Culture medium suitable for differentiation and culture of intestinal organoids

Abstract

The present application relates to a culture medium for culturing intestinal organoids, which is characterized by containing neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). The present application also relates to a method of using this medium for culturing intestinal organoids.

Description

Technical Field

[0001] The present invention relates to a culture medium suitable for long-term or short-term in vitro culture of intestinal organoid cells.

Background Art

[0002] The intestinal tract is an organ in the human body that has the largest contact area with the external environment and has functions essential for maintaining life such as digestion and absorption. Most of the functions of the intestinal tract are performed by the intestinal epithelium covering its inner layer. The intestinal epithelium is composed of two compartments: villi consisting of three differentiated cells (mucus-producing cells, absorptive epithelial cells, and endocrine cells) and crypts mainly composed of undifferentiated proliferating cells. In the small intestinal crypts, Paneth cells that produce antibacterial peptides are present at the bottom of the crypts. Recently, molecular genetic cell lineage analysis has revealed that Lgr5-positive cells (also called "crypt base columnar (CBC) cells") sandwiched between Paneth cells are intestinal epithelial stem cells. Lgr5-positive intestinal epithelial stem cells produce progenitor cells called transit amplifying cells, but these progenitor cells do not have permanent self-renewal ability and their differentiation ability is limited to 1 to 3 lineages. Transit amplifying cells differentiate by dividing 2 to 4 times in the crypts and reach terminal differentiation in the villi. These differentiated cells are shed at the tip of the villi and die by apoptosis. The intestinal epithelium is a tissue with a high metabolic rate and moves from the crypt stem cells to the tip of the villi in 4 to 5 days. Unlike other differentiated cells, Paneth cells move to the bottom of the crypts in a differentiated state and have a long cell lifespan of about 2 months.

[0003] The self-renewal mechanism of intestinal epithelial stem cells has been found from the results of several genetically modified mice to be controlled by Wnt signal and bone morphogenetic protein (BMP) signal. Intestinal epithelial-specific knockout mice of adenomatous polyposis coli (APC), which is a suppressor molecule of Wnt signal, show hyperplasia of intestinal epithelial cells and adenoma formation. In addition, ectopic crypt formation is observed in mice overexpressing Noggin, which is an inhibitory protein of BMP, in intestinal epithelial cells. Therefore, it has been suggested that BMP signal acts inhibitory on intestinal epithelial stem cells.

[0004] In addition, long-term culture of intestinal epithelial cells has been impossible for a long time. This is thought to be due to the fact that growth factors necessary for the maintenance of intestinal epithelial stem cells were not known. In recent years, success in long-term maintenance of intestinal epithelial stem cells has been achieved by culturing the cells in the presence of a cell culture medium containing a basal medium for animal or human cells with the intestinal epithelial stem cells adhered to the extracellular matrix and BMP inhibitor, mitogenic growth factor, and Wnt agonist added.

[0005] Another means for the study of the intestine is the study of intestinal organoids. Organoids are small self-organizing three-dimensional tissue cultures derived from stem cells. Such cultures can be made to express their selected aspects, such as reproducing most of a complex organ or producing only a specific type of cell. Organoid formation generally requires culturing stem cells or progenitor cells in an organ / tissue-specific medium. Summary of the Invention

[0006] Through research, the inventors of the present invention have surprisingly succeeded in defining a culture medium suitable for long-term or short-term in vitro culture of intestinal organoid cells.

[0007] The claims thus include a culture medium for culturing intestinal organoids, characterized in that it contains neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). This culture medium of the present invention is suitable for culturing small intestine organoids, colon organoids, colorectal cancer organoids, Crohn's disease organoids or ulcerative colitis organoids.

[0008] The concentration of NRG1 in the medium can be between 1 ng / ml and 100 ng / ml. The concentration of atRA in the medium can be between 10 nM and 10 μM. In some embodiments, the culture medium of the present invention is free of animal serum. In some embodiments, the culture medium of the present invention further contains any one of R-Spondin, recombinant Noggin, NRG1, IGF1, FGF2 and gastrin. All of these factors, including NRG1 and atRA, can be human or mouse and recombinant or purified natural proteins. The culture medium of the present invention can also contain Wnt NGS (Next Generation Surrogate).

[0009] The claims also include a composition comprising the medium of the present invention together with a 3D matrix that mimics the extracellular matrix by its interaction with the extracellular matrix or cell membrane proteins. Such a 3D matrix extracellular matrix can be a synthetic hydrogel or Matrigel (trademark). Alternatively, in particular, UltriMatrix (Culturex Ultimatrix RGF basement membrane extract) and BME (Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear) and collagen-1 or fibronectin can be used. In some embodiments, the extracellular matrix or 3D matrix is washed away after an initial period.

[0010] The claims further include the use of the medium of the present invention or the composition of the present invention for culturing intestinal organoids.

[0011] The claims are directed to a method for generating intestinal organoids, which further includes the steps of providing a suspension of single cells or fragments of intestinal tissue, incubating the suspension of single cells or fragments of intestinal tissue with a first medium for at least one day, and then replacing the first medium with the medium of the present invention as described above. In some embodiments, the first medium can contain atRA at a concentration of 10 nM. The single cells or fragments of intestinal tissue used in the method of the present invention can be cells or tissue fragments obtained from the small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue, or can be definitive endoderm cells.

[0012] In the method of the present invention, the first medium can contain an inhibitor of Rho-associated kinase (ROCK), such as the ROCK inhibitor Y27632. In the method of the present invention, the first medium can contain an inhibitor of the activin / NODAL / TGF-β pathway, such as the inhibitor A83-01.

[0013] In some embodiments of the method of the present invention, the cells can be incubated in the first medium for at least three days.

[0014] The first and second media of the method of the present invention can be adapted to the nature of the single cells or fragments of intestinal tissue used in the method of the present invention. For example, for small intestine cells and / or tissue fragments, the appropriate concentration of NRG1 in the first medium is 1 ng / ml and there is no atRA, while the concentrations of NRG1 and atRA in the second medium, i.e., the medium of the present invention, are 10 ng / ml and 2.5 μM, respectively. For cells and tissue fragments derived from the colon, the appropriate concentration of NRG1 in the first medium is 1 ng / ml and there is no atRA, and the concentrations of NRG1 and atRA in the second medium are 10 ng / ml and 10 nM, respectively.

[0015] The claims also encompass intestinal organoids obtained using the method of the present invention.

[0016] The following drawings are for the purpose of explaining embodiments of the present invention and do not limit the scope of the present invention encompassed by the claims.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0018] Through research, the inventors of the present invention have surprisingly succeeded in defining a culture medium suitable for long-term or short-term in vitro culture of intestinal organoid cells.

[0019] The claims thus include a culture medium for culturing intestinal organoids, characterized by containing neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). This culture medium of the present invention is suitable for culturing small intestinal organoids, colonic organoids, colorectal cancer organoids, Crohn's disease organoids or ulcerative colitis organoids.

[0020] The concentration of NRG1 in the medium can be between 1 ng / ml and 100 ng / ml. The concentration of atRA in the medium can be between 10 nM and 10 μM. In some embodiments, the culture medium of the present invention is free of animal serum. In some embodiments, the culture medium of the present invention further contains any one of R-Spondin, recombinant Noggin, NRG1, IGF1, FGF2 and gastrin. All of these factors including NRG1 and atRA can be human or mouse and recombinant or purified natural proteins. The culture medium of the present invention can also contain Wnt NGS (next-generation surrogate).

[0021] The claims also include a composition comprising the medium of the present invention together with a 3D matrix that mimics the extracellular matrix by its interaction with the extracellular matrix or cell membrane proteins. Such a 3D matrix extracellular matrix can be a synthetic hydrogel or Matrigel (trademark). Alternatively, among other things, UltriMatrix (Culturex Ultimatrix RGF basement membrane extract) and BME (Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear) and collagen-1 or fibronectin can be used. In some embodiments, the extracellular matrix or 3D matrix is washed away after an initial period.

[0022] The claims further include the use of the medium or composition of the present invention for culturing intestinal organoids.

[0023] The claim is a method for generating intestinal organoids, comprising the steps of providing a suspension of single cells or fragments of intestinal tissue, incubating the suspension of single cells or fragments of intestinal tissue with a first medium for at least one day, and then replacing the first medium with the medium of the present invention described above. In some embodiments, the first medium can contain atRA at a concentration of 10 nM. The single cells or fragments of intestinal tissue used in the method of the present invention can be cells or tissue fragments obtained from the small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue or can be embryonic endoderm cells.

[0024] In the method of the present invention, the first medium can contain an inhibitor of Rho-associated kinase (ROCK), such as the ROCK inhibitor Y27632. In the method of the present invention, the first medium can contain an inhibitor of the activin / NODAL / TGF-β pathway, such as the inhibitor A83-01.

[0025] In some embodiments of the method of the present invention, the cells can be incubated in the first medium for at least three days.

