Culture medium suitable for differentiation and culture of intestinal organs

Abstract

The present application relates to a culture medium for culturing intestinal organoids, characterized in that the culture medium comprises neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). The invention also relates to a method for culturing intestinal organs by using the culture medium.

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 intestine is the organ with the largest contact area with the external environment in the human body, and has indispensable functions for maintaining life such as digestion and absorption. Most of the intestinal functions are undertaken by the intestinal epithelium covering its inner layer. The intestinal epithelium consists of two compartments: villi composed of three differentiated cells (mucus-producing cells, absorptive epithelial cells and endocrine cells), and crypts composed mainly of undifferentiated proliferative cells. In the small intestinal crypts, Paneth cells that produce antimicrobial peptides are present at the bottom of the crypts. Recently, molecular genetic cell lineage analysis revealed that Lgr5-positive cells sandwiched between Paneth cells (also called "crypt base columnar (CBC) cells") are intestinal epithelial stem cells. Lgr5-positive intestinal epithelial stem cells produce progenitor cells called transit amplification cells, but these progenitor cells do not have permanent self-renewal ability, and the differentiation potential is also limited to 1 to 3 lineages. Transit amplification cells differentiate 2 to 4 times in the crypts and finally differentiate in the villi. These differentiated cells fall off at the top of the villi and die by apoptosis. The intestinal epithelium is a rapidly metabolic tissue that migrates from crypt stem cells to the villus tip within 4 to 5 days. Unlike other differentiated cells, Paneth cells migrate to the crypt bottom during differentiation and have a long lifespan of up to 2 months.

[0003] From the results of several genetically modified mice, it is known that the self-renewal mechanism of intestinal epithelial stem cells is controlled by Wnt signals and bone morphogenetic protein (BMP) signals. Mice with intestinal epithelial-specific knockout of the adenomatous polyposis coli gene (APC), a suppressor gene molecule of Wnt signals, exhibited intestinal epithelial cell hyperproliferation and adenoma formation. In addition, the formation of ectopic crypts was observed in mice that overexpressed Noggin (an inhibitory protein of BMP) in intestinal epithelial cells, and thus indicated that BMP signals had an inhibitory effect on intestinal epithelial stem cells.

[0004] In addition, the long-term culture of intestinal epithelial cells has been unable to be achieved for a long time. Since it is not clear that the necessary growth factors for maintaining the growth of intestinal epithelial stem cells are necessary, it is believed that it cannot be achieved. In recent years, the long-term maintenance of intestinal epithelial stem cells has been successfully achieved in the following ways: intestinal epithelial stem cells are adhered to the extracellular matrix, and cultured in the presence of a cell culture medium containing a basal culture medium for animal or human cells (wherein BMP inhibitors, mitogenic growth factors and Wnt agonists have been added). Another tool for studying the intestine is to study intestinal organoids. Organoids are tiny, self-organized three-dimensional tissue cultures derived from stem cells. Such cultures can be prepared to replicate most of the complexity of an organ, or to express the selected aspects of an organ, such as only producing certain types of cells. The formation of organoids usually requires culturing stem cells or progenitor cells in a culture medium specific to an organ / tissue. Summary of the invention

[0005] Through their work, 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.

[0006] Thus, the claims cover a culture medium for culturing intestinal organoids, characterized in that the culture medium comprises neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). This culture medium of the invention is suitable for culturing small intestinal organoids, colon organoids, colorectal cancer organoids, Crohn's disease organoids or ulcerative colitis organoids.

[0007] The concentration of NRG1 in the culture medium may be between 1 ng / ml and 100 ng / ml. The concentration of atRA in the culture medium may be between 10 nM and 10 μM. In some embodiments, the culture medium of the present invention does not contain animal serum. In some embodiments, the culture medium of the present invention further comprises any one of R-Spondin, recombinant Noggin, NRG1, IGF1, FGF2 and gastrin. All of these factors (including NRG1 and atRA) may be human or mouse, as well as recombinant or purified natural proteins. The culture medium of the present invention may also comprise Wnt NGS (Next Generation Surrogate). The claims also cover a composition comprising the culture medium of the present invention and an extracellular matrix or a 3D matrix that simulates the extracellular matrix through the interaction of the extracellular matrix with cell membrane proteins. Such a 3D matrix extracellular matrix may be a synthetic hydrogel or Matrigel TMAlternatively, among others, UltriMatrix (Culturex Ultimatrix RGF Basement Membrane Extract) and BME (Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear), as well as collagen-1 or fibronectin, may be used. In some embodiments, the extracellular matrix or 3D matrix is ​​washed off after an initial period of time.

[0008] The claims further encompass the use of a culture medium according to the invention or a composition according to the invention for culturing intestinal organoids.

[0009] The claims further encompass a method of generating intestinal organoids, the method 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 culture medium for at least one day, and then replacing the first culture medium with a culture medium of the present invention as described above. In some embodiments, the first culture medium may contain atRA at a concentration of 10 nM. The single cells or fragments of intestinal tissue used in the methods of the present invention may be cells or tissue fragments obtained from the small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue, or may be definitive endoderm cells.

[0010] In the method of the present invention, the first culture medium may contain an inhibitor of Rho-associated kinase (ROCK), such as the ROCK inhibitor Y27632. In the method of the present invention, the first culture medium may contain an inhibitor of the activin / NODAL / TGF-β pathway, such as the inhibitor A 83-01.

[0011] In some embodiments of the methods of the invention, the cells can be incubated in the first culture medium for at least three days.

[0012] The first and second culture media of the method of the present invention can be adapted to the properties of the single cells or fragments of intestinal tissue used in the method of the present invention. For example, for small intestinal cells and / or tissue fragments, the appropriate concentration of NRG1 in the first culture medium is 1 ng / ml and does not contain atRA, while the concentrations of NRG1 and atRA in the second culture medium (i.e., the culture medium of the present invention) are 10 ng / ml and 2.5 μM, respectively. For cells and tissue fragments from the colon, the appropriate concentration of NRG1 in the first culture medium is 1 ng / ml and does not contain atRA, while the concentrations of NRG1 and atRA in the second culture medium are 10 ng / ml and 10 nM, respectively.

[0013] The claims also cover intestinal organoids obtained using the methods of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings illustrate embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0015] Figure 1 Image-based media screening for human large and small intestinal organoids from single cells to steady-state mature organoids. A) Schematic representation of the workflow, where 100 single cells / well are initially seeded from trypsinized mature colon organoids. For the initial culture medium, 2 different EGF / NRG1 combinations were tested. On day 4, the culture medium was changed to one of 16 different media with different concentrations of EGF, hNRG1, WNT-NGS, for a total of 32 combinations. Organoids were fixed on day 10 and shape features were extracted from 5848 organoids segmented based on maximum intensity projections of the DAPI channel (RDCNET ref). B) Force-directed layout (FDL, eccentricity: left and log) of 5848 human colon organoids calculated based on morphological organoid features. 10 (Area): right). C) PhenoGraph clustering represented by FDL, with subpopulations highlighted (clusters 0 to 15). Representative organoids of the three clusters are illustrated below (cluster 0: blue, cluster 7: orange-red, cluster 4: dark green). D) Heat map quantifying the performance of all 32 combinations of culture media tested in human colon organoids. Scores for important parameters used to evaluate culture media performance are shown. The scores are normalized according to the relative performance of the culture media (0: worst performer, 1: best performer). Detailed culture media composition is shown in Table 1. Condition 2.3.3 (green squares) is the selected condition for future experiments. E) Force-directed layout of 5533 human small intestinal organoids calculated based on morphological organoid features (FDL, eccentricity: left and log 10 (Area): right). F) PhenoGraph clustering represented in FDL with subpopulations highlighted (clusters 0 to 15). Representative organoids of three clusters are illustrated below (cluster 3: light orange, cluster 11: light brown, cluster 4: dark green). G) Heatmap quantifying the performance of the eight combinations tested in human small intestinal organoids similar to D. In contrast to human colon organoids, small intestinal organoids were independent of WNT-NGS after day 4. Detailed media composition is shown in Table 1. Condition 2.3.1 (green squares) is the selected condition for future experiments. H) Bright field images of human healthy colon and colorectal cancer (CRC) organoid lines from the same patient in 384-well plates at fixed time points on days 2, 4, 6, 8, 10 and 13. For the time course of both organoids, the selected media composition was used (see Figure 1 D). I) Antibody staining of major mature cell types in mature colon (upper panel) and small intestine (lower panel) organoids grown in selected media compositions (see Figure 1 D. Figure 1 G). Panels from left to right: stem cells (OLFM4; green) and enterocytes (FABP1; red), goblet cells (MUC2; red), transient amplifying cells (NUSAP1; red), tuft cells (POU2F3; red), M cells (RANKL; red), proliferation marker (KI67; red), enteroendocrine cells (TPH1; red), deep secretory cells (REG4; red), and Paneth cells (LYZ1; red). The nuclear marker in all organoids is DAPI (white).

