin, aspirate the culture medium and wash the cells with 10 mL of DPBS (Dulbecco phosphate-buffered saline). Then aspirate the DPBS. Add 3 mL of TrypLE to the flask before shaking to ensure complete coverage. Then incubate the flask at 37°C for 2 min.

[0544] The TrypLE cells containing fibroblasts were then transferred to fresh Ad-DF+++ medium (5 ml Ad-DF+++ / 1 ml TrypLE) for neutralization, followed by centrifugation at 1500 rpm (450 xg) for 5 minutes. Finally, the supernatant was aspirated, and the cells were resuspended in 1 mL Ad-DF+++ medium and counted.

[0545] Organoid preparation

[0546] Biopsies from human colon tissue were collected in 50 mL standard tubes containing 10–15 mL of ice-cold, fully advanced DMEM / F12 medium containing penicillin / streptomycin (100× stock solution of 10,000 U / mL penicillin and 10 K μM / mL streptomycin), HEPES (100× stock solution of 1 M), GlutaMAX (100× stock solution; all from Gibco™), and the Rho kinase inhibitor Y-27632 (Sigma-Aldrich). Biopsies were stored on ice and processed immediately or could be stored at 4°C for up to 24 hours until separation began.

[0547] The colonic mucosa was treated with EDTA to release crypts and derive colonic organoids. The muscle layer and fat were removed using surgical scissors and forceps under a dissecting microscope. The cleaned tissue was cut into strips approximately 1–2 mm thick. One strip was fixed in 4% formaldehyde (Sigma-Aldrich) for histological analysis, and another strip was flash-frozen (in dry ice or liquid nitrogen) and stored at -80°C for gene and / or protein analysis.

[0548] The remaining strips were washed three times with a 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 from Sigma-Aldrich). The washed strips were incubated for 30 minutes at 4°C in a rotating wheel (cold chamber) with a chelation solution prepared with 2 mM EDTA (internal) and 0.5 mM DL-dithiothreitol (DTT; Sigma-Aldrich).

[0549] Vigorously shake the test tube to release the colonic crypts from the mesenchyme. If the crypts are not visible, repeat the incubation with fresh, fully chelated solution. Allow the tissue fragments to settle for 1–2 minutes, and transfer the supernatant containing the crypts to a new tube. Add 5–10 mL of fetal bovine serum (FCS; Sigma-Aldrich), and centrifuge the crypts at 300 × g for 5 minutes at 4°C.

[0550] The crypts were washed three times in fully purified DMEM / F12. The crypts were resuspended in a mixture of Matrigel / BME and 70% culture medium, plated in dome-shaped layers at varying densities, and incubated at 37°C and 5% CO2 for 30 minutes. After Matrigel / BME solidification, CNM supplemented with the Rho kinase inhibitor Y-27632 was added. Organoids formed from the crypts were passaged every 7–10 days.

[0551] Subsequently, whole-genome sequencing, mRNA sequencing, and peptidomimetics can be used to perform preliminary analysis of organoid cultures.

[0552] Organoids of division

[0553] Organoid cultures were disrupted (“fractured”) by pipetting Matrigel / BME droplets onto growth medium using 1 mL micropipettes (P1000 Gilson). The disrupted organoids were centrifuged at 500 × g for 5 min. The precipitated organoids were aspirated into excess complete high-grade DMEM / F12 and centrifuged at 500 × g for 5 min. The organoid fragments were replated at the desired density in 70% Matrigel in CNM containing the Rho kinase inhibitor Y-27632 (1 / 1000) and incubated in a humidified incubator at 37 °C and 5% CO2.

[0554] The culture medium should be refreshed every 3-4 days. Organoids should be passaged every 7-10 days.