[0026] The first and second media of the method of the present invention can be adapted to the nature of the single cells or fragments of intestinal tissue used in the method of the present invention. For example, for small intestine cells and / or tissue fragments, the appropriate concentration of NRG1 in the first medium is 1 ng / ml and there is no atRA, while the concentrations of NRG1 and atRA in the second medium, i.e., the medium of the present invention, are 10 ng / ml and 2.5 μM, respectively. For cells and tissue fragments derived from the colon, the appropriate concentration of NRG1 in the first medium is 1 ng / ml and there is no atRA, and the concentrations of NRG1 and atRA in the second medium are 10 ng / ml and 10 nM, respectively.

[0027] The claim also includes intestinal organoids obtained using the method of the present invention.

[0028] The following definitions are provided to facilitate the understanding of certain terms used throughout this specification.

[0029] As used herein, the term "totipotent stem cell" (also known as a pluripotent stem cell) is a stem cell that can differentiate into embryonic and extraembryonic cell types. From such cells, a complete and viable organism can be constructed. These cells are produced from the fusion of an egg and a sperm. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0030] As used herein, the term "pluripotent stem cell (PSC)", which is also commonly known as a PS cell, encompasses any cell that can differentiate into cells derived from any of the three germ layers (germ epithelium), namely, the endoderm (inner lining of the stomach, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissue and nervous system), including almost all cells. PSCs can be descendants of totipotent cells obtained by derivation from embryonic stem cells (including embryonic germ cells) or through the induction of non-pluripotent cells such as adult somatic cells by forcing the expression of specific genes.

[0031] As used herein, the term "induced pluripotent stem cell (iPSC)", which is also commonly abbreviated as an iPS cell, refers to a type of pluripotent stem cell that is artificially induced from normally non-pluripotent cells such as adult somatic cells by inducing the "forced" expression of specific genes.

[0032] As used herein, the term "embryonic stem cell (ESC)", which is also commonly abbreviated as an ES cell, refers to a pluripotent cell derived from the inner cell mass of a blastocyst, an early embryo. For the purposes of the present invention, the term "ESC" is sometimes used broadly to also include embryonic germ cells.

[0033] As used herein, the term "progenitor cell" encompasses any cell that can be used in the methods described herein in which one or more progenitor cells are capable of self-renewing or acquiring the ability to differentiate into one or more specialized cell types. In some embodiments, the progenitor cells are pluripotent or have the potential to become pluripotent. In some embodiments, the progenitor cells are subjected to treatment with exogenous factors (e.g., growth factors) to acquire pluripotency. In some embodiments, the progenitor cells can be totipotent (or omnipotent) stem cells; pluripotent stem cells (induced or non-induced); multipotent stem cells; oligopotent stem cells and unipotent stem cells. In some embodiments, the progenitor cells can be derived from embryos, infants, children or adults. In some embodiments, the progenitor cells can be somatic cells that are subjected to treatments such that pluripotency is conferred via genetic manipulation or protein / peptide treatment. In developmental biology, cell differentiation is the process by which less-specialized cells become more-specialized cell types. As used herein, the term "directed differentiation" describes the process by which less-specialized cells become a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable method that can be used to direct or alter the fate of the progenitor cells. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment and nucleic acid treatment.

[0034] As used herein, the term "cellular component" refers to, for example, individual genes, proteins, mRNAs that express genes, and / or any other various cellular components or protein activities such as the degree of protein modification (e.g., phosphorylation) typically measured by those skilled in the art in biological experiments (e.g., by microarray or immunohistochemical examination). Important discoveries regarding biological systems, the complex network of biochemical processes underlying normal human diseases, as well as gene discovery and structure determination can now be considered to be due to the utilization of large amounts of data on cellular components as part of the investigation process. Large amounts of data on cellular components can help identify biomarkers, distinguish disease subtypes, and identify mechanisms of toxicity.

[0035] As described herein, the methods and systems are established using a temporal series of growth factor manipulations to mimic intestinal embryogenesis in culture. In particular, the methods and systems are established to direct in vitro differentiation of both PSCs, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs) into intestinal tissue.

[0036] The generation of gastric and small intestinal organoids from pluripotent stem cells (PSCs) has revolutionized the study of human gastrointestinal (GI) development and disease. However, attempts to generate large intestinal organoids have lagged, with the molecular understanding of posterior intestinal development being challenging.

[0037] In certain embodiments of the present invention, "about" or "approximately" refers to a number that varies from the referenced number by up to 5% or in other embodiments up to 10%, and in other embodiments up to 25%.

[0038] As used herein, the term "serum-free" refers to the fact that the medium is essentially free of serum. In certain embodiments, the total serum in the subject medium is 0% (none at all) or less than about 0.001%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 1.0% or 10.0%. The most common types of serum include various forms of bovine serum (calf serum, fetal bovine serum, calf serum, donor calf serum, neonatal calf serum, etc.), horse serum, and human serum.

[0039] "Chemically defined" means that the structure, chemical formula, and percentage of the various individual components within a chemical composition are known or can be specified. Various tissue extracts, such as bovine pituitary extracts, are not chemically defined because at least in some cases, not all of the individual components of the extract are known. For those known components, the amounts and relative percentages of the various components can vary from batch to batch (and usually do). This is caused in part by the fact that individual animals may inherently have different levels of various chemical compositions in the same tissue even in response to factors such as health, nutrition, mood, pathological infections, trauma, etc.

[0040] In certain embodiments, the medium of the present invention contains no animal serum products prepared for the purpose of tissue culture. It also contains no tissue extracts with unknown / unspecified chemical components. Instead, all of the essential components necessary to maintain the desired growth / proliferation of the desired cell type are chemically defined. Most, if not all, of these individual components can be purchased off-the-shelf from various vendors such as Sigma-Aldrich Corp. (St. Louis, MO), GIBCO-Invitrogen Corp. (Carlsbad, CA); Calbiochem and / or BD Biosciences (San Jose, CA), etc.

[0041] In certain other embodiments, the presence in the media of the subject serum and / or tissue extracts, even in trace amounts, will not substantially interfere with the characteristics of the media.

[0042] The present invention also provides a method for carrying out the discovery and development of pharmaceutical / biotechnology products, including creating enteroid organoid models derived from various cell types isolated and expanded using the media and methods of the present invention and screening a library of drug molecules or lead compounds to identify molecules that affect the enteroid organoid models.

[0043] The terms "cell culture medium," "culture medium" (the plural in each case being "media"), and "medium formulation" refer to a nutrient solution for culturing cells and may be used interchangeably.

[0044] The cell culture medium of the present invention is aqueous (however, it can be reconstituted from dry powder and / or frozen components) and contains many components in a solution, preferably a solution of deionized water and / or distilled water.

[0045] The term "component" refers to any compound that can be used in a cell culture medium to maintain or promote the growth of cell proliferation, regardless of whether it is of chemical origin or biological origin. The terms "constituent," "nutrient," and "component" can be used interchangeably and are all intended to refer to such compounds. Typical components used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc. Other components that promote or maintain cell culture ex vivo can be selected by those skilled in the art according to specific needs.

[0046] "Cell culture" or "culturing" means the maintenance of cells in an artificial in vitro environment. However, the term "cell culture" is a general term and may be used to encompass the culture of not only individual cells but also tissues, organs, organ systems, or whole organisms. It should be understood that the terms "tissue culture", "organ culture", "organ system culture", "organoid culture", or "organotypic culture" may in some cases be used interchangeably with the term "cell culture".

[0047] Certain cells, such as human cells, must have sufficient amounts of nine amino acids to survive. These so-called "essential" amino acids cannot be synthesized from other precursors. However, cysteine can partially satisfy the requirement for methionine (both contain sulfur), and tyrosine can partially substitute for phenylalanine. Such essential amino acids include histidine, isoleucine, leucine, lysine, methionine (and / or cysteine), phenylalanine (and / or tyrosine), threonine, tryptophan, and valine. In certain embodiments, only histidine, isoleucine, leucine, lysine, threonine, tryptophan, and valine are included.

[0048] Some or all of the components can be mixed together in solution to form a "basal medium". To this basal medium, other components such as at least one nucleotide synthesis and / or salvage pathway precursor (e.g., hypoxanthine), epidermal growth factor (EGF), an agent that increases the intracellular cyclic adenosine monophosphate (cAMP) level, and an antioxidant can be added to formulate the complete culture medium of the present invention. These latter added components, such as EGF and the agent that increases cAMP, may be added to the freshly formulated basal medium or they may be mixed as in a stock solution and preferably stored frozen at about -20°C to about -70°C until added to the basal medium to formulate the complete culture medium of the present invention.

[0049] To the extent that the components do not substantially affect the performance of the medium with respect to the culture of intestinal organoids, in certain embodiments, the subject medium may contain one or more of such components and may tolerate their presence.