[0016] Scale bars: 100 μm in I, 50 μm in C and F.

[0017] Figure 2 Comparison of different culture medium schemes from single cells to mature organoids. A) shows the bright field image of the outgrowth efficiency of human colon organoids under different culture medium schemes on day 10 of the same organoid system. 250 single cells were inoculated on day 0. B) Quantification of the outgrowth efficiency of human colon organoids in different culture media on days 4 and 8. CD) Human colon organoid imaging time course (i.e., days 2, 4, 6, 8, and 10) stained for (C) stem cell marker OLFM4 (green) and intestinal epithelial cell marker FABP1 (red) or (D) goblet cell marker MUC2 (red) under 4 different culture medium schemes. E) On days 2, 4, 6, 8, 10, and 13, fluorescent images of organoid development from different biopsy locations of the colon (indicated on the left in each small figure) were captured using the developing culture medium scheme. Each developmental trajectory represents a different patient, showing enteroendocrine cells (TPH1; magenta), goblet cells (MUC2; red), and transient amplification cells (NUSAP1; green). The nuclear marker in all organoids is DAPI (white). Scale bar: 50 μm.

[0018] Figure 3 Pseudotime of intestinal organoids reveals the transition from regeneration to homeostatic equilibrium using the FMI protocol. A) Schematic overview of human colon organoid seeding and time point sampling. B) After segmentation of individual organoids ( Figure 1 A) Individual organoids approximating maturation progression were sorted using features based on shape features in pseudochronology (palantir, Setty et al., 2019). C) Pseudochronology of organoid development divided into 10 equal bins for DAPI, OLFM4, FABP1, MUC2, YAP1, and TPH1. Fluorescence images (inferno LUT) represent each bin. On the right, the average intensity of each marker and important shape features for all imaged organoids is plotted relative to the 10 pseudochronological bins.

[0019] Figure 4 Single-cell RNA sequencing time course of regeneration in human colon and small intestine organoids. A and D) t-distributed stochastic neighbor embedding (t-SNE) projections color-coded for experimental time and B and E) cell type. Left colon, right small intestine. C and F) Relative expression of known cell type marker genes in regenerating and mature cell types highlighted in the t-SNE plots.

[0020] Figure 5 Individual demonstration cases of organoid culture using the FMI protocol. A) One-week-old human organoid generated from a fresh biopsy from an inflammatory bowel disease (IBD) patient. Brightfield images of Crohn’s disease colon (left) and healthy colon (middle) plus diseased colon (right) from a patient with ulcerative colitis. B-C) ​​Brightfield images of mouse small intestinal organoids plated at different densities in (B) at day 7. C) Brightfield magnified views showing organoid development from single cells to budding organoids using the FMI protocol. D) Schematic overview of outgrowth of Lgr5-negative or -positive mouse colon cells using the FMI protocol. Adapted from Serra et al., 2019. E) Representative fluorescent images of Lgr5-positive (green) (upper panel) and negative (lower panel) organoid development. F) Shows proliferative areas (Ki67 = red) and stem cells (Lgr5 = green) over time. G) Three panels showing the Wnt target genes Sox9 (top; red), MUC2 (middle; goblet cell marker), and FABP2 (bottom; intestinal epithelial cell marker) in addition to Lgr5 (green). The nuclear marker in all organoids was DAPI (blue). DETAILED DESCRIPTION

[0021] Through their work, 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.

[0022] Thus, the claims cover a culture medium for culturing intestinal organoids, characterized in that the culture medium comprises neuregulin 1 (NRG1) and all-trans retinoic acid (atRA). This culture medium of the invention is suitable for culturing small intestinal organoids, colon organoids, colorectal cancer organoids, Crohn's disease organoids or ulcerative colitis organoids.

[0023] The concentration of NRG1 in the culture medium may be between 1 ng / ml and 100 ng / ml. The concentration of atRA in the culture medium may be between 10 nM and 10 μM. In some embodiments, the culture medium of the present invention does not contain animal serum. In some embodiments, the culture medium of the present invention further comprises any one of R-Spondin, recombinant Noggin, NRG1, IGF1, FGF2 and gastrin. All of these factors (including NRG1 and atRA) may be human or mouse, as well as recombinant or purified natural proteins. The culture medium of the present invention may also comprise Wnt NGS (Next Generation Substitute).

[0024] The claims also cover compositions comprising the culture medium of the present invention and an extracellular matrix or a 3D matrix that mimics the extracellular matrix through the interaction of the extracellular matrix with cell membrane proteins. Such a 3D matrix extracellular matrix can be a synthetic hydrogel or Matrigel TM Alternatively, among others, UltriMatrix (Culturex Ultimatrix RGF Basement Membrane Extract) and BME (Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear), as well as collagen-1 or fibronectin, may be used. In some embodiments, the extracellular matrix or 3D matrix is ​​washed off after an initial period of time.

[0025] The claims further encompass the use of a culture medium according to the invention or a composition according to the invention for culturing intestinal organoids.

[0026] The claims further encompass a method of generating intestinal organoids, the method 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 culture medium for at least one day, and then replacing the first culture medium with a culture medium of the present invention as described above. In some embodiments, the first culture medium may contain atRA at a concentration of 10 nM. The single cells or fragments of intestinal tissue used in the methods of the present invention may be cells or tissue fragments obtained from the small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue, or may be definitive endoderm cells.

[0027] In the method of the present invention, the first culture medium may contain an inhibitor of Rho-associated kinase (ROCK), such as the ROCK inhibitor Y27632. In the method of the present invention, the first culture medium may contain an inhibitor of the activin / NODAL / TGF-β pathway, such as the inhibitor A 83-01.

[0028] In some embodiments of the methods of the invention, the cells can be incubated in the first culture medium for at least three days.

[0029] The first and second culture media of the method of the present invention can be adapted to the properties of the single cells or fragments of intestinal tissue used in the method of the present invention. For example, for small intestinal cells and / or tissue fragments, the appropriate concentration of NRG1 in the first culture medium is 1 ng / ml and does not contain atRA, while the concentrations of NRG1 and atRA in the second culture medium (i.e., the culture medium of the present invention) are 10 ng / ml and 2.5 μM, respectively. For cells and tissue fragments from the colon, the appropriate concentration of NRG1 in the first culture medium is 1 ng / ml and does not contain atRA, while the concentrations of NRG1 and atRA in the second culture medium are 10 ng / ml and 10 nM, respectively.

[0030] The claims also cover intestinal organoids obtained using the methods of the invention.

[0031] The following definitions are provided to aid understanding of certain terms used throughout this specification.

[0032] As used herein, "totipotent stem cells" (also called omnipotent stem cells) refer to stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells can build a complete, viable organism. These cells are produced by the fusion of egg cells and sperm cells. The cells produced by the first few divisions of the fertilized egg are also omnipotent.