[0555] organoid collection

[0556] Before incubating the plate at 37°C and 5% CO2 for 30 minutes, add 20 μL of 100x dispersant enzyme solution to each well of a 6-well plate (2 ml) containing the organoids prepared above. Collect organoids from all wells by pipetting through a 100 μm filter (pre-wetted twice with 10 mL AD-DF+++ medium) using a P1000 pipette into a 50 mL plastic tube. Then wash the 100 μm filter containing organoids twice with 10 mL AD-DF+++ medium. Filter the flow-through through a 20 μm filter (pre-wetted twice with 10 mL AD-DF+++ medium). Invert the 20 μm filter (containing organoids) and wash with Ad-DF+++ medium supplemented with the Rho kinase inhibitor Y-27632 (four times the volume of the flow-through), and centrifuge the flow-through at 1500 rpm (450 x g) for 3 min at 8°C.

[0557] Finally, the supernatant was carefully aspirated and the organoid pellet was resuspended in 1 mL CCDM+serum / filled 6-well plate before counting.

[0558] Example 2: Formation of co-culture

[0559] 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 and 5% Matrigel and the Rho kinase inhibitor Y-27632. The solution was kept cold, and 100–200 µl was dispensed into each well of a 96-well plate.

[0560] The co-culture was released from the BME using cell recovery solution and fixed in 4% paraformaldehyde. Whole-sample slides fixed with phalloidin were stained to label polymerized actin and DAPI to label cell nuclei. The whole-sample slides were fixed in ProLong Gold anti-fading fixative on glass slides and imaged on a Leica SP8X confocal microscope (data not shown).

[0561] Example 3: Induction of a pro-inflammatory spectrum in co-cultures

[0562] The co-cultures prepared as described in Examples 1-2 were induced to exhibit a pro-inflammatory spectrum upon exposure to pro-inflammatory stimuli. Figure 1 The process diagrams for this embodiment and subsequent embodiments are shown.

[0563] Specifically, stock solutions of OSM and IL-1β were diluted 1:1 with 0.6% Tween in PBS to achieve 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.

[0564] In Examples 4-11, fibroblast cultures, organoid cultures, and co-cultures of fibroblasts and organoids were prepared according to Examples 1-2, unless otherwise stated. OSM (1 ng / mL) and IL-1β (1 ng / mL) were added to the co-cultures according to Example 3, wherein “pro-inflammatory stimulant” or “pro-inflammatory stimulus” is as described in Examples 4-11. The co-cultures were incubated on plates at 37°C and 5% CO2 for 72 hours prior to cytokine or caspase analysis, or on plates at 37°C and 5% CO2 for 96 hours prior to imaging analysis.

[0565] Example 4: Imaging shows increased organoid size when co-cultures are prepared under pro-inflammatory stimuli.

[0566] This embodiment uses imaging analysis to evaluate the morphology of co-cultured organoids. It demonstrates that organoids and fibroblasts can be successfully co-cultured and exhibit normal organoid morphology consistent with that of organoids cultured in cCDM (e.g., expected size, area, and appearance under bright-field imaging), and show increased aggregation due to fibroblast-mediated cell-cell interactions.

[0567] This embodiment also shows that pro-inflammatory stimulation of organoids leads to an increase in organoid area and affects aggregation (relative to cocultures without pro-inflammatory stimulation), indicating that organoid size is an indicator of the pro-inflammatory spectrum of cocultures.

[0568] Organoids in the co-culture were first stained with calcein AM. Forty-eight hours after combining organoids and fibroblasts in the co-culture, 50 μL of DMSO was added to a vial of calcein AM and mixed. 88 μL of the mixture was added to 4,312 μL of Ad-DF+++ medium. Before shaking in a Tecan D300 for 5–10 seconds, 10 μL of the final calcein AM mixture was added to the co-culture. The plate was then incubated at 37°C and 5% CO2 for 2 hours. Imaging was then performed to assess organoid morphology.

[0569] like Figure 2 As shown in Figure A, co-cultures exposed to pro-inflammatory stimuli exhibited increased cystic morphology compared to the small, compact morphology of organoids cultured alone or co-cultures not exposed to pro-inflammatory stimuli. 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 with pro-inflammatory stimuli.

[0570] like Figure 2 As shown in B, organoid area is significantly increased in co-cultures with a pro-inflammatory spectrum.