[0050] One or more components of the medium may also be replaced, if necessary, by other chemicals with similar properties. Such modified media having no one or more non-essential / unnecessary components are within the scope of the present invention. Similarly, one of ordinary skill in the art can determine, for each of the listed components, the optimal level of any given component for a particular cell type by testing ranges of concentrations (e.g., 10%, 25%, 50%, 75%, 100%, 2, 5, 10, 20, 50, 100, 200, 500, 1000 times higher or 10%, 25%, 50%, 75%, 100%, 2, 5, 10, 20, 50, 100, 200, 500, 1000 times lower) based on or starting from the concentrations listed for that particular component. Some components have concentrations within the listed ranges. The appropriate or optimal concentration for any particular cell type can also be determined, similarly, starting from the listed concentrations. In conducting such tests, the initial broad concentration tests may be reduced later based on the results of the first experiment. For example, for the first test, the concentration of a certain component of interest may be changed to 10 -3 、10 -2 、10 -1 、10-fold, 100-fold, and 1000-fold. If the test at 10 -2 still maintains the desired growth and the test at 10 -3 does not, then from 10 -2 to 10 -3For a 10-fold concentration difference between them, it may be further investigated in a second test to accurately identify the optimal range. Thus, a medium so optimized for a particular cell type is also within the scope of the present invention. As will be immediately apparent to those skilled in the art, the concentration of a given component can be increased or decreased beyond the disclosed range, and the effects of the increased or decreased concentration can be determined using only routine experimentation. Optimization of the media formulations of the present invention for any particular cell type can be carried out using the approaches described by Ham (Ham, Methods for Preparation of Media, Supplements and Substrata for Serum-Free Animal Culture, Alan R. Liss, Inc., New York, pp. 3-21, 1984) and Waymouth (Waymouth, C, Methods for Preparation of Media, Supplements and Substrata for Serum-Free Animal Culture, Alan R. Liss, Inc., New York, pp. 23-68, 1984). The optimal final concentration for a media component is typically identified by empirical studies, in single component titrations, or by interpretation of past and current scientific literature. In a single component titration, animal cells are used where the concentration of a single media component is varied while keeping all other components and variables constant, and the effect of the single component on the survival, growth, or continued health of the animal cells is measured.

[0051] The specific factors, vitamins, and hormones listed in this specification can exist in various forms, as is known in the art (e.g., various naturally occurring or non-naturally occurring forms), and it will be understood that they can be used as alternatives to each other. It should also be recognized that when this application discloses a vitamin or hormone, embodiments in which any form of such vitamin or hormone having similar biological activity (or a compound that can be modified or metabolized in a cell culture medium or within a cell to provide a biologically active form) is used in the media and / or methods of the present invention are encompassed by the present invention.

[0052] The medium components can be dissolved in a liquid carrier or maintained in a dry form. When dissolved in a liquid carrier at the preferred concentrations shown above (i.e., "1x formulation"), the pH of the medium should be adjusted to about 7.0 to 7.6, such as about 7.1 to 7.5 or about 7.2 to 7.4. The osmolality of the medium can also be adjusted to about 275 to 350 mOsm, such as about 285 to 325 mOsm or about 280 to 310 mOsm. The type of liquid carrier and the method used to dissolve the components in the solution vary and can be determined by one of ordinary skill in the art through routine experimentation. Typically, the medium components can be added in any order.

[0053] Cell culture media are composed of many components, and these components vary from medium to medium. "1X formulation" is intended to refer to any aqueous solution containing some or all of the components found in cell culture media at their effective concentrations. "1X formulation" can refer to, for example, a cell culture medium or any subgroup of the components of that medium. The concentration of a component in a 1X solution is approximately the same as the concentration of that component found in a cell culture formulation used to maintain or culture cells in vitro. By definition, a cell culture medium used for in vitro culture of cells is a 1X formulation. When there are many components, each component in a 1X formulation has a concentration approximately equal to the concentration of those components in the cell culture medium. For example, RPMI-1640 culture medium contains, among other components, 0.2 g / L L-arginine, 0.05 g / L L-asparagine and 0.02 g / L L-aspartic acid. The "1X formulation" of these amino acids contains these components at approximately the same concentration in solution. Thus, when referring to a "1X formulation", it is intended that each component in the solution have the same or approximately the same concentration as that found in the stated cell culture medium. The concentrations of the components in a 1X formulation of a cell culture medium are well known to those skilled in the art. See Methods for preparation of media, Supplements and Substrate For Serum-Free Animal Cell Culture Allen R.Liss, N.Y. (1984). The osmolality and / or pH, however, may differ in the 1X formulation compared to the culture medium, especially when fewer components are included in the 1X formulation.

[0054] "10× formulation" is intended to refer to a solution in which each component is approximately 10 times more concentrated than the same component in cell culture medium. For example, a 10× formulation of RPMI-1640 culture medium may contain, among other components, 2.0 g / L L-arginine, 0.5 g / L L-asparagine, and 0.2 g / L L-aspartic acid (compared to the 1× formulation above). A "10× formulation" may contain many additional components at a concentration approximately 10 times that found in 1× culture medium. As will be immediately apparent, "25× formulation", "50× formulation", "100× formulation", "500× formulation", and "1000× formulation" represent solutions containing components at approximately 25, 50, 100, 500, or 1000 times the concentration, respectively, compared to 1× cell culture medium. Again, the osmolality and pH of the medium formulation and concentrated solution may vary. Preferably, the solution containing the components is more concentrated than the concentration of the same components in the 1× medium formulation. The components can be concentrated 10-fold (10× formulation), 25-fold (25× formulation), 50-fold (50× formulation), or 100-fold (100× formulation). More highly concentrated formulations can be made, provided that the components remain soluble and stable. See, for example, U.S. Patent No. 5,474, which relates to methods for solubilizing high concentrations of culture medium constituents.

[0055] When medium components are prepared as separate concentrated solutions, appropriate (sufficient) amounts of each concentrate are combined with a diluent to produce a 1× medium formulation. Typically, the diluent used is water, although other solutions, including aqueous buffers, aqueous saline, or other aqueous solutions, may be used according to the present invention.

[0056] The culture medium of the present invention is typically sterilized to prevent unwanted contamination. Sterilization can be achieved, for example, by filtration through a low protein-binding filtration membrane with a pore size of about 0.1 - 1.0 μm (e.g., commercially available from Millipore, Bedford, Mass.) after mixing the concentrated components to produce the sterilized culture medium. Alternatively, a concentrated subgroup of the components may be filter sterilized and stored as a sterile solution. These sterilized concentrates can then be mixed with a sterile diluent under sterile conditions to produce a concentrated 1× sterilized culture formulation. Autoclaving or other high-temperature-based methods of sterilization are not preferred because many of the components of the culture medium of the present invention are heat-labile and are irreversibly degraded by temperatures such as those achieved during most heat sterilization methods.

[0057] Many tissue culture media typically contain one or more antibiotics, which are not necessary for cell growth / proliferation itself but are present to inhibit the growth of other harmful microorganisms such as bacteria and / or fungi. Antibiotics are relatively low molecular weight natural chemical substances produced by various species of microorganisms such as bacteria (including species of the genus Bacillus), actinomycetes (including the genus Streptomyces), and fungi, which inhibit or destroy the growth of other microorganisms. Substances with similar structures and modes of action may be chemically synthesized or natural compounds may be modified to produce semi-synthetic antibiotics. These biosynthetic and semi-synthetic derivatives are also effective as antibiotics. The major classes of antibiotics are: (1) β-lactams, including penicillins, cephalosporins, and monobactams; (2) aminoglycosides, such as gentamicin, tobramycin, netilmicin sulfate, and amikacin; (3) tetracyclines; (4) sulfonamides and trimethoprim; (5) fluoroquinolones, such as ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) macrolides, including for example erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, such as polymyxin, chloramphenicol, and lincosamide.Antibiotics achieve their antibacterial effects through several mechanisms of action, which can generally be classified as follows: (1) agents that act on the bacterial cell wall, such as bacitracin, cephalosporins, cycloserine, fosfomycin, penicillins, ristocetin, and vancomycin; (2) agents that affect the cell membrane or exert a washing effect, such as colistin, novobiocin, and polymyxins; (3) agents that affect cellular mechanisms such as protein synthesis through their effects on replication, information transfer, and ribosomes, for example aminoglycosides, tetracyclines, chloramphenicol, clindamycin, cycloheximide, fusidic acid, lincomycin, puromycin, rifampicin, other streptomycins, and macrolide antibiotics such as erythromycin and oleandomycin; (4) agents that affect nucleic acid metabolism, such as fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, gliotoxin, rifamycin; and (5) drugs that affect intermediate metabolism, such as sulfonamides, trimethoprim, and the antituberculosis agents isoniazid and para-aminosalicylic acid. Some agents may have more than one main mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of bacterial cells often occur after the main effect of the antibacterial agent.