[0033] As used herein, the term "pluripotent stem cell (PSC)", also commonly referred to as PS cell, encompasses any cell that can differentiate into almost any cell, i.e., cells derived from any of the three germ layers (reproductive epithelium) (including endoderm (stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital system), and ectoderm (epidermal tissue and nervous system). PSCs can be descendants of totipotent cells, derived from embryonic stem cells (including embryonic germ cells), or obtained by inducing non-pluripotent cells (e.g., adult somatic cells) by forcibly expressing certain genes.

[0034] As used herein, the term "induced pluripotent stem cell (iPSC)" (also often abbreviated as iPS cell) refers to a type of pluripotent stem cell artificially derived from normal non-pluripotent cells (eg, adult somatic cells) by inducing "forced" expression of certain genes.

[0035] As used herein, the term "embryonic stem cell (ESC)" (also often abbreviated as ES cell) refers to cells that are pluripotent and derived from the inner cell mass of a blastocyst (early embryo). For the purposes of the present invention, the term "ESC" is sometimes also used broadly to encompass embryonic germ cells.

[0036] As used herein, the term "precursor cell" encompasses any cell that can be used in the methods described herein, by which one or more precursor cells obtain the ability to self-renew or differentiate into one or more specialized cell types. In some aspects, the precursor cell is pluripotent or has the ability to become pluripotent. In some aspects, the precursor cell is subjected to the treatment of external factors (e.g., growth factors) to obtain pluripotency. In some aspects, the precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cell and a unipotent stem cell. In some aspects, the precursor cell can be from an embryo, an infant, a child or an adult. In some aspects, the precursor cell can be a treated somatic cell so that pluripotency is conferred by genetic manipulation or protein / peptide treatment.

[0037] 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 specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0038] As used herein, the term "cellular component" is a single gene, protein, mRNA expressed gene and / or any other variable cellular component or protein activity, such as the degree of protein modification (e.g., phosphorylation), such as typically measured by those skilled in the art in biological experiments (e.g., by microarrays or immunohistochemistry). Now, major discoveries related to the complex network of biochemical processes behind life systems, common human diseases, and gene discovery and structure determination can be attributed to the application of cellular component abundance data as part of the research process. Cellular component abundance data can help identify biomarkers, distinguish disease subtypes, and identify toxic mechanisms.

[0039] As described herein, methods and systems are established using time-series growth factor manipulation to mimic embryonic intestinal development in culture. In particular, methods and systems are established to direct the in vitro differentiation of PSCs (human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) into intestinal tissue.

[0040] Generation of gastric and small intestinal organoids from pluripotent stem cells (PSCs) has revolutionized the study of human gastrointestinal (GI) development and disease. However, work on generating large intestinal organoids has lagged, in part due to a poor molecular understanding of hindgut development.

[0041] In certain embodiments of the invention, "about" or "approximately" refers to a number that varies by up to 5%, or in other embodiments by up to 10%, and in other embodiments by up to 25% from the recited number.

[0042] As used herein, the term "serum-free" refers to a culture medium that is substantially free of serum. In certain embodiments, there is 0% (completely free) or less than about 0.001%, 0.005%, 0.01%, 0.025%, 0.05%, 0.1%, 1.0% or 10.0% total serum in the target culture medium. The most common types of serum include: various forms of bovine serum (calf serum, fetal bovine serum, bovine calf serum, donor bovine calf serum, newborn calf serum, etc.), horse serum and human serum.

[0043] "Chemically defined" means that the structures, chemical formulas, and percentages of the various individual components of a chemical composition are known or can be determined. The chemical composition of various tissue extracts (e.g., bovine pituitary extracts) is not determined, at least in part because all of the individual components of the extract are not known. For those components that are known, the amounts and relative percentages of the various components can (and typically will) vary from batch to batch. This is in part due to the fact that even within the same tissue, individual animals may have inherently different levels of various chemical compositions, depending on factors such as general health, nutrition, mood, pathological infections, trauma, etc.

[0044] In certain embodiments, the culture medium of the present invention does not contain any animal serum products prepared for tissue culture purposes. The culture medium does not contain any chemical composition unknown / undetermined tissue extracts either. On the contrary, all necessary components required for the growth / proliferation of the desired cell type are chemically determined. Most (if not all) of these individual components can be easily purchased from various suppliers (such as Sigma-Aldrich Corp. (St. Louis, MO), GIBCO-Invitrogen Corp. (Carlsbad, CA), Calbiochem and / or BD Biosciences (BD Biosciences) (San Jose, CA, San Jose, CA) etc.) as commercial products.

[0045] In certain other embodiments, the presence of serum and / or tissue extracts in the target culture medium, particularly in trace amounts, does not substantially perturb the characteristics of the culture medium.

[0046] The present invention also provides methods for conducting drug / biotech product discovery and development, which methods include creating intestinal organoid models derived from various cell types isolated and propagated using the culture media and methods of the present invention, and screening libraries of drug molecules or lead compounds to identify molecules that affect them.

[0047] The phrases "cell culture medium," "culture medium" (plural "culture media" in each case), and "culture medium formulation" refer to nutrient solutions used to cultivate cells and are used interchangeably.

[0048] The cell culture media of the present invention are water-based (but can be reconstituted from dry powders and / or frozen components) and comprise the various ingredients in a solution of preferably deionized and / or distilled water.

[0049] The term "ingredient" refers to any compound, whether chemical or biological, that can be used in a cell culture medium to maintain or promote cell growth or proliferation. The terms "component," "nutrient," and "ingredient" are used interchangeably and all refer to such compounds. Typical ingredients used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other ingredients that promote or maintain ex vivo cell culture can be selected by those skilled in the art according to specific needs.

[0050] "Cell culture" or "culture" refers to the maintenance of cells in an artificial in vitro environment. However, it should be understood that the term "cell culture" is a general term and can be used to cover not only the culture of single cells, but also the culture of tissues, organs, organ systems or whole organisms, and the terms "tissue culture", "organ culture", "organ system culture", "organoid culture" or "organotype culture" can be used interchangeably with the term "cell culture" in special cases.

[0051] Some cells (e.g., human cells) must have a sufficient amount of 9 kinds of amino acids to survive. These so-called "essential" amino acids cannot be synthesized by other precursors. However, cysteine ​​can partially meet the needs of methionine (they all contain sulfur), and tyrosine can partially replace 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.

[0052] When mixed together in solution, some or all of the ingredients may form a "basal medium". Other components (e.g., at least one nucleotide synthesis and / or salvage pathway precursor (e.g., hypoxanthine), epidermal growth factor (EGF), agents that increase intracellular cyclic adenosine monophosphate (cAMP) levels, and antioxidants) may be added to the basal medium to prepare a complete medium of the present invention. These later added components (e.g., EGF and cAMP increasing agents) may be added to a freshly prepared basal medium, or these components may be mixed into a stock solution that is stored frozen (preferably at about -20°C to about -70°C) until the stock solution is added to the basal medium to prepare a complete medium of the present invention.

[0053] To the extent that the components do not substantially affect the performance of the culture medium with respect to intestinal organoid culture, in certain embodiments, the subject culture medium can include and tolerate the presence of one or more of such components.