[0571] like Figure 2 As shown in C, exposure to alternative stimulants such as IL-6 (20 ng / mL), LIGHT (20 ng / mL), or a combination of both did not cause changes in organoid size.

[0572] like Figure 2 As shown in D and 2E, similar results were obtained using alternative primary organoids “A”, “B”, and “C” (each derived from the small intestine).

[0573] like Figure 2 As shown in F, similar results were obtained when testing the immortalized and primary fibroblast cell lines in the absence ("-") and presence ("+") of pro-inflammatory stimuli (see Table 1 above).

[0574] like Figure 2 As shown in G and 2H, similar results were obtained for different ratios of fibroblasts to organoids (“F / O”) when the culture duration was extended to day 3 (“D3”) and day 6 (“D6”).

[0575] Example 5: When co-cultures were prepared with pro-inflammatory stimuli, expression measurements showed increased secretion of markers indicating a pro-inflammatory spectrum.

[0576] Centrifuge the plate contents at 1500 rpm for 5 minutes and collect the supernatant. Collect 100 μL, and centrifuge the remaining supernatant again. Collect another 30 μL, for a total of 130 μL of supernatant. Seal the supernatant container and store at -80°C until use. Analyze IL-6 and CXCL2 expression separately by HTRF (CisBio / Revvity, “Human IL-6HTRF Kit”, https: / / uk.cisbio.eu / human-il6-kit-40419) and ELISA (Bio-Techne Ltd., “Human CXCL2 / GRO beta DuoSet ELISA”) according to the supplier's protocol. https: / / www.rndsystems.com / products / human-cxcl2-gro-beta-duoset-elisa_dy276-05 ).

[0577] like Figure 3 As shown in Figure A, no significant IL-6 expression was observed in organoids, fibroblasts, or co-cultures in the absence of pro-inflammatory stimuli. Pro-inflammatory stimuli induced IL-6 expression in cultured fibroblasts alone and in co-cultures, but not in cultured organoids alone.

[0578] like Figure 3 As shown in B, pro-inflammatory stimulation promoted some expression of CXCL2 in fibroblasts and organoids (compared to fibroblasts and organoids cultured alone without pro-inflammatory stimulation), but showed significant synergistic induction in co-cultures with pro-inflammatory stimulation (compared to co-cultures without pro-inflammatory stimulation).

[0579] like Figure 3 As shown in C and 3D, similar results were obtained in the different fibroblast cell lines shown in Table 1 above, in the absence ("-") and presence ("+") of pro-inflammatory stimuli.

[0580] like Figure 3 As shown in E, similar CXCL2 results were observed in three different primary organoids, “A”, “B”, and “C” (derived from the small intestine).

[0581] like Figure 3 As shown in F and 3G, a higher fibroblast-organoid ratio led to increased secretion of IL6 and CXCL2, independent of the concentration of pro-inflammatory stimulants (IL-1β and OSM, respectively, from 0 ng / ml to 5 ng / ml, and then to 15 ng / ml). Therefore, the presence of increased fibroblasts relative to organoids when prepared under pro-inflammatory stimuli results in an increased pro-inflammatory spectrum in the co-culture.

[0582] Example 6: Caspase assays showed increased apoptosis when co-cultures were prepared with pro-inflammatory stimuli and challenged with the inflammatory stimuli.

[0583] Seventy-two hours after organoids and fibroblasts were combined in a co-culture, apoptosis in the co-culture was triggered using an inflammatory stimulant (in the form of damage-inducing cytokines), and measurements were taken 24 hours later in co-cultures prepared with and without the pro-inflammatory stimulant.

[0584] Stock solutions of TNF and IFNγ were diluted 1:1 with 0.6% Tween in PBS 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.

[0585] Apoptosis was assessed using a caspase-Glo assay (e.g., Promega) to determine relative caspase 3 / 7 activity. Briefly, 95 μL of room temperature caspase reagent was added to each plate. The plates were then shaken for 1 minute and incubated in the dark at room temperature for 30 minutes. A 160 μL sample from each plate was then transferred to a black plate for analysis.