[0058] Accordingly, for convenience and other practical reasons, one or more antibiotics or other substances that inhibit the growth / proliferation of harmful bacteria / fungi / viruses may be supplemented as an addition to the subject medium. In other embodiments, however, the subject medium may be without any antibiotics in order to ensure optimal growth of primary cells. When handling cells that grow in a medium without antibiotics, special care should be taken to avoid possible contamination.

[0059] The medium of the present invention can be made from individual components purchased separately from various chemical vendors. Alternatively, certain commercially available media may be conveniently mixed and supplemented with additional components to produce the subject medium. The present invention thus provides a method for making a tissue culture medium, the method comprising supplementing a commercially available cell culture medium or a mixture of two or more such media by adding one or more of the components disclosed herein.

[0060] The present invention provides a tissue culture medium for intestinal organoids containing components sufficient for cell growth. The composition of these media may vary. For example, the concentration of any of the components may independently vary up to 10%, 20%, 30%, 40% or 50% or up to a factor of 2 to 3 times compared to the original concentration. In one embodiment, the concentration of each component varies by 10% or less from the listed value. In one embodiment, the concentration of each component varies by 25% or less from the listed value. Unless otherwise indicated, as used herein, a variation of X% or less means a variation of ±X% with respect to the listed value. For example, if the listed value is 100 ng / ml, a 25% variation can be taken to mean that the value is in the range between 75 ng / ml and 125 ng / ml (i.e., 75 - 125 ng / ml). Unless otherwise indicated, when a range of values is disclosed, the endpoints are included within the range. Further, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, a value expressed as a range is assumed, in various embodiments of the present invention, to include any specific value or sub-range within the stated range down to one tenth of the unit of the lower limit of the range. It should also be understood that when a series of numerical values is recited herein, the present invention includes embodiments relating to any intermediate value or range defined by any two values in the series, and the lowest value may be taken as the minimum value and the largest value may be taken as the maximum value. For any embodiment of the present invention in which the term "about" or "approximately" is placed before a value, the present invention includes embodiments in which the exact value is recited. For any embodiment of the present invention in which the term "about" or "approximately" is not placed before a numerical value, the present invention includes embodiments in which "about" or "approximately" is placed before the value.

[0061] Some of the components may be provided as salts, esters, biologically active metabolites or derivatives, or as precursors that are metabolized, processed or degraded by cells or in the medium to produce some of the biologically active forms of the components disclosed herein. "Biologically active" in this context refers to the ability of a component to exert its desired effect on cells when present in a cell culture medium.

[0062] The medium of the present invention may be in liquid or solid powder form or a combination of both. The liquid form may be a complete medium, which contains all the components sufficient to maintain the growth / proliferation of the target cells. Alternatively, the liquid medium may be stored as separate packages such that each individual package is stored under its appropriate conditions (temperature, humidity, etc.). For example, most components can be pre-dissolved in a single solution and stored under appropriate conditions (e.g., 4°C in the dark and a dry place, etc.) if it is desired that they be present in the medium of the present invention. Other components that may become unstable under the storage conditions for other components, or may react slowly with other components, or are otherwise better kept as separate stocks, may be stored under a different set of conditions (e.g., -20°C or -80°C, etc.). Immediately before or just prior to use, these separately stored components are combined to make up the whole medium. Each separate package may be sold or marketed separately or as various concentrated stocks (e.g., 2×, 5×, 10×, 100×, 1000×, etc.). In some embodiments, the medium of the present invention is sold or marketed together with one or more cell lines (e.g., one or more of the cell lines disclosed herein), and the medium is suitable for culturing the cell line.

[0063] Similarly, the complete medium or individual components, their packages, can be in the form of a dry powder, which, when reconstituted with an aqueous solution (such as water), will result in the desired medium or its concentrated stock (2×, 5× or 10×, etc.).

[0064] Components that can be made into separate stocks shortly before use or can be better maintained as separate stocks include growth factors (such as epidermal growth factor), hormones (such as estrogen, progesterone, testosterone), other unstable enzymes / proteins (such as transferrin, insulin, cholera toxin, etc.), steroids (such as hydrocortisone, cholesterol), vitamins (vitamin A, Bi2, K3), pH indicators (such as phenol red), one or more buffer components (such as sodium bicarbonate, HEPES) and other chemicals (such as glutathione, 17-β-estradiol, O-phosphorylethanolamine, etc.).

[0065] In certain embodiments, at least some or all of the components of the medium are in liquid / aqueous form. In other embodiments, at least some or all of the components of the medium are in solid / powder form.

[0066] The medium of the present invention is suitable for a variety of primary cells derived from various mammals including birds, reptiles, humans and other non-human mammals. The latter further includes non-human primates (such as monkeys, gorillas, etc.), mice, rats, rabbits, cattle as livestock, horses, pigs, sheep, goats, dogs and cats.

[0067] As used herein, "substantially free" refers to a population of desired cells that is at least about 80% pure, preferably 85%, 90%, 95%, 99% or more pure in the total cell population.

[0068] The present invention also provides an in vitro method for identifying an agent that enhances or otherwise affects one or more characteristics of intestinal organoids, including differentiation, apoptosis, sensitivity to chemotherapy / radiotherapy, or aging, the method comprising: (1) contacting a culture of intestinal organoids in the medium of the present invention with a candidate agent to be evaluated for its ability to enhance or otherwise affect one or more characteristics of these organoids, under appropriate conditions for the agent to enter the cells; (2) determining the extent to which the characteristics are enhanced or otherwise affected, for example, in the presence of the candidate agent to be evaluated; and (3) comparing the determined extent with the characteristics of the intestinal organoids in the absence of the candidate agent to be evaluated, under the same conditions, wherein if the characteristics are substantially enhanced or otherwise affected in the presence of the candidate agent to be evaluated as compared to in the absence of the candidate agent, the candidate agent to be evaluated is an agent that enhances or otherwise affects one or more characteristics of the intestinal organoids.

[0069] The present invention also provides, similarly, an in vivo method for identifying an agent that inhibits or otherwise negatively affects one or more characteristics of intestinal organoids.

[0070] In certain embodiments, the agent is an RNAi molecule. In certain embodiments, the agent is a siRNA molecule. In certain embodiments, the agent is a chemical compound.

[0071] In the present invention, "isolated" refers to a material that has been removed from its original environment (e.g., the natural environment if it occurs naturally), and thus has been "artificially" altered from its natural state. For example, an isolated polynucleotide can be part of a vector or composition of matter or can be contained within a cell and still be "isolated" because the vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term "isolated" does not refer to genomic libraries, cDNA libraries, total cellular RNA preparations, mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto a blot), sheared total cellular genomic DNA preparations, or other compositions in which the art has not demonstrated the distinguishing characteristics of the polynucleotides / sequences of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or (partially or substantially) purified DNA molecules in solution. Examples of isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, nucleic acids contained within clones that are members of a library (e.g., a genomic or cDNA library) that have not been isolated from other members of the library (e.g., in the form of a homogeneous solution containing that clone and other members of the library), or within chromosomes removed from cells or cell lysates (e.g., "chromosome spreads" as in a karyotype), or within preparations of randomly sheared genomic DNA or genomic DNA digested with one or more restriction enzymes are not "isolated" in the present invention. As further discussed herein, isolated nucleic acid molecules according to the present invention can be produced naturally, recombinantly, or synthetically.

[0072] In the present invention, the "secreted" protein refers to a protein that can be directed to the ER, secretory vesicles, or extracellular space by a signal sequence and a protein that is not necessarily contained in the signal sequence but is released into the extracellular space. When the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can be caused by many mechanisms including exocytosis and protein cleavage.

[0073] "Polynucleotide" can be composed of single-stranded DNA and double-stranded DNA, DNA which is a mixture of single-stranded regions and double-stranded regions, single-stranded RNA and double-stranded RNA, and RNA which is a mixture of single-stranded regions and double-stranded regions, and hybrid molecules which can be single-stranded, or more typically double-stranded, or a mixture of single-stranded regions and double-stranded regions of DNA and RNA. Furthermore, the polynucleotide can be composed of triple-stranded regions containing RNA or DNA or both RNA and DNA. The polynucleotide can also contain one or more modified bases or a DNA or RNA backbone modified for stability or for other reasons. Examples of "modified" bases include, for example, tritylated bases and abnormal bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0074] The expression "polynucleotide encoding a polypeptide" encompasses a polynucleotide containing only the coding sequence for that polypeptide as well as a polynucleotide containing additional coding sequences and / or non-coding sequences.

[0075] The term "stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution containing 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared salmon sperm DNA, followed by washing of the filter in 0.1×SSC at approximately 50°C. Changes in the stringency of hybridization and signal detection are primarily achieved through manipulation of the formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution containing 6×SSPE (20×SSPE = 3 M NaCl; 0.2 M NaH2PO4; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm blocking DNA, followed by washing in 1×SSPE, 0.1% SDS at 50°C. In addition, to achieve even lower stringency, washes performed after stringent hybridization can be done at higher salt concentrations (e.g., 5×SSC). Variations in the above conditions can be achieved through the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of the specified blocking reagents may require modification of the above hybridization conditions due to compatibility issues.