[0054] If necessary, one or more components of the culture medium can also be replaced with other chemicals with similar properties. Such modified culture medium without one or more non-essential / unnecessary components is within the scope of the present invention. Similarly, the technician can also determine the optimal level of any given component of a specific cell type by, for example, testing a series of concentrations of each listed component (e.g., high 10%, 25%, 50%, 75%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times based on or starting from the listed concentration of a specific component; or low 10%, 25%, 50%, 75%, 100%, 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500, 1 / 1000) based on or starting from the listed concentration of a specific component. Some components have a listed concentration range. The appropriate or optimal concentration for any specific cell type can also be similarly determined starting from the listed concentration. When conducting such tests, an initially broad range of concentrations may be tested and then gradually narrowed based on the results of the initial experiments. For example, for initial testing, the concentration of a component of interest may be changed to 10% of the initial concentration. -3 , 10 -2 , 10 -1 , 10 times, 100 times and 1000 times. -2 Testing still supports the expected growth, while 10 -3 If not, you can further explore 10 in the second round of testing. -2 and 10 -3The optimal range is determined by a 10-fold concentration difference between the two groups. Thus, culture media so optimized for specific cell types are also within the scope of the present invention. As will be apparent to one of ordinary skill in the art, the concentration of a given component may be increased or decreased beyond the disclosed ranges, and the effect of the increased or decreased concentration may be determined using only routine experimentation. The medium formulations of the invention for any particular cell type can be optimized using the methods 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 concentrations of the medium components are typically determined by empirical studies, single component titration studies, or by reading the historical and current scientific literature. In single-component titration studies using animal cells, the concentration of a single media component is varied while keeping all other ingredients and variables constant, and the effect of the single component on the viability, growth, or ongoing health of the animal cells is measured.

[0055] It should be understood that some of the factors (vitamins and hormones) listed herein may exist in different forms known in the art (e.g., different naturally occurring or non-naturally occurring forms) and may be used as substitutes for each other. It should also be understood that where vitamins or hormones are disclosed herein, it should be understood that the invention encompasses embodiments in which such vitamins or hormones in any form having similar biological activity (or compounds that can be modified or metabolized in cell culture media or in cells to provide biologically active forms) are used in the culture media and / or methods of the invention.

[0056] The medium components can be dissolved in a liquid carrier or maintained in a dry form. If dissolved in a liquid carrier (i.e., a "1-fold preparation") at the preferred concentrations shown above, the pH of the medium should be adjusted to about 7.0-7.6, such as about 7.1-7.5, or about 7.2-7.4. The osmotic pressure of the medium can also be adjusted to about 275-350mOsm, such as about 285-325mOsm, or about 280-310mOsm. The type of liquid carrier and the method of dissolving the components into solutions vary, and those of ordinary skill in the art can determine by routine experiments. Typically, the medium components can be added in any order.

[0057] Cell culture medium is made up of multiple components, and these components vary from culture medium to culture medium." 1 times preparation " refers to any aqueous solution containing some or all of the components found in the cell culture medium at the concentration used. " 1 times preparation " can refer to, for example, a cell culture medium or any component subgroup used for the culture medium. The concentration of the component in 1 times solution is about the same as the concentration of the component found in the cell culture preparation for maintaining or culturing cells in vitro. According to definition, the cell culture medium used for cell in vitro culture is 1 times preparation. When there are multiple components, the concentration of each component in 1 times preparation is approximately equal to the concentration of those components in the cell culture medium. For example, RPMI-1640 culture medium contains components such as 0.2g / L L-arginine, 0.05g / L L-asparagine and 0.02g / L L-aspartic acid. " 1 times preparation " of these amino acids contains these components of approximately the same concentration in solution. Therefore, when referring to " 1 times preparation ", it is expected that each component in the solution has the same or approximately the same concentration found in the cell culture medium. The concentrations of components in 1x preparations of cell culture media are well known to those of ordinary skill in the art. See Methods for preparation of media, Supplements and Substrate For Serum-Free Animal Cell Culture, Allen R. Liss, New York (1984). However, the osmotic pressure and / or pH in a 1x preparation may be different compared to culture medium, particularly when the 1x preparation contains fewer components.

[0058] "10 times of preparations" refers to a solution in which the concentration of each component in the solution is about 10 times of the same component in the cell culture medium. For example, 10 times of preparations of RPMI-1640 culture medium can contain components such as 2.0g / L L-arginine, 0.5g / L L-asparagine and 0.2g / L L-aspartic acid (compared with 1 times of preparations above). "10 times of preparations" can contain a variety of other components, and its concentration is about 10 times of the concentration in 1 times of culture medium. It will be apparent that "25 times of preparations", "50 times of preparations", "100 times of preparations", "500 times of preparations" and "1000 times of preparations" respectively represent solutions containing components of about 25 times, 50 times, 100 times, 500 times or 1000 times of concentration compared with 1 times of cell culture medium. Similarly, the osmotic pressure and pH of culture medium preparations and concentrated solutions can vary. Preferably, the solution comprising a component is higher than the concentration of the same component in 1 times of culture medium preparations. The ingredients can be 10 times concentrated (10 times formulation), 25 times concentrated (25 times formulation), 50 times concentrated (50 times formulation), or 100 times concentrated (100 times formulation). Formulations of higher concentrations can be prepared provided that the ingredients remain soluble and stable. See, e.g., U.S. Pat. No. 5,474, which relates to methods of solubilizing high concentration culture medium components.

[0059] If the media components are prepared as separate concentrated solutions, an appropriate (sufficient) amount of each concentrate is combined with a diluent to produce a 1x media formulation. Typically, the diluent used is water, but other solutions including aqueous buffers, aqueous saline solutions or other aqueous solutions may be used according to the present invention.

[0060] Substratum of the present invention is typically sterilized to prevent undesirable contamination. Sterilization can be accomplished, for example, by filtering through a low protein binding membrane filter (commercially available from, for example, Millipore, Bedford, Mass.) of about 0.1-1.0 μm pore size, and then the concentrated components are mixed to produce sterile culture medium. Alternatively, concentrated component subgroups can be sterilized by filtration and stored as sterile solutions. These sterile concentrates can then be mixed with sterile diluents under aseptic conditions to produce 1 times of concentrated sterile culture medium preparations. Autoclaving or other sterilization methods based on high temperature are unwelcome because many components of current culture medium are heat unstable, and irreversible degradation will occur at the temperature reached during, for example, most of the thermal sterilization methods.

[0061] Many tissue culture media typically contain one or more antibiotics, which are not necessary for cell growth / proliferation per se, but are present to inhibit the growth of other undesirable microorganisms (e.g., bacteria and / or fungi). Antibiotics are relatively low-molecular-weight natural chemicals produced by various microorganisms (e.g., bacteria (including Bacillus species), actinomycetes (including Streptomyces) and fungi) species that inhibit the growth of other microorganisms or destroy other microorganisms. Materials similar to structures and modes of action can be chemically synthesized, or natural compounds can 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, and amikacin; (3) tetracyclines; (4) sulfonamides and trimethoprim; (5) fluoroquinolones, such as ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) macrolides, which include, for example, erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, such as polymyxins, chloramphenicol, and lincosamides. Antibiotics achieve their antibacterial effects through several mechanisms of action and can generally be grouped 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 detergent effect, such as colistin, novobiocin, and polymyxin; (3) agents that affect cell replication, information transmission, and protein synthesis machinery by acting on the ribosome, such as 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, griseofulvin, and rifamycins; and (5) drugs that affect intermediary metabolism, such as sulfonamides, trimethoprim, and the antituberculosis agents isoniazid and para-aminosalicylic acid. Some agents may have more than one primary mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of the bacterial cell often occur after the primary action of the antimicrobial drug.

[0062] Therefore, for convenience and other practical reasons, the target culture medium may be additionally supplemented with one or more antibiotics or other substances that inhibit the growth / proliferation of undesirable bacteria / fungi / viruses. However, in other embodiments, the target culture medium may not contain any antibiotics to ensure optimal growth of primary cells. Extra care should be taken when handling cells grown in antibiotic-free culture medium to avoid possible contamination.

[0063] The culture medium of the present invention can be made from individual components purchased separately from various chemical suppliers. Alternatively, certain commercial culture media can be conveniently mixed and supplemented with additional components to prepare the target culture medium. Therefore, the present invention provides methods for preparing tissue culture media, which methods include supplementing commercially available cell culture media or mixtures of two or more such culture media by adding one or more components disclosed herein.