[0586] like Figure 4 As shown in Figure A, in the absence of pro-inflammatory stimuli, fibroblasts in the co-culture are protected from increased caspase activity induced by inflammatory stimuli. This protection is lost when the co-culture is prepared with pro-inflammatory stimuli, leading to increased apoptosis compared to co-cultures prepared without pro-inflammatory stimuli. Furthermore, pro-inflammatory stimuli increase the baseline caspase activity (i.e., apoptosis in the presence of pro-inflammatory stimuli but not in the absence of inflammatory stimuli).

[0587] like Figure 4 As shown in B, similar results were obtained in different fibroblast cell lines, as shown in Table 1 above.

[0588] like Figure 4 As shown in C, similar results were obtained using three alternative primary organoids, “A”, “B”, and “C” (all derived from the small intestine).

[0589] Caspase activity was measured using 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). Unsurprisingly, co-cultures exposed to pro-inflammatory stimuli followed by exposure to inflammatory stimuli showed a greater degree of increased apoptosis than co-cultures exposed to inflammatory stimuli alone.

[0590] Example 7: Pro-inflammatory stimuli exert separable effects on apoptosis and organoid size; tofacitinib ameliorate increased apoptosis in co-cultures with a pro-inflammatory spectrum upon exposure to inflammatory stimuli.

[0591] Tofacitinib is a small-molecule JAK inhibitor used to treat inflammatory diseases, including ulcerative colitis (UC). The effect of 10 µM tofacitinib on the measurement parameters in Examples 4 and 6 was evaluated.

[0592] like Figure 5 As shown in A, tofacitinib treatment resulted in minor morphological changes (measured according to Example 4), but as Figure 5 As shown in B, tofacitinib significantly reduced caspase activity upon exposure to inflammatory cytokines (measured according to Example 6).

[0593] from Figure 5 As can be clearly seen in A-5B, the pro-inflammatory stimulant IL-1β appears to play a dominant role in morphological changes. Figure 5 A), while OSM appears to play a dominant role in apoptosis ( Figure 5 (B) This indicates that different pro-inflammatory stimuli exert their effects through non-redundant pathways, leading to different phenotypic changes in co-cultures. This has implications for the simulation, diagnosis, and treatment of inflammatory conditions.

[0594] Example 8: Conditioned culture medium from fibroblasts prepared under pro-inflammatory stimulation triggers a pro-inflammatory spectrum in organoids cultured alone.

[0595] The inventors hypothesized that the effects of pro-inflammatory stimuli on co-cultures could be mediated, at least in part, by soluble effectors secreted by fibroblasts into the surrounding culture medium (“conditioned medium”). Therefore, conditioned medium was isolated from fibroblasts cultured solely under pro-inflammatory stimuli and combined with organoids to determine whether the organoid morphology measured as in Example 4 could be reproduced without combining organoids and fibroblasts.

[0596] like Figure 6 As shown, when organoids are exposed to conditioned medium derived from fibroblasts cultured under pro-inflammatory stimuli, the increase in organoid area and aggregation is significant, comparable to that when organoids and fibroblasts are co-cultured under pro-inflammatory stimuli. This suggests that the inflammatory response in the co-culture is mediated at least in part by soluble factors derived from fibroblasts.

[0597] Example 9: Gene expression analysis of co-cultured organoids

[0598] Following the method in Example 8, the gene expression levels of several gene types in organoids cultured in expansion medium (CNM) or differentiation medium (cCDM) + serum were investigated, with or without conditioned medium from fibroblasts cultured under pro-inflammatory stimuli.

[0599] like Figure 7 As shown, the expression profiles of key inflammatory genes were altered.

[0600] Example 10: The effect of alternative culture medium on co-culture measurements

[0601] The inventors investigated alternative culture media for use in the methods of Examples 4-6 above.

[0602] The methods of Examples 1-3 were repeated, with the following used as co-culture media: cCDM + serum; cCDM; cCDM + serum without PD0325901; cCDM + serum without DAPT; CNM; or eCDM. The methods of Examples 4-6 were then performed as follows.