[0076] The terms "fragment", "derivative", and "analog" when referring to a polypeptide mean a polypeptide that retains either substantially the same biological function or activity as such polypeptide. Examples of analogs include proproteins that can be activated by cleavage of the proprotein moiety to produce an active mature polypeptide.

[0077] The term "gene" means a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).

[0078] A polypeptide can be composed of amino acids linked to each other by peptide bonds or amino acids linked to each other by modified peptide bonds, i.e., peptide isosteres, and can contain amino acids other than the 20 genetically encoded amino acids. A polypeptide can be modified by either natural processes such as post-translational processing or chemical modification techniques well known in the art. Such modifications are well described in basic textbooks and more detailed monographs as well as numerous research papers. Modifications can occur at any location in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It is recognized that the same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Furthermore, a given polypeptide can contain many types of modifications. Polypeptides can be, for example, branched as a result of ubiquitination, and they can be cyclic, with or without branches. Cyclic, branched, and branched cyclic polypeptides can result from natural post-translational processes or can be produced by synthetic methods.Modifications include, but are not limited to, acetylation, acylation, biotinylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, derivatization with known protecting / blocking groups, disulfide bond formation, demethylation, formation of covalent cross-links, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, conjugation to an antibody molecule or other cell ligand, methylation, myristoylation, oxidation, pegylation, proteolytic processing (e.g., cleavage), phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of an amino acid to a protein, e.g., arginylation and ubiquitination (see, e.g., PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B.C. Johnson, Ed., Academic Press, New York, pgs. I-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

[0079] A "biologically active" polypeptide fragment refers to a polypeptide that exhibits an activity, which may or may not be dose-dependent, measured in a specific biological assay and that is similar, but not necessarily identical, to the activity of the original polypeptide, for example, the activity of the mature form. If dose-dependence exists, it need not be identical to that of the polypeptide, but rather is substantially similar in dose-dependence for a given activity compared to the original polypeptide (i.e., the candidate polypeptide exhibits high activity or at least 25-fold less, in some embodiments at least 10-fold less or at least 3-fold less activity than the original polypeptide).

[0080] Species homologs can be isolated and identified by generating suitable probes or primers from the sequences provided herein and screening suitable nucleic acid resources for the desired homologs.

[0081] A "variant" refers to a polynucleotide or polypeptide that differs from the original polynucleotide or polypeptide but retains its essential properties. Generally, a variant is overall closely similar to the original polynucleotide or polypeptide and is identical in many regions.

[0082] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequences of the present invention can conventionally be determined using known computer programs. A preferred method for determining the best overall match between a query sequence (the sequence of the present invention) and a target sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In sequence alignment, both the query sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The result of the global sequence alignment is in terms of percent identity. Preferred parameters used for FASTDB alignment of DNA sequences to calculate percent identity are as follows: matrix = unitary, k-tuple = 4, mismatch penalty = -1, join penalty = -30, randomization group length = 0, cutoff score = 1, gap penalty = -5, gap size penalty = 0.05, window size = 500 or the shorter of the length of the target nucleotide sequence. If the target sequence is shorter than the query sequence due to a 5' or 3' deletion rather than an internal deletion, the result must be corrected manually. This is because the FASTDB program does not consider 5' and 3' truncations of the target sequence when calculating percent identity. For a target sequence truncated at the 5' or 3' end relative to the query sequence, the percent identity is corrected as the percent of all bases of the query sequence by calculating the number of bases of the query sequence at the 5' and 3' of the target sequence that are not matched / aligned. Whether a nucleotide is matched / aligned is determined by the result of the FASTDB sequence alignment. This ratio is then subtracted from the percent identity calculated by the above FASTDB program using the defined parameters to reach the final percent identity score.This corrected score is the one used for the present invention. Only the bases outside the 5' and 3' bases of the target sequence displayed by the FASTDB alignment that are not matched / aligned with the query sequence are calculated for the purpose of manually adjusting the percent identity score. For example, a 90-base target sequence is aligned with a 100-base query sequence to determine the percent identity. A deletion occurs at the 5' end of the target sequence, and thus the FASTDB alignment does not show a match / alignment for the first 10 bases at the 5' end. These 10 damaged bases represent 10% of the sequence (the number of bases at the unmatched 5' and 3' ends / the total number of bases in the query sequence), and thus 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases are completely matched, the final percent identity is 90%. In another example, a 90-base target sequence is compared with a 100-base query sequence. In this case, since the deletion is an internal deletion, there are no bases at either the 5' or 3' of the target sequence that are not matched / aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the 5' and 3' bases of the target sequence that are not matched / aligned with the query sequence are manually corrected.

[0083] The amino acid sequence of the target polypeptide is intended to be identical to the query sequence, except that it may contain up to 5 amino acid changes per 100 amino acids of the query sequence, by a polypeptide having at least, for example, 95% "identical" amino acid sequence to the query amino acid sequence of the present invention. In other words, up to 5% of the amino acid residues in the target sequence can be inserted, deleted, or substituted with another amino acid in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence. These changes to the reference sequence can occur individually or as one or more continuous groups within the reference sequence, anywhere between residues in the reference amino acid sequence, at the amino or carboxy terminal positions of the reference amino acid sequence, or between those terminal positions.

[0084] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to, for example, the amino acid sequence shown in the sequence or the amino acid sequence encoded by the deposited DNA clone, can conventionally be determined using known computer programs. A preferred method for determining the best overall match between a query sequence (the sequence of the present invention) and a target sequence, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp.App.Biosci. (1990) 6:237-245). In sequence alignment, both the query sequence and the target sequence can be either nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is in terms of percent identity. Preferred parameters used for FASTDB amino acid alignment are as follows: matrix = PAM0, k-tuple = 2, mismatch penalty = -1, join penalty = 20, randomization group length = 0, cutoff score = 1, window size = sequence length, gap penalty = -5, gap size penalty = -0.05, window size = the shorter of 500 or the length of the target amino acid sequence. If the target sequence is shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the result must be corrected manually. This is because the FASTDB program does not consider N- and C-terminal truncations of the target sequence when calculating global percent identity. For a target sequence truncated at the N- and C-termini relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are at the N- and C-termini of the target sequence that do not match / align with the corresponding target residues, as a percentage of the total bases of the query sequence. Whether a residue is matched / aligned is determined by the result of the FASTDB sequence alignment. This ratio is then subtracted from the percent identity calculated by the above FASTDB program using the defined parameters to reach the final percent identity score.This final percent identity score is the one used for the present invention. Only the residues at the N and C termini of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. It is only the query residue positions outside the outermost N and C terminal residues of the subject sequence. Only the residue positions outside the N and C termini of the subject sequence that are displayed in the FASTDB alignment and that are not matched / aligned with the query sequence are corrected manually. No other manual corrections should be made for the present invention.

[0085] Naturally occurring protein variants are called "allelic variants" and refer to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Genes 11, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide level and / or the polypeptide level. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0086] Using known methods of protein engineering and recombinant DNA technology, variants may be produced to improve or alter polypeptide characteristics. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantially losing biological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993) reported that the mutant KGF protein had heparin binding activity even after deleting 3, 8 or 27 amino-terminal amino acid residues. Similarly, interferon gamma showed up to 10-fold higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)). Furthermore, there is ample evidence demonstrating that variants often continue to maintain biological activity similar to that of the naturally occurring protein. For example, Gayle and co-workers (J. Biol. Chem 268:22105-22111 (1993)) performed extensive mutagenesis analysis on human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants with an average of 2.5 amino acids altered per mutant over the full length of the molecule. A large number of mutations were examined at every possible amino acid position. These researchers discovered that “most molecules can be altered without significantly affecting either [binding activity or biological activity].” (See the abstract.) In fact, out of the over 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins with activity significantly different from the wild type. Additionally, even if deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion mutant to induce and / or bind to an antibody that recognizes the secreted form will probably be retained if less than half of the residues of the secreted form are removed from the N-terminus or C-terminus.Whether a particular polypeptide lacking the N- or C-terminal residue of a protein continues to possess such immunogenic activity can be readily determined by routine methods described herein or otherwise known in the art.

[0087] By "biological sample" is meant an individual, a body fluid, a cell line, a tissue culture, or any biological sample obtained from other sources containing the polypeptide or mRNA of the present invention. As shown, biological samples include body fluids (e.g., semen, lymph, serum, plasma, urine, synovial fluid, and cerebrospinal fluid) and other tissue sources known to express the polypeptide of the present invention. Methods for obtaining tissue biopsy materials and body fluids from mammals are well known in the art. When the biological sample is to contain mRNA, tissue biopsy material is a preferred source.