[0064] The present invention provides a tissue culture medium for intestinal organoids, which includes components sufficient to grow cells. The composition of these culture media may be different. For example, relative to the original concentration, the concentration of any component can independently change up to 10%, 20%, 30%, 40% or 50%, or up to 2-3 times. In one embodiment, the concentration of each component differs from the listed value by no more than 10%. In one embodiment, the concentration of each component differs from the listed value by no more than 25%. Unless otherwise indicated, as used herein, up to X% change means relative to the listed value ± X% change. For example, if the listed value is 100ng / ml, 25% change means that the value can change between 75ng / ml and 125ng / ml (i.e., 75-125ng / ml). Unless otherwise indicated, when disclosing a numerical range, the endpoint is also included in the range. In addition, it should be understood that, unless otherwise indicated or otherwise apparent from the context and understanding of those of ordinary skill in the art, the numerical values ​​expressed as ranges in different embodiments of the present invention may take any specific value or sub-range within the stated range to one tenth of the lower limit unit of the range, unless otherwise clearly defined by the context. It should also be understood that, in the case of a series of numerical values ​​stated herein, the present invention includes embodiments involving any intermediate values ​​or ranges defined by any two values ​​in the series, wherein the lowest value may be considered a minimum value, and the highest value may be considered a maximum value. For any embodiment of the present invention in which a value begins with the term "about" or "approximately", the present invention includes embodiments in which the exact value is listed. For any embodiment of the present invention in which a numerical value does not begin with "about" or "approximately", the present invention includes embodiments in which a value begins with "about" or "approximately".

[0065] It will be understood that certain components can be provided as salts, esters, biologically active metabolites or derivatives, or as precursors that are metabolized, processed or broken down by cells or in the culture medium to produce biologically active forms of certain components disclosed herein. In this context, "biological activity" refers to the ability of a component to exert its desired effect on a cell when present in a cell culture medium.

[0066] The culture medium of the present invention can be a liquid or solid powder, or a combination of the two. The liquid form can be a complete culture medium, which contains all components sufficient to support the growth / proliferation of target cells. Alternatively, the liquid culture medium can be stored as a separate package so that each separate package can be stored under its appropriate conditions (temperature, humidity, etc.). For example, if most components are required to be included in the culture medium of the present invention, it can be pre-dissolved in a single solution and stored under appropriate conditions (e.g., 4°C, in a dark and dry place, etc.). Other components may be unstable under the storage conditions of other components, or may react slowly with other components, or it is best to store them as separate stock solutions, so they can be stored under different conditions (e.g., -20°C or -80°C, etc.). Only shortly before use or when about to be used, these separately stored components will be combined together to form the entire culture medium. Each separate package can be commercially sold or sold separately, or as a stock solution of different concentrations (e.g., 2 times, 5 times, 10 times, 100 times, 1000 times, etc.). In some embodiments, the culture medium of the present invention is commercially sold or sold together with one or more cell lines (e.g., one or more cell lines disclosed herein) suitable for culturing with the culture medium.

[0067] Similarly, complete medium or its individual components, packaging may be in the form of a dry powder, which upon reconstitution with an aqueous solution (eg, water) will yield the desired medium or a concentrated stock solution thereof (2-fold, 5-fold, or 10-fold, etc.).

[0068] Components that can be stored or are preferably stored as separate stock solutions prior to use include: growth factors (e.g., epidermal growth factor), hormones (e.g., estrogen, progesterone, testosterone), other unstable enzymes / proteins (e.g., transferrin, insulin, cholera toxin, etc.), steroids (e.g., cortisol, cholesterol), vitamins (vitamin A, Bi2, K3), pH indicators (e.g., phenol red), one or more buffer components (e.g., sodium bicarbonate, HEPES), and other chemicals (e.g., glutathione, 17-β-estradiol, O-phosphoethanolamine, etc.).

[0069] In certain embodiments, at least some or all components of the culture medium are in liquid / aqueous form. In other embodiments, at least some or all components of the culture medium are in solid / powder form.

[0070] The culture medium of the present invention is suitable for a variety of primary cells from different mammals (including birds, reptiles, humans and other non-human mammals). Mammals also include: non-human primates (such as monkeys, gorillas, etc.), mice, rats, rabbits, cattle, horses, pigs, sheep, goats, dogs and cats.

[0071] As used herein, "substantially free" refers to a desired cell population that is at least about 80% pure, preferably 85%, 90%, 95%, 99% or more pure, among the entire cell population.

[0072] The present invention also provides an in vitro method for identifying an agent that enhances or positively affects one or more characteristics of intestinal organoids, including differentiation, apoptosis, sensitivity to chemotherapy / radiotherapy, or senescence, the method comprising: (1) contacting a culture of intestinal organoids in a culture medium of the present invention with a candidate agent to be evaluated for its ability to, for example, enhance or positively affect one or more characteristics of the organoids under conditions suitable for entry of the agent into the cells; (2) determining the extent to which the characteristic is, for example, enhanced or positively affected in the presence of the candidate agent to be evaluated; and, (3) comparing the determined extent with the characteristic of the intestinal organoids under the same conditions but in the absence of the candidate agent to be evaluated, wherein if the characteristic is significantly enhanced or positively affected in the presence of the candidate agent to be evaluated than in its absence, then the candidate agent to be evaluated is an agent that enhances or positively affects one or more characteristics of the intestinal organoids.

[0073] Similarly, the present invention also provides in vivo methods for identifying agents that inhibit or negatively affect one or more characteristics of intestinal organoids.

[0074] In certain embodiments, the agent is an RNAi molecule. In certain embodiments, the agent is an siRNA molecule. In certain embodiments, the agent is a chemical compound.

[0075] In the present invention, "isolated" refers to materials removed from their original environment (e.g., if the original environment is naturally occurring, it is a natural environment), and thus "by artificial" changes from their natural state. For example, an isolated polynucleotide can be part of a vector or composition of matter, or can be contained in a cell, and still be "isolated" because the vector, composition of matter, or specific cell is not the original environment of the polynucleotide. The term "isolated" does not refer to a genome or cDNA library, a whole cell population or mRNA preparation, a genomic DNA preparation (including those separated by electrophoresis and transferred to a blot), a sheared whole cell genomic DNA preparation, or other compositions in which the art does not show the distinguishing characteristics of the polynucleotide / sequence of the present invention. Other examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules of the present invention. However, for the purposes of the present invention, nucleic acids contained in clones that are members of a library (e.g., a genomic or cDNA library) but have not been separated from other members of the library (e.g., in the form of a homogenous solution containing the clone and other members of the library), or chromosomes removed from cells or cell lysates (e.g., "chromosome spreads," as in a karyotype), or preparations of randomly sheared genomic DNA, or preparations of genomic DNA cleaved by one or more restriction enzymes are not "isolated." As further discussed herein, isolated nucleic acid molecules according to the present invention can be produced naturally, recombinantly, or synthetically.

[0076] In the present invention, a "secreted" protein refers to a protein that can be directed to the ER, secretory vesicles, or extracellular space due to a signal sequence, as well as a protein that is released into the extracellular space without necessarily containing a signal sequence. If a secreted protein is released into the extracellular space, the secreted protein may undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by a number of mechanisms, including exocytosis and proteolytic cleavage.

[0077] "Polynucleotides" may consist of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides may consist of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides may also contain one or more modified bases or a DNA or RNA backbone modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. DNA and RNA may be modified in a variety of ways; therefore, "polynucleotides" include chemically, enzymatically, or metabolically modified forms.

[0078] The expression "a polynucleotide encoding a polypeptide" encompasses polynucleotides comprising only the coding sequence for the polypeptide as well as polynucleotides comprising additional coding and / or non-coding sequences.