[0603] Imaging

[0604] According to Example 4, organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example were imaged. Results are shown below. Figure 8 The results for organoids are only seen Figure 8 A, and the results of organoid-fibroblast coculture are shown in [see...]. Figure 8 B.

[0605] The imaging results show that, after the application of pro-inflammatory stimulation, the culture medium allowed for the observation of increased organoid size and cystic morphology relative to the control. Figure 8 B). In organoids lacking fibroblasts, no major morphological changes were observed when any culture medium was used. Figure 8 A).

[0606] Expression measurement

[0607] According to Example 5, IL-6 and CXCL2 expression in organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example were measured. The results for IL-6 showed... Figure 9 The results of A,CXCL2 show Figure 9 B.

[0608] Expression measurements showed that the culture medium allowed for the identification of changes in the secretion of markers associated with the presence of fibroblasts and pro-inflammatory stimuli. However, the induction of expression of CNM, CXCL2, and IL-6 was much lower. Serum samples from cCDM and cCDM+ alone revealed increased induction of the markers IL-6 and CXCL2.

[0609] Caspase Measurement

[0610] According to Example 6, caspase levels were measured in organoids and organoid-fibroblast co-cultures prepared using the culture medium described in this example. The results showed... Figure 10 .

[0611] The caspase assays demonstrated that the culture medium allowed for 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 such as OSM and IL-1β, cCDM+ serum particularly highlighted the potential of fibroblasts to be protected against anti-inflammatory stimuli such as IFN and TNF.

[0612] Example 11: RNA-seq experiments indicating further biomarkers of the pro-inflammatory profile

[0613] To identify potential novel biomarkers for the pro-inflammatory profile in indicator cocultures, RNA-seq was performed using cCDM, CM, and CNM media as described in Example 10, or organoid and organoid-fibroblast cocultures prepared with (“INF”) or without (“Control”) pro-inflammatory stimulation of OSM (1 ng / ml) and IL-1β (1 ng / ml), or using conditioned media (“CM”) as described in Example 8. RNA extraction was performed using Qiagen QIASymphony SP, and library preparation was performed using the TruSeq® RNA Chain polyA Kit, both performed according to the standard protocols of the respective manufacturers. All samples were collected three days post-inoculation. The mean normalized expression of different RNAs is shown in Table 2. Normalization was performed using the ratio median method (more information on normalization methods can be found at https: / / hbctraining.github.io / DGE_workshop / lessons / 02_DGE_count_normalization.html).

[0614] As shown in Table 2, the expression of IL-6, CXCL2, IGFBP1, WNT7A, MMP7, and HIF3A was particularly upregulated in cCDM co-cultures with pro-inflammatory stimuli, but not in cCDM organoid cultures with pro-inflammatory stimuli. This is contrary to the expression profiles of some other biomarkers, which were differentially upregulated in monocultures and co-cultures with and without pro-inflammatory stimuli.

[0615]

[0616] Using the same RNA-seq protocol, additional markers of significant downregulation or upregulation were detected between cultures prepared differently. The results for the following culture types are shown in the table below: Table 3: Fibroblast-organoid cocultures in cCDM with or without pro-inflammatory stimulation from IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose significant changes (upregulation or downregulation) in expression are associated with pro-inflammatory stimulation in cCDM cocultures.

[0617] Table 4: Organoid cultures in cCDM without fibroblasts, with or without pro-inflammatory stimulation from IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose significant changes (upregulation or downregulation) in expression are associated with pro-inflammatory stimulation of cCDM organoids in the absence of fibroblasts.

[0618] Table 5: Organoid cultures in CNM without fibroblasts, with or without pro-inflammatory stimulation from IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose significant changes (upregulation or downregulation) in expression are associated with pro-inflammatory stimulation of CNM organoids in the absence of fibroblasts.

[0619] Table 6: Fibroblast cultures in cCDM without organoids, with or without pro-inflammatory stimulation from IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose significant changes (upregulation or downregulation) in expression are associated with pro-inflammatory stimulation in cCDM fibroblasts in the absence of organoids.