[0088] 「Matrigel」 is the trade name of a gel-like protein mixture secreted by mouse tumor cells and sold by BD Biosciences. This mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. In a typical experimental procedure, a small volume of chilled (4°C) Matrigel is dispensed onto plastic tissue culture ware. When incubated at 37°C (body temperature), the Matrigel proteins self-assemble to produce a thin film that covers the surface of the ware. Cells cultured on Matrigel exhibit complex cell behaviors that would not be observable under experimental conditions on plastic. For example, endothelial cells form a spider web-like network that penetrates the surface coated with Matrigel, but not on a plastic surface. Such a network structure strongly resembles the capillary system of microvessels that line living tissues with blood. Therefore, the process by which endothelial cells construct such a network structure is of great interest to biological researchers, and Matrigel enables researchers to observe this. In some cases, researchers use a larger volume of Matrigel to produce a thick three-dimensional gel. The usefulness of the thick gel is that cells are induced to move from the surface of the gel into the interior by the gel. This migratory behavior is being studied by researchers as a model of tumor cell metastasis. Pharmaceutical scientists use Matrigel to screen drug molecules. A typical experiment consists of adding a test molecule to Matrigel and observing cell behavior. Test molecules that promote endothelial cell network formation are candidates for tissue regeneration therapy, while test molecules that inhibit endothelial cell network formation are candidates for anti-cancer therapy. Similarly, test molecules that inhibit tumor cell migration may also have the potential as anti-cancer drugs. Matrigel's ability to stimulate complex cell behaviors is the result of its heterogeneous composition. The main components of Matrigel are structural proteins such as laminin and collagen, which present adhesive peptide sequences that cultured cells would encounter in their natural environment. Growth factors that promote the differentiation and proliferation of many cell types are also present.Matrigel contains a small amount of a number of other proteins and its exact composition is not known. Matrigel is also used as an attachment substrate in embryonic stem cell culture. When growing embryonic stem cells in the absence of feeder cells, extracellular matrix components such as Matrigel are necessary to maintain pluripotency and the undifferentiated state (self-renewal).

[0089] Neuregulin 1, also known as NRG1, ARIA, GGF, GGF2, HGL, HRG, HRG1, HRGA, MST131, MSTP131, NDF, NRG1-IT2 or SMDF, is a member of the epidermal growth factor family encoded by the NRG1 gene in humans. NRG1 is one of four proteins in the neuregulin family that act on receptors of the EGFR family. Neuregulin 1 produces a number of isoforms by alternative splicing, by which neuregulin 1 can perform a wide variety of functions. A suitable NRG1 is the NRG1 sold by R&D Systems (Cat# 5898-NR-050; human neuregulin-1 / NRG1 protein derived from NS0, Ser20-Lys241, with a C-terminal 6-His tag).

[0090] All-trans retinoic acid, also known as atRA, NSC122758, retinoic acid, trans-retinoic acid, tretinoin and vitamin A acid, is a derivative of vitamin A that functions as a ligand for the retinoic acid receptor (RAR, IC 50 =14 nM). RAR heterodimerizes with the retinoid X receptor (RXR), binds to the retinoic acid response element (RARE) in DNA, acts as a transcription factor and changes gene expression.

[0091] RSPO1, also known as CRISTIN3 and R-spondin-1, is a secreted protein encoded in humans by the Rspo1 gene, which is found on chromosome 1. In humans, R-spondin-1 interacts with WNT4 during the process of female development. Loss of function can cause sex reversal from female to male. Additionally, R-spondin-1 promotes canonical WNT / β-catenin signaling.

[0092] Noggin, also known as NOG, Nog, SYM1, SYNS1, and SYNS1A, is a protein involved in the development of many body tissues, including nervous tissue, muscle, and bone. In humans, noggin is encoded by the NOG gene. The amino acid sequence of human noggin is highly homologous to that of rat, mouse, and Xenopus laevis. Noggin is an inhibitor of several bone morphogenetic proteins (BMPs): Noggin inhibits at least BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14.

[0093] Insulin-like growth factor 1 (IGF-1), also called somatomedin C, IGF-I, IGF1A, IGFI, and MGF, is a hormone with a molecular structure similar to insulin that plays an important role in childhood growth and has anabolic effects in adults. IGF-1 is a protein encoded in humans by the IGF1 gene. IGF-1 consists of a single chain of 70 amino acids with three intramolecular disulfide bridges. IGF-1 has a molecular weight of 7,649 daltons. IGF-1 is produced mainly by the liver. Production is stimulated by growth hormone (GH). Most IGF-1 binds to one of six binding proteins (IGF-BP). IGFBP-1 is regulated by insulin.

[0094] FGF2, also known as BFGF, FGF-2, FGFB, HBGF-2, fibroblast growth factor 2, basic fibroblast growth factor (bFGF), and FGF-β, is a growth factor and a signaling protein encoded by the FGF2 gene. FGF2 binds to specific fibroblast growth factor receptor (FGFR) proteins, a family of molecules that are closely related to each other, and exerts its effects through this binding.

[0095] Gastrin, also known as GAST and GAS, is a peptide hormone that promotes the secretion of gastric acid (HCl) by the parietal cells of the stomach and aids in gastric motility. Gastrin is released by G cells in the pyloric antrum of the stomach, duodenum, and pancreas. Gastrin binds to the cholecystokinin B receptor, stimulates the release of histamine in enterochromaffin-like cells, and gastrin induces the insertion of the K+ / H+ ATPase pump into the apical membrane of parietal cells (which in turn increases the release of H+ into the gastric lumen). Its release is stimulated by peptides in the gastric lumen.

[0096] The WNT gene family consists of structurally related genes that encode secreted signaling proteins. These proteins have been implicated in several developmental processes, including tumor formation and the regulation of cell fate and pattern formation during embryogenesis. This gene is a member of the WNT gene family. This gene is highly conserved during evolution, and the protein encoded by this gene is known to be 98% identical to the mouse Wnt1 protein at the amino acid level.

[0097] Miao et al. (Volume 27, Issue 5, 5 November 2020, Pages 840 - 851.e6) devised and designed Wnt NGS. Wnt NGS is a water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnt that heterodimerizes with Fzd and Lrp6. NGS Wnt maintains long-term expansion of various types of organoids, including kidney, colon, hepatocytes, ovary, and breast. NGS Wnt is superior to Wnt3a (another potential option for the medium of the present invention) conditioned medium in organoid expansion and single-cell organoid growth.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described below. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Additionally, it should be noted that despite the fact that the foregoing description focuses on medical uses, the methods and agents of the present invention are also suitable for any non-medical use.

Examples

[0099] Example of a protocol for the preparation of various media for the culture of intestinal organoids: Small intestine medium composition 1× formulation First culture medium for the first, for example, 4-day culture 0.5 nM Wnt-NGS 1 μg / ml recombinant R-Spondin-1 100 ng / ml recombinant Noggin 1× B27™ supplement 1.25 mM NAC 50 ng / ml hEGF 1 ng / ml hNRG1 0.5 μM A83-01 100 ng / ml hIGF1 50 ng / ml hFGF2 25 nM Gastrin 10 μM ROCK inhibitor (Y-27632) In DMEM / F12 + 15 mM HEPES, 1×GlutaMax (1:100 #35050-038) and 100 μg / ml PenStrep (#15140-122) For example, the second medium after 4 days of culture 0.5 μg / ml recombinant R-Spondin1 100 ng / ml recombinant Noggin 1×B27™ Supplement 1.25 mM NAC 5 ng / ml hEGF 10 ng / ml hNRG1 100 ng / ml hIGF1 50 ng / ml hFGF2 25 nM Gastrin 2.5 μM atRA (all-trans retinoic acid) In DMEM / F12 + 15 mM HEPES, 1×GlutaMax (1:100 #35050-038) and 100 μg / ml PenStrep (#15140-122) Colon medium composition 1× formulation The first culture medium for the first, for example, 4-day culture 0.5 nM Wnt (NGS) 1 μg / ml recombinant R-Spondin1 100 ng / ml recombinant Noggin 1×B27™ Supplement 1.25 mM 50 ng / ml hEGF 1 ng / ml hNRG1 0.5 μM A83-01 100 ng / ml hIGF1 50 ng / ml hFGF2 25 nM Gastrin 10 μM ROCK inhibitor (Y-27632) In DMEM / F12 + 15 mM HEPES, 1× GlutaMax (1:100 #35050 - 038), and 100 μg / ml PenStrep (#15140 - 122) For example, the second medium after 4 days of culture 0.1 nM Wnt (NGS) 0.5 μg / ml recombinant R - Spondin1 100 ng / ml recombinant Noggin 1× B27™ Supplement 1.25 mM NAC 5 ng / ml hEGF 10 ng / ml hNRG1 100 ng / ml hIGF1 50 ng / ml hFGF2 25 nM gastrin 25 nM 0.1 μM atRA In DMEM / F12 + 15 mM HEPES, 1× GlutaMax (1:100 #35050 - 038), and 100 μg / ml PenStrep (#15140 - 122)

[0100] Materials and Methods Human organoid lines All patients provided consent for additional biopsy samples to be taken for research purposes while undergoing surveillance colonoscopy. Human healthy colon (W18 - 50157; HUB - 02 - A2 - 040) and small intestine (D1n (HUB - 04 - A2 - 001)) organoids were provided by HUB Organoids, collaborators of the inventors located in Utrecht, Netherlands.