[0079] "Stringent hybridization conditions" refers to 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 the filter in 0.1× SSC at about 50° C. Alterations in the stringency of hybridization and signal detection are primarily achieved by controlling formamide concentration (lower percentages of formamide result in reduced stringency); salt conditions or temperature. For example, moderately high stringency conditions include overnight incubation at 37°C in a solution containing 6X SSPE (20XSSPE=3M NaCl; 0.2MNaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm to block DNA; followed by washing with 1XSSPE, 0.1% SDS at 50°C. In addition, to achieve even lower stringency, washes performed after stringent hybridization can be performed at higher salt concentrations (e.g., 5X SSC). Variations on the above conditions can be achieved by including and / or replacing alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhart's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of a specific blocking reagent may require modification of the above hybridization conditions due to compatibility issues.

[0080] When referring to polypeptides, the terms "fragment", "derivative" and "analog" mean a polypeptide that retains substantially the same biological function or activity as such polypeptide. Analogs include proproteins that can be activated to produce active mature polypeptides by cleaving the proprotein portion.

[0081] The term "gene" means the DNA segment involved in producing a polypeptide chain; it includes the regions preceding and following the coding region, the "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).

[0082] Polypeptides can be composed of amino acids connected to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain amino acids other than 20 kinds of genetically encoded amino acids. Polypeptides can be modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. Such modifications are fully described in basic textbooks and more detailed monographs as well as a large number of research documents. Modifications can occur anywhere in a polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It should be recognized that the same type of modification can exist at several sites in a given polypeptide to the same or different extents. Moreover, a given polypeptide can contain many types of modifications. For example, polypeptides can be branched, for example, due to ubiquitination, and they can be cyclic, with or without branches. Circular, branched, and branched cyclic polypeptides can be produced by natural processes after translation, or can be prepared 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 a phosphatidylinositol, cross-linking, cyclization, derivatization by known protecting / blocking groups, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, attachment to antibody molecules or other molecular ligands, methylation, myristoylation, oxidation, pegylation, proteolytic processing (e.g., cleavage), phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination. (See, e.g., PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd ed., TeCrighton, WH Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Johnson, ed., Academic Press, New York, pp. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

[0083] A "biologically active" polypeptide fragment refers to a polypeptide that exhibits an activity similar to, but not necessarily identical to, the activity of the original polypeptide (including mature forms), as measured in a specific biological assay, with or without dose dependence. Where dose dependence does exist, it need not be the same as the dose dependence of the polypeptide, but rather substantially similar to the dose dependence in a given activity compared to the original polypeptide (i.e., relative to the original polypeptide, the candidate polypeptide will exhibit a stronger activity or no more than about 25 times, and in some embodiments, no more than about one-tenth, or no more than about one-third of the activity).

[0084] Species homologs can be isolated and identified by preparing appropriate probes or primers from the sequences provided herein and screening an appropriate nucleic acid source for the desired homolog.

[0085] "Variant" refers to a polynucleotide or polypeptide that is different from an original polynucleotide or polypeptide but retains its essential properties. Typically, a variant is very similar to the original polynucleotide or polypeptide overall, and in many regions, is identical to the original polynucleotide or polypeptide.

[0086] In fact, whether any specific nucleic acid molecule or polypeptide has at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of the present invention can be determined using known computer programs routinely. The preferred method for determining the best overall match between the query sequence (sequence of the present invention) and the target sequence, also referred to as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of the people such as Brutlag (Comp.App.Blosci.[Computer Bioscience Applications] (1990) 6:237-245). In the sequence alignment, the query sequence and the target sequence are both DNA sequences. RNA sequences can be compared by converting U to T. The result of the global sequence alignment is the percentage identity. The preferred parameters for calculating the percent identity of the FASTDB comparison of DNA sequences are: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 500 or the length of the target nucleotide sequence (whichever is shorter). If the target sequence is shorter than the query sequence due to 5' or 3' deletions, then the result must be manually corrected. This is because the FASTDB program does not consider the 5' and 3' brachymes of the target sequence when calculating the percent identity. For the target sequence truncated at 5' or 3' ends relative to the query sequence, the percentage identity is corrected by calculating the percentage of bases that are not matched / aligned at 5' and 3' of the target sequence in the query sequence as the total number of bases of the query sequence. Determine whether the nucleotide is matched / aligned by the result of the FASTDB sequence comparison. Then deduct the percentage from the percentage identity calculated using the specified parameters of the above-mentioned FASTDB program to derive the final percentage identity score. The score after this correction is the score for the purpose of the present invention. In order to manually adjust the percentage identity score, only the bases that are not matched / aligned with the query sequence (as shown by the FASTDB comparison) outside the 5' and 3' bases of the target sequence are calculated. For example, the target sequence of 90 bases is compared with the query sequence of 100 bases to determine the percentage identity. The deletion occurs at the 5' end of the target sequence, so the FASTDB comparison does not show the matching / alignment of the first 10 bases of the 5' end.These 10 damaged bases account for 10% of the sequence (the total number of bases in the 5' and 3' ends that do not match / query sequence), so 10% is subtracted from the percentage identity score calculated by the FASTDB program. If the remaining 90 bases are fully matched, the final percentage identity is 90%. In another example, the target sequence of 90 bases is compared with the query sequence of 100 bases. This time the deletion is an internal deletion, so there is no base that does not match / align with the query sequence on the 5' or 3' of the target sequence. In this case, the percentage identity calculated by FASTDB is not manually corrected. Again, only the bases that do not match / align with the query sequence at 5' and 3' of the target sequence are manually corrected.

[0087] By a polypeptide having an amino acid sequence that is at least (for example) 95% "identical" to a query amino acid sequence of the invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid changes in every 100 amino acids of the query amino acid sequence. In other words, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These changes to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0088] In fact, known computer programs can be used to determine in a conventional manner whether any particular polypeptide has at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity to, for example, the amino acid sequence shown in the sequence or to the amino acid sequence encoded by the deposited DNA clone. The preferred method for determining the best overall match between the query sequence (sequence of the present invention) and the target sequence, also known as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. [Computer Biological Science Applications] (1990) 6: 237-245). In the sequence alignment, the query sequence and the target sequence are both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is the percentage identity. The preferred parameters for FASTDB amino acid comparison are: matrix=PAM 0, k-tuple=2, mismatch penalty--1, connection penalty=20, random group length=0, cut-off score=1, window size=sequence length, gap penalty--5, gap size penalty--0.05, window size=500 or the length of the target amino acid sequence (whichever is shorter). If the target sequence is shorter than the query sequence due to N- or C-terminal deletions, rather than due to internal deletions, the result must be manually corrected. This is because the FASTDB program does not consider the N- and C-terminal truncations of the target sequence when calculating global percentage identity. For the target sequence truncated at the N- and C-terminals relative to the query sequence, the percentage identity is corrected by calculating the percentage of the number of residues in the query sequence that are not matched / aligned with the corresponding target residues at the target sequence N- and C-terminals to the total number of bases in the query sequence. Determine whether the residues are matched / aligned by the result of the FASTDB sequence comparison. The percentage is then subtracted from the percentage identity calculated using the specified parameters of the above-mentioned FASTDB program to derive a final percentage identity score. This final percentage identity score is a score for the purpose of the present invention. In order to manually adjust the percentage identity score, only the bases that the target sequence N- and C-terminals do not match / align with the query sequence are considered. In other words, only the residue positions outside the farthest N- and C-terminal residues of the target sequence are queried. Only the residue positions that do not match / align with the query sequence outside the target sequence N- and C-terminals (as shown in the FASTDB comparison) are manually corrected. For purposes of the present invention, it is not necessary to carry out other manual corrections.