[0620] Table 7: Organoid cultures without fibroblasts and organoid-fibroblast co-cultures in cCDM, both exhibiting pro-inflammatory stimulation with IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose expression (upregulation or downregulation) is significantly altered in organoid co-cultures with fibroblasts, compared to organoids without fibroblasts, both in cCDM and both exhibiting pro-inflammatory stimulation.

[0621] Table 8: Organoid cultures without fibroblasts and organoid-fibroblast co-cultures in cCDM, both without pro-inflammatory stimulation. Therefore, this table lists biomarkers whose expression (upregulation or downregulation) is significantly altered in organoid co-cultures with fibroblasts compared to organoids without fibroblasts, both in cCDM and without pro-inflammatory stimulation.

[0622] Table 9: Organoid cultures without fibroblasts in cCDM and organoid cultures without fibroblasts in conditioned medium (CM), both without pro-inflammatory stimulation. Therefore, this table lists biomarkers whose expression changes significantly (up- or down-regulated) in relation to changes in the culture medium from cCDM to CM for organoids in the absence of fibroblasts and pro-inflammatory stimulation.

[0623] Table 10: Organoid cultures without fibroblasts in cCDM and CM, both with pro-inflammatory stimulation of IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists the biomarkers whose expression changes (upregulation or downregulation) are correlated with changes in the culture medium from cCDM to CM for organoids in the absence of fibroblasts and with pro-inflammatory stimulation.

[0624] Table 11: Organoid cultures in CM without fibroblasts, with or without pro-inflammatory stimulation from IL-1β (1 ng / ml) and OSM (1 ng / ml). Therefore, this table lists biomarkers whose significant changes (upregulation or downregulation) in expression are associated with pro-inflammatory stimulation of organoids in CM in the absence of fibroblasts.

[0625] Several biomarkers 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 biomarkers were less frequently observed in organoid cultures alone (Tables 4 and 5). This suggests that fibroblasts play an important role in the response to pro-inflammatory stimuli.

[0626] Table 3 - Changes in expression of co-cultured proteins (cCDM) after pro-inflammatory stimulation

[0627] Table 4 - Expression changes in non-fibroblast organoids (cCDM) after pro-inflammatory stimulation.

[0628] Table 5 - Expression changes in pro-inflammatory organoids (CNMs) without fibroblasts after pro-inflammatory stimulation.

[0629] Table 6 - Expression changes of organoid fibroblasts (cCDM) after pro-inflammatory stimulation

[0630] Table 7 - Changes in expression of pro-inflammatory substances in co-cultured organisms (cCDM) after pro-inflammatory stimulation compared to those in organoids without fibroblasts (cCDM).

[0631] Table 8 - Expression changes in fibroblast-free organoids (cCDM) compared to co-cultured organisms (cCDM), both without pro-inflammatory stimulation.

[0632] Table 9 - Expression changes of fibroblast-free organoids in cCDM compared to those in conditioned medium (CM), with no pro-inflammatory stimulation in either culture medium.

[0633] Table 10 - Expression changes in organoids without fibroblasts in cCDM after pro-inflammatory stimulation compared to organoids without fibroblasts in CM.

[0634] Table 11 - Expression changes in organoids without fibroblasts in CM under non-pro-inflammatory stimulation compared to organoids without fibroblasts under pro-inflammatory stimulation.

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, wherein the method comprises: combining at least one organoid with at least one stromal cell in a co-culture medium to form the co-culture; and determining the presence or absence of the at least one change in the co-culture, optionally wherein: (a) the method comprises preparing the at least one stromal cell by culturing stromal cells in a stromal cell medium; and / or (b) the method comprises preparing the at least one organoid by culturing epithelial cells in an organoid medium.

2. A method of testing at least one therapeutic agent for a disease, 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 the at least one therapeutic agent to the co-culture; and determining the presence or absence of at least one change in the co-culture, optionally wherein: (a) the method comprises preparing the at least one stromal cell by culturing stromal cells in a stromal cell medium; and / or (b) the method comprises preparing the at least one organoid by culturing epithelial cells in an organoid medium.