[0101] Mouse organoid lines All animal experiments were approved by the Basel Cantonal Veterinary Authorities and were conducted in accordance with the Guide for Care and Use of Laboratory Animals. Male and female non-inbred mice between 7 and 15 weeks of age were used in all experiments. Mouse strains used: C57BL / 6 wild type (Charles River Laboratories) and Lgr5::DTR-EGFP (Genentech, de Sauvage laboratory).

[0102] Human organoid culture Organoids grown from primary tissue were generated from crypts isolated from human biopsy material collected by colonoscopy, as previously described 1。The organoids were maintained in FMI-START medium supplemented with 10 mM Y27632 (Rock inhibitor, Stem Cell technologies) for 4 days after trypsin treatment. The medium was supplemented with 15 mM HEPES (STEMCELL Technologies)-containing advanced DMEM / F-12, 100 μg / ml penicillin-streptomycin, 1× Glutamax (Thermo Fisher Scientific), 1× B27 (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine (Sigma), 1 μg / ml recombinant human R-spondin-1 (gift from Novartis), 100 ng / ml Noggin (PeproTech), 0.5 nM WNT-NGS (U-Protein Express), 0.5 μM A83-01 (Tocris), 100 ng / ml human IGF1 (R&D Systems), 50 ng / ml human FGF2 (R&D systems), 25 nM gastrin (Sigma), 1 ng / ml human NRG1 (R&D Systems), and 50 ng / ml human or mouse EGF (Thermo Scientific).After 4 days, the organoids were overlaid with FMI-BALANCE colon (advanced DMEM / F-12 containing 15 mM HEPES (STEMCELL Technologies), 100 μg / ml penicillin-streptomycin, 1× Glutamax (Thermo Fisher Scientific), 1× B27 (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine (Sigma), 0.5 μg / ml recombinant human R-spondin-1 (gift from Novartis), 100 ng / ml Noggin (PeproTech), 0.1 nM WNT-NGS (U-Protein Express), 100 ng / ml human IGF1 (R&D Systems), 50 ng / ml human FGF2 (R&D systems), 25 nM gastrin (Sigma), 10 ng / ml human NRG1 (R&D Systems), 10 μM (Figs. 1A–G) / 0.1 μM (others) atRA (Sigma-Aldrich), and 5 ng / ml human or mouse EGF (Thermo Scientific)) or FMI-BALANCE small intestine (advanced DMEM / F-12 containing 15 mM HEPES (STEMCELL Technologies), 100 μg / ml penicillin-streptomycin, 1× Glutamax (Thermo Fisher Scientific), 1× B27 (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine (Sigma), 0.5 μg / ml recombinant human R-spondin-1 (gift from Novartis), 100 ng / ml Noggin (PeproTech), 100 ng / ml human IGF1 (R&D Systems), 50 ng / ml human FGF2 (R&D systems), 25 nM gastrin (Sigma), 10 ng / ml human NRG1 (R&D Systems), 10 μM (Figs. 1A–G) / 1 μM (Fig. 1I) / 2.5 μM (others) atRA (Sigma-Aldrich), and 5 ng / ml human or mouse EGF (Thermo Scientific)) according to their origin. The medium was replaced on day 7 and again on day 10 because the maturation time for small intestine-derived organoids was long.

[0103] Mouse Organoid Culture Mouse organoids were generated from crypts isolated from the mouse small intestine as previously described 2 The organoids were maintained in FMI-START medium supplemented with 10 μM Y27632 (ROCK inhibitor, STEMCELL Technologies) for 3 days after trypsin treatment. After 3 days, the organoids were maintained in FMI-BALANCE colon or FMI-BALANCE small intestine, and the medium was replaced once again on day 5.

[0104] Image-Based Screening Assay Mouse small intestine and colon organoids were collected 5 - 7 days after passage and digested with (0.05%) trypsin-EDTA (Gibco) at 37 °C for 5 minutes. Human small intestine and colon organoids were collected 10 - 13 days after passage and digested with (0.05%) trypsin-EDTA (Gibco) at 37 °C for 5 - 10 minutes. The dissociated cells were passed through a cell strainer with a 40-μm pore size. For the experiments shown, live single cells were sorted by FACS (Becton Dickinson FACSAria or SONY MA900 cell sorter). Forward scatter and side scatter properties were used to remove cell doublets and dead cells. Dead cells were also filtered out using DRAQ7 stain (1.5 μM, Thermo Fisher Scientific). Mouse organoid lines were derived from C57BL / 6 wild-type mice unless otherwise indicated.

[0105] Imaging-based experiments were performed in 384-well plates (CellCarrier-384, PerkinElmer, catalog number 6007550) or 96-well plates (μClear, Greiner, catalog number 655090).

[0106] For experiments in 384-well plates, cells were resuspended in FMI-START containing 10 μM Y27632 (ROCK inhibitor, STEMCELL Technologies) to reach a density of approximately 6 - 50 cells / μl depending on the experiment and line used. Next, the wells of the 384-well plate were covered with 10 μl / well of ice-cold 2:1 Matrigel (Corning):FMI-START mixture using an Assist Plus pipetting robot (Integra) under constant cooling to cover the entire bottom of the well. The plate was centrifuged to flatten the layer of Matrigel mixture and then incubated at RT for 10 minutes to slightly solidify. Thereafter, 40 μl of the cell mixture containing 10 μM Y27632 (ROCK inhibitor, STEMCELL Technologies) was carefully overlaid onto the prepared wells. The cells were carefully centrifuged using a low-speed (50 - 70 rcf for 5 seconds) centrifuge and placed into the Matrigel. Finally, the plate was placed in an incubator (37 °C, 5% CO2) for culture.

[0107] For experiments in 96-well plates, cells were resuspended in ice-cold 2:1 Matrigel (Corning):FMI-START mixture containing 10 μM Y27632 (ROCK inhibitor, STEMCELL Technologies) to reach a density of approximately 6 - 50 cells / μl (depending on the experiment and line used) and seeded as 5 μl droplets in the center of the well. The plate was incubated at 37 °C for 20 minutes to solidify the Matrigel. Thereafter, 100 μl of FMI-START supplemented with 10 μM Y27632 (ROCK inhibitor, STEMCELL Technologies) was added for culture in an incubator (37 °C, 5% CO2).

[0108] To test various medium compositions, as previously described 1,3* it was decided to simultaneously change the medium on days 4 and 7 with respect to the FMI-medium composition. For the medium composition of Fujii et al. 3 in the case of Beumer et al.1 and Stem Cell Technologies * No differentiation medium was required compared to the composition of 1 and Stem Cell Technologies. Therefore, the inventors cultured the organoids in the same medium throughout with the composition of Fujii et al. and refreshed it on day 4 and day 7.

[0109] During the experiment, the medium was changed to FMI-BALANCE on day 4 and refreshed once on day 7. The small intestine organoids had the medium refreshed additionally on day 10 to grow until maturation on day 13.

[0110] For mouse organoids, the cells were mixed with Matrigel (Corning) at a 1:1 medium to Matrigel ratio. 5 μl droplets with various densities were seeded into each well of a 96-well plate (μClear, Greiner, catalog number 655090). After 20 minutes of coagulation at 37°C, 100 μl of medium was overlaid.

[0111] Fixed sample preparation and imaging To enable imaging of all organoids within a similar z-range, each well plate was centrifuged at 1000 rcf for 10 minutes in a centrifuge pre-cooled to 10°C before fixation. The organoids were fixed in 4% PFA (Electron Microscopy Sciences) in PBS at room temperature for the indicated time points.

[0112] For image-based screening assays, organoids were permeabilized and blocked with 0.5% Triton X-100 (Sigma-Aldrich), 3% donkey serum (Sigma-Aldrich), 100 mM NH4Cl in PBS for 3 hours (human) or 1 hour (mouse) at RT. Primary and secondary antibodies were diluted in antibody buffer (0.1% Triton X-100, 3% donkey serum in PBS) and added as described in Table 2. For nuclear staining, 0.2 μg / ml DAPI (4’,6-diamidino-2-phenylindole, Invitrogen) was added to the secondary antibody staining mix. Fixation, blocking / permeabilization, and wash steps were performed using an EL406 Combination Washer Dispenser (BioTek Instruments).

[0113] High-throughput imaging was performed on one of two automated spinning disk microscopes (Molecular Devices ImageXpress Micro Confocal or Yokogawa CellVoyager 7000S). The Molecular Devices ImageXpress Micro Confocal combined with a Nikon 20x Plan Apo 0.75 NA objective lens was used for imaging. Z-planes covering the size of the organoids were acquired for each site. A 5-μm z-step was used in all experiments unless otherwise indicated.