[0089] Naturally occurring protein variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism. (Genes 11, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants may vary at the polynucleotide and / or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

[0090] Using known protein engineering methods and recombinant DNA technology, variants can be generated to improve or change the characteristics of polypeptides. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without causing substantial loss of biological function. Ron et al., J.Biol.Chem. [Journal of Biological Chemistry] 268: 2984-2988 (1993) reported variant KGF proteins that still had heparin binding activity even after the deletion of 3, 8 or 27 amino terminal amino acid residues. Similarly, interferon gamma exhibited up to ten times the activity after the deletion of 8-10 amino acid residues from the carboxyl terminus of the protein (Dobeli et al., J.Biotechnology [Journal of Biological Technology] 7: 199-216 (1988)). In addition, there is ample evidence that variants generally retain biological activity similar to that of naturally occurring proteins. For example, Gayle and colleagues (J.Biol.Chem [Journal of Biological Chemistry] 268: 22105-22111 (1993)) conducted extensive mutation analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants, with each variant having an average of 2.5 amino acid changes over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The researchers found that "most of the molecules could be altered with little or no effect on [binding or biological activity]." (See, Abstract). In fact, of the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type. In addition, even if one or more amino acids are missing from the N-terminus or C-terminus of a polypeptide, resulting in modification or loss of one or more biological functions, other biological activities may still be retained. For example, when less than the majority of the secreted form's residues are removed from the N-terminus or C-terminus, the ability of the deletion variant to induce and / or bind to antibodies that recognize the secreted form may be retained. Whether a particular polypeptide lacking the N- or C-terminal residues of a protein retains such immunogenic activity can be readily determined by conventional methods described herein and known in the art.

[0091] "Biological sample" refers to any biological sample containing a polypeptide or mRNA of the present invention obtained from an individual, body fluid, cell line, tissue culture, or other source. As noted, biological samples include body fluids (e.g., semen, lymph, serum, plasma, urine, synovial fluid, and spinal fluid) and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. In the case where the biological sample includes mRNA, tissue biopsies are a preferred source.

[0092] "Matrigel" is the trade name for a gelatinous protein mixture secreted by mouse tumor cells and commercially 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. A common laboratory procedure is to dispense a small volume of frozen (4°C) Matrigel onto plastic tissue culture labware. When incubated at 37°C (body temperature), Matrigel proteins self-assemble to produce a film covering the surface of the labware. Cells cultured on Matrigel exhibit complex cell behaviors that are impossible to observe under laboratory conditions. For example, endothelial cells produce complex spider web-like networks on Matrigel-coated surfaces, but not on plastic surfaces. Such a network is very similar to the microvascular capillary system that supplies blood to living tissues. Therefore, the process by which endothelial cells build such a network is of great interest to biological researchers, and Matrigel allows them to observe this. In some cases, researchers use larger volumes of Matrigel to produce thick three-dimensional gels. The utility of thick gels is that thick gels induce cells to migrate from the surface of the gel to the interior. Researchers study this migratory behavior as a model for tumor cell metastasis. Pharmaceutical scientists use Matrigel to screen drug molecules. A typical experiment consists of adding test molecules to Matrigel and observing cell behavior. Test molecules that promote endothelial cell network formation are candidates for tissue regeneration therapies, while test molecules that inhibit endothelial cell network formation are candidates for anti-cancer therapies. Similarly, test molecules that inhibit tumor cell migration may also have potential as anti-cancer drugs. Matrigel's ability to stimulate complex cell behaviors is a result of its heterogeneous composition. The main components of Matrigel are structural proteins (such as laminin and collagen), which provide the cultured cells with adhesion peptide sequences that the cells would encounter in their natural environment. There are also growth factors that promote differentiation and proliferation of many cell types. Matrigel contains small amounts of many other proteins, and its exact composition is not clear. Matrigel can also be used as an attachment substrate in embryonic stem cell culture. When embryonic stem cells are grown in the absence of feeder cells, extracellular matrix components (such as Matrigel) are required to maintain a pluripotent, undifferentiated state (self-renewal).

[0093] 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 the EGFR family of receptors. Neuregulin 1 is produced in multiple isoforms by alternative splicing, which allows it to perform multiple functions. A suitable NRG1 is sold by R&D Systems (Catalog No. 5898-NR-050; NS0-derived human neuregulin-1 / NRG1 protein, Ser20-Lys241, with a C-terminal 6-His tag).

[0094] All-trans retinoic acid (also known as atRA, NSC 122758, retinoic acid, trans-retinoic acid, retinoic acid, and retinoic acid) is a derivative of vitamin A that functions as a kinase inhibitor for the retinoic acid receptor (RAR, IC 50 =14 nM). RAR heterodimerizes with the retinoid X receptor (RXR) and binds to the retinoic acid response element (RARE) in DNA and acts as a transcription factor, thereby altering gene expression.

[0095] R-spondin-1 (also known as RSPO1, CRISTIN3, and RSPO) is a secreted protein encoded in humans by the Rspo1 gene found on chromosome 1. In humans, it interacts with WNT4 during female sexual development. Loss of function may lead to sex reversal from female to male. In addition, it promotes canonical WNT / β-catenin signaling.

[0096] 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 African clawed frogs. Noggin is an inhibitor of several bone morphogenetic proteins (BMPs): it inhibits at least BMP2, BMP4, BMP5, BMP6, BMP7, BMP13, and BMP14.

[0097] Insulin-like growth factor 1 (IGF-1) (also known as 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 by the IGF1 gene in humans. IGF-1 consists of 70 amino acids in a single chain with three intramolecular disulfide bridges. The molecular weight of IGF-1 is 7,649 Daltons. IGF-1 is mainly produced by the liver. Production is stimulated by growth hormone (GH). Most IGF-1 binds to one of the six binding proteins (IGF-BP). IGFBP-1 is regulated by insulin.

[0098] 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 signaling protein encoded by the FGF2 gene. It binds to and exerts its effects on specific fibroblast growth factor receptor (FGFR) proteins, which themselves are a family of closely related molecules.

[0099] Gastrin (also known as GAST and GAS) is a peptide hormone that stimulates gastric parietal cells to secrete gastric acid (HCl) and contributes to gastric motility. It is released by G cells in the stomach, duodenum, and pyloric antrum of the pancreas. Gastrin binds to the cholecystokinin B receptor to stimulate the release of histamine in enterochromaffin-like cells, and it induces the insertion of the K+ / H+ ATPase pump into the apical membrane of the parietal cells (which in turn increases the release of H+ into the gastric lumen). Its release is stimulated by peptides in the gastric lumen.

[0100] The WNT gene family consists of structurally related genes that encode secreted signaling proteins. These proteins have been implicated in tumorigenesis and several developmental processes, including the regulation of cell fate and patterning during embryogenesis. This gene is a member of the WNT gene family. It is evolutionarily well conserved, and the protein encoded by this gene is known to be 98% identical to the mouse Wnt1 protein at the amino acid level.

[0101] Miao et al. (Vol. 27, No. 5, November 5, 2020, pp. 840-851.e6) designed and engineered WntNGS. Wnt NGS is a water-soluble, Fzd isoform-specific "next generation replacement" (NGS) Wnt that heterodimerizes Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocytes, ovary, and breast. NGS Wnt outperforms Wnt3a (another possible alternative to the culture medium of the present invention) conditioned medium in organoid expansion and single-cell organoid outgrowth.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. In addition, it should be noted that although the above description focuses on medical use, the method and medicament of the present invention are also suitable for any non-medical use.

[0103] Examples

[0104] Example protocol for preparing different media for culturing intestinal organoids:

[0105] Small Intestine Culture Medium Solution 1x Preparation

[0106] First culture medium for the first culture (e.g., day 4)

[0107] 0.5nM Wnt-NGS

[0108] 1μg / ml recombinant R-Spondin-1

[0109] 100ng / ml recombinant Noggin

[0110] 1x B27 TM Supplements

[0111] 1.25mM NAC

[0112] 50ng / ml hEGF

[0113] 1ng / ml hNRG1

[0114] 0.5μM A83-01

[0115] 100ng / ml hIGF1

[0116] 50ng / ml hFGF2

[0117] 25nM gastrin

[0118] 10μM ROCK inhibitor (Y-27632)

[0119] In DMEM / F12 + 15mM HEPES, 1x GlutaMax (1:100 #35050-038) and 100μg / ml PenStrep (#15140-122)

[0120] Second culture medium after 4 days of culture

[0121] 0.5 μg / ml recombinant R-Spondin1

[0122] 100ng / ml recombinant Noggin

[0123] 1x B27 TM Supplements

[0124] 1.25mM NAC

[0125] 5ng / ml hEGF

[0126] 10ng / ml hNRG1

[0127] 100ng / ml hIGF1

[0128] 50ng / ml hFGF2

[0129] 25nM gastrin

[0130] 2.5μM atRA (all-trans retinoic acid)

[0131] In DMEM / F12 + 15mM HEPES, 1x GlutaMax (1:100 #35050-038) and 100μg / ml PenStrep (#15140-122)

[0132] Colon culture medium 1x preparation

[0133] First culture medium for the first culture (e.g., day 4)

[0134] 0.5nM Wnt(NGS)

[0135] 1μg / ml recombinant R-Spondin1

[0136] 100ng / ml recombinant Noggin

[0137] 1x B27 TM Supplements

[0138] 1.25mM

[0139] 50ng / ml hEGF

[0140] 1ng / ml hNRG1

[0141] 0.5μM A83-01

[0142] 100ng / ml hIGF1

[0143] 50ng / ml hFGF2

[0144] 25nM gastrin

[0145] 10μM ROCK inhibitor (Y-27632)

[0146] In DMEM / F12 + 15mM HEPES, 1x GlutaMax (1:100 #35050-038) and 100μg / ml PenStrep (#15140-122)

[0147] Second culture medium after 4 days of culture

[0148] 0.1nM Wnt(NGS)

[0149] 0.5 μg / ml recombinant R-Spondin1

[0150] 100ng / ml recombinant Noggin

[0151] 1x B27 TM Supplements

[0152] 1.25mM NAC

[0153] 5ng / ml hEGF

[0154] 10ng / ml hNRG1

[0155] 100ng / ml hIGF1

[0156] 50ng / ml hFGF2

[0157] 25nMGastrin25nM

[0158] 0.1 μM atRA

[0159] In DMEM / F12 + 15mM HEPES, 1x GlutaMax (1:100 #35050-038) and 100μg / ml PenStrep (#15140-122)

[0160] Materials and Methods

[0161] Human Organ System

[0162] All patients gave informed consent for additional biopsy samples to be collected for research purposes during elective colonoscopy. Human healthy colon (W18-50157; HUB-02-A2-040) and small intestine (D1n(HUB-04-A2-001)) organoids were provided by our collaborators, HUB Organoids in Utrecht, the Netherlands.

[0163] Mouse organoid lines

[0164] All animal experiments were approved by the Basel Cantonal Veterinary Authorities and performed according to the Guide for Care and Use of Laboratory Animals. Male and female outbred mice aged 7 to 15 weeks were used for all experiments. Mouse strains used: C57BL / 6 wild type (Charles River Laboratories) and Lgr5::DTR-EGFP (Genentech, de Sauvage Laboratory).

[0165] Human organ culture

[0166] Organoids grown from primary tissue were generated from isolated crypts from human biopsies collected by colonoscopy as previously described 1After trypsinization, organoids were maintained in FMI-START medium (with 15 mM HEPES (Stem Cell technologies) supplemented with 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 (kindly 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 (Advanced DMEM / F-12 from Thermo Scientific) for 4 days. After 4 days, organoids were treated with FMI-BALANCE Colon (with 15 mM HEPES (Stem Cell Technologies) supplemented with 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 (kindly provided by Novartis AG), 100 ng / ml Noggin (Paptech), 0.1 nM WNT-NGS (Ipressa Technologies), 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 ( Figure 1 A- Figure 1G) / 0.1 μM (other) atRA (Sigma-Aldrich) and 5 ng / ml human or mouse EGF (Thermo Scientific) in Advanced DMEM / F-12) or FMI-BALANCE small intestine (with 15 mM HEPES (Stem Cell Technologies) supplemented with 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 (kindly gift from Novartis AG), 100 ng / ml Noggin (Paptech), 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 ( Figure 1 A- Figure 1 G) / 1μM( Figure 1 I) / 2.5 μM (other) atRA (Sigma-Aldrich) and 5 ng / ml human or mouse EGF (Thermo Scientific) in Advanced DMEM / F-12. For small intestine-derived organoids, the medium was changed again on days 7 and 10 due to the extended maturation time.

[0167] Mouse organoid culture

[0168] Mouse organoids were generated from isolated crypts of the murine small intestine as previously described. 2 After trypsinization, organoids were maintained in FMI-START medium supplemented with 10 μM Y27632 (ROCK inhibitor, Stem Cell Technologies) for 3 days. After 3 days, organoids were maintained in FMI-BALANCE Colon or FMI-BALANCE Small Intestine and the medium was changed again on day 5.

[0169] Image-based screening assays

[0170] Mouse small intestine and colon organoids were collected 5-7 days after passage and digested with (0.05%) trypsin EDTA (Gibco) for 5 min at 37 ° C. Human small intestine and colon organoids were collected 10-13 days after passage and digested with (0.05%) trypsin EDTA (Gibco) for 5-10 min at 37 ° C. The dissociated cells were passed through a cell filter with a pore size of 40 μm. For the indicated experiments, single living cells were sorted by FACS (Becton Dickinson FACSAria or SONYMA900 cell sorter). Cell doublets and dead cells were removed using forward scatter and side scatter properties. Dead cells were also filtered out by DRAQ7 stain (1.5 μM, Thermo Fisher Scientific). Unless otherwise stated, mouse organoids are derived from C57BL / 6 wild-type mice.

[0171] Imaging-based experiments were performed in 384-well plates (CellCarrier-384, PerkinElmer, Cat. No. 6007550) or 96-well plates (μClear, Greiner, Cat. No. 655090).

[0172] For experiments in 384-well plates, cells were resuspended in FMI-START containing 10 μM Y27632 (ROCK inhibitor, Stem Cell Technology) to a density of about 6-50 cells / μl, depending on the experiment and cell line used. Next, under constant cooling, the wells of the 384-well plate were covered with 10 μl / well ice-cold 2:1 Matrigel (Corning): FMI-START mixture using an Assist Plus pipetting robot (Integra), covering the entire bottom of the well. The plate was centrifuged to allow the Matrigel mixture to reach a flat layer, and then incubated at RT for 10 min to slightly solidify. Afterwards, 40 μl of a cell mixture containing 10 μM Y27632 (ROCK inhibitor, Stem Cell Technology) was carefully covered on the prepared wells. The cells were carefully centrifuged into Matrigel using a low-speed (50-70 rcf, 5 s) centrifuge. Finally, the plate was placed in an incubator (37 ° C, 5% CO2) for culture.

[0173] For experiments in 96-well plates, cells were resuspended in an ice-cold 2:1 Matrigel (Corning): FMI-START mixture containing 10 μM Y27632 (ROCK inhibitor, Stem Cell Technology) to a density of approximately 6-50 cells / μl (depending on the experiment and cell line used) and seeded in 5 μl drops to the center of the well. The plate was incubated at 37°C for 20 minutes to allow the Matrigel to solidify. Thereafter, 100 μl of FMI-START supplemented with 10 μM Y27632 (ROCK inhibitor, Stem Cell Technology) was added for culture in an incubator (37°C, 5% CO2).

[0174] To test different culture media protocols described previously 1,3 *, it was decided to change the medium at the same time as the FMI medium regimen on days 4 and 7. In the medium regimen of Fujii et al. 3 In, with Beumer et al. 1 Compared to the Stem Cell Technologies* protocol, no differentiation medium is required. Therefore, the inventors continued to culture organoids in the same medium according to the Fujii et al. protocol, changing the medium at days 4 and 7.

[0175] During the experiment, the medium was changed to FMI-BALANCE on day 4 and again on day 7. Small intestinal organoids received an additional medium change on day 10 to allow growth until