3. A method for determining the presence or absence of a disease diagnosis and / or prognosis 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; and determining the presence or absence of at least one change in the co-culture, optionally wherein: (a) the method comprises preparing the at least one stromal cell by culturing stromal cells in a stromal cell medium; and / or (b) the method comprises preparing the at least one organoid by culturing epithelial cells in an organoid medium.

4. The method of any preceding claim, wherein determining the presence or absence of at least one change in the co-culture comprises: (a) determining the presence or absence of stromal cells in the co-culture, 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 the presence or absence of an inflammatory response, optionally wherein: i. the at least one inflammatory stimulus comprises a cytokine, optionally wherein the cytokine comprises TNF and / or IFNy; and / or ii. the inflammatory response comprises increased caspase activity and / or apoptosis in the co-culture; and / or (c) adding at least one pro-inflammatory stimulus to the co-culture medium, optionally wherein: i. the at least one pro-inflammatory stimulus is added to the co-culture medium prior to the addition of any inflammatory stimulus to the co-culture medium; and / or ii. the at least one pro-inflammatory stimulus comprises at least one cytokine, optionally wherein the at least one cytokine comprises IL-1 b and / or oncostatin M (OSM).

5. The method of any preceding claim, wherein determining the presence or absence of 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 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 of any one of claims 2-5, wherein the disease is an inflammatory disease or a fibrotic disease, optionally wherein the disease is an inflammatory disease, optionally wherein the inflammatory disease is a gastrointestinal inflammatory disease, optionally wherein the inflammatory disease is inflammatory bowel disease (IBD), optionally wherein the IBD comprises ulcerative colitis (UC) or Crohn’s disease (CD).

7. The method of any preceding claim, wherein the at least one change comprises 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 apoptosis, an increase in caspase activity, and / or a change in expression of one or more genes, optionally wherein the change in expression of one or more genes comprises a change in one or more disease biomarkers, one or more fibrosis biomarkers, and / or one or more inflammation biomarkers.

8. The method of any preceding claim, wherein the at least one change comprises a change in secretome profile, optionally a change in cytokine secretion, optionally a change in IL-6 and / or CXCL2 secretion.

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

10. The method of any preceding claim, wherein the presence or absence of the at least one change is determined relative to a reference organoid, optionally wherein the reference organoid does not comprise stromal cells.

11. The method of any preceding claim, wherein the at least one organoid is a patient-derived organoid.

12. The method of any preceding claim, wherein the at least one stromal cell comprises a fibroblast cell, optionally wherein the fibroblast cell is an intestinal fibroblast cell, e.g., a colon fibroblast cell or a small intestinal fibroblast cell, optionally wherein the fibroblast cell is an immortalized human colon fibroblast cell, a human colon fibroblast cell, an immortalized human small intestinal fibroblast cell, or a human small intestinal fibroblast cell.

13. The method of any preceding claim, wherein the co-culture, the at least one organoid, and / or the at least one stromal cell: (a) is derived from the subject; and / or (b) is derived from lung tissue, kidney tissue, pancreas tissue, or liver tissue; and / or (c) is derived from fibrotic tissue; and / or (d) is derived from inflamed tissue; and / or (e) comprises or consists of: a mammalian cell, optionally a human cell; and / or (f) is derived from the intestine, optionally the colon.

14. The method of any preceding claim, wherein the at least one organoid and the at least one stromal cell are derived from the same subject, optionally from (a) the same sample from the subject and / or (b) the same tissue of the same subject.

15. The method of any preceding claim, wherein the co-culture medium comprises a mitogenic growth factor, a BMP inhibitor, and R-spondin, optionally wherein the co-culture medium comprises a Wnt agonist, a mitogenic growth factor, a BMP inhibitor, and R-spondin, optionally wherein the co-culture medium comprises N-Ac, a TGF-β inhibitor, a mitogenic growth factor, gastrin, a BMP inhibitor, an antibiotic, R-spondin, a Notch pathway inhibitor, an ERK inhibitor, and a Wnt agonist.

16. The method of any preceding claim, wherein the co-culture medium comprises serum, optionally fetal bovine serum, optionally 5% fetal bovine serum.

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