[0114] Instead, the CellVoyager 7000S combined with an Olympus 20x UPLSAPO 20x 0.75 NA objective lens was also used for imaging. Here, a search first imaging approach was used (the Search First module of the Wako Software Suite). For this purpose, in each well, one field of view was acquired at 4× resolution to cover the entire well area. This whole image was then used to segment the organoids during programming by a custom ImageJ macro that outputs coordinates for the position of the organoids. These coordinates were used to generate a map of the locations for high-resolution re-imaging (20×, NA = 0.75). For each site, a z-plane covering the size of the organoid was acquired. A 5-μm z-step was used in all experiments unless otherwise indicated.

[0115] Image analysis The individually acquired fields of view were first combined to generate a whole-well image for each well. Next, maximum intensity projection images were generated for all whole-well images and channels. The DAPI channel was subsequently used to segment individual organoids using the adapted RDCNet Network 4 After segmentation, the area of a single organoid was extracted from the whole-well image based on the segmentation. All cropped regions were subjected to a filtering step to remove mis-segmented organoids and debris based on size, DAPI brightness, or aspect ratio. In addition, organoids cut by the imaging boundary were excluded from further analysis. The features of the remaining organoids were mainly processed computationally using the regionprops function of the python package scikit-image. Furthermore, outliers were removed by a quantile filter with 0.01 and 0.99 as the lower and upper limits, respectively.

[0116] For the experiments shown, pseudotime was calculated using the shape-feature space. Briefly, a subset of shape-features was carefully selected to model the temporal development of the organoids. This subset was then used to calculate the pseudotime of all organoids using the palantir python package 5 To do this, organoids at the earliest and latest time points were randomly selected, and trajectories between them were determined using 500 random intermediate points in between. This process was repeated 5 times, and the average pseudotime was calculated for each organoid.

[0117] Single-cell RNA sequencing (scRNAseq) and raw data processing Single cells were isolated from the organoids at the indicated time point, passed through a cell strainer with a pore size of 40 μm, and used for FACS sorting to remove debris and dead cells. The cell suspension was loaded onto the 10x Genomics Chromium Single Cell instrument to generate single cell GEMs. The single cell RNA-Seq library was prepared using the GemCode Single Cell 3’ Gel Bead and Library Kit according to CG00052_SingleCell3’ReagentKitv2UserGuide_RevB. GEM RT was performed in a Bio-Rad PTC-200 Thermal Cycler with a semi-skirted 96-well plate (Eppendorf P / N 0030 128.605): 53 °C for 45 minutes, 85 °C for 5 minutes; hold at 4 °C. After RT, the GEMs were disrupted and the single-stranded cDNA was cleaned up with DynaBeads® MyOneTM Silane Beads (Life Technologies P / N 37002D). The cDNA was amplified using a Bio-Rad PTC-200 Thermal cycler with 0.2 ml 8-strip nonFlex PCR tubes with flat caps (STARLAB P / N I1402-3700): 98 °C for 3 minutes; 12 × cycles: 98 °C for 15 seconds, 67 °C for 20 seconds, and 72 °C for 1 minute; 72 °C for 1 minute; hold at 4 °C. The amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6X SPRI).After these steps, an indexed sequencing library was constructed using the reagents of Chromium Single Cell 3’ library kit V2 (10x Genomics P / N 120237): 1) fragmentation, end repair and A-tailing; 2) after fragmentation, end repair and A-tailing, double-sided size selection with SPRIselect 10 Reagent Kit (0.6X SPRI and 0.8X SPRI); 3) adapter ligation; 4) after ligation, cleanup with SPRIselect (0.8X SPRI); 5) sample index PCR using Chromium Multiplex kit (10x Genomics P / N -120262); 6) after sample indexing, double-sided size selection with SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI). The barcode sequencing library was quantified using Qubit 2.0 with Qubit TM dsDNA HS Assay Kit (Invitrogen P / N Q32854), and the quality of the library was examined on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent P / N 5067-4626). The sequencing library was loaded at 10 pM onto an Illumina HiSeq2500 using a 2×50 paired-end kit with the following read lengths: 26 cycles Read1, 8 cycles i7 index, and 98 cycles Read2. The CellRanger suite (1.3.0) was used to generate a pooled gene expression matrix from the BCL files generated by the sequencer based on the mm10 Cell Ranger human genome annotation file.

[0118] scRNA sequencing analysis To exclude low-quality cells, the inventors removed cells with fewer than 4,500 reads (4,000 for the small intestine dataset) or cells in which more than 26% of the transcripts were derived from mitochondrial genes. To exclude low-quality features, the inventors removed transcripts detected in fewer than 27 cells. After removing unwanted cells and features from the dataset, the inventors corrected the data by total expression, multiplied it by the mean of the library size distribution, and log-transformed the results. The inventors further excluded types of features in the dataset that did not sufficiently contribute to the total UMI count (including non-coding RNAs, pseudogenes, ribosomal proteins, snoRNAs, and snRNAs). After preprocessing, when the inventors' dataset was counted, there were 18,469 transcripts in 53,640 cells sampled at 9 time points for colon organoids and 17,195 transcripts in 26,453 cells sampled at 5 time points for small intestine organoids.

[0119] The inventors used the modelGeneVar and getTopHVGs functions of the scran package in Bioconductor, blocked the time points, and identified variable genes using a threshold of 0.05 for the false discovery rate. The inventors performed density equalizing principal component analysis using sketchR, an R implementation of geometric sketching, proposed by Hie et al. 2019. 6

[0120] To visualize the data, the inventors further reduced the dimensionality of the dataset and projected the cells into 2D space using the t-distributed stochastic neighbor embedding (t-SNE) based on the first 50 principal components. The inventors then used the Seurat R package to perform graph-based clustering. To visualize the expression of known cell type markers in the clusters, the inventors used the DotPlot function of the Seurat package. The inventors assigned clusters to known cell types based on relative gene expression. *https: / / www.stemcell.com / products / intesticult-organoid-differentiation-medium-human.html#section-product-documents

[0121] References 1. Beumer, J. et al. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells. Cell 181, 1291 - 1306.e19 (2020). 2. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262 - 265 (2009). 3. Fujii, M. et al. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell 23, 787 - 793.e6 (2018). 4. Ortiz, R., De Medeiros, G., Peters, A.H.F.M., Liberali, P. & Rempfler, M. RDCNet: Instance segmentation with a minimalist recurrent residual network. 5. Setty, M. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat. Biotechnol. 37, 451 - 460 (2019). 6. Hie, B., Cho, H., DeMeo, B., Bryson, B. & Berger, B. Geometric Sketching Compactly Summarizes the Single-Cell Transcriptomic Landscape. Cell Syst. 8, 483-493.e7 (2019).

[0122] [Table 1]

[0123] [Table 2]

[0124] [Table 3]

Claims

1. A culture medium for culturing intestinal organoids, characterized by containing neuregulin 1 (NRG1) and all-trans retinoic acid (atRA).

2. The culture medium according to claim 1, wherein the intestinal organoids are small intestine organoids, colon organoids, colorectal cancer organoids, Crohn's disease organoids or ulcerative colitis organoids.

3. The culture medium according to claim 1 or 2, wherein the concentration of NRG1 is between 1 ng / ml and 100 ng / ml, and the concentration of atRA is between 10 nM and 10 μM.

4. The culture medium according to any one of claims 1 to 3, characterized by the absence of animal serum.

5. The culture medium according to any one of claims 1 to 4, further comprising recombinant R-Spondin, recombinant Noggin, NRG1, IGF1, FGF2 and gastrin.

6. The culture medium according to any one of claims 1 to 5, further comprising Wnt NGS.

7. A composition comprising the medium according to any one of claims 1 to 6 and an extracellular matrix or a 3D matrix that mimics the extracellular matrix by its interaction with cell membrane proteins.

8. The composition according to claim 7, wherein the 3D matrix extracellular matrix is a synthetic hydrogel or Matrigel (trademark).

9. Use of the medium or composition according to any one of claims 1 to 8 for culturing intestinal organoids.

10. A method for generating intestinal organoids, comprising the steps of providing a suspension of single cells or fragments of intestinal tissue, incubating the suspension of single cells or fragments of intestinal tissue with a first medium for at least one day, and then replacing the first medium with the medium according to claims 1 to 6.

11. The method for generating intestinal organoids according to claim 10, wherein the single cells or fragments of intestinal tissue are obtained from small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue or embryonic endoderm cells.

12. The method according to claim 10 or 11, wherein the first medium comprises an inhibitor of Rho-associated kinase (ROCK), such as the ROCK inhibitor Y27632, and an inhibitor of the activin / Nodal / TGF-β pathway, such as the inhibitor A83-01.

13. The method according to any one of claims 10 to 12, wherein the cells are incubated in the first medium for at least 3 days.

14. An intestinal organoid obtained by using the method according to any one of claims 10 to 13.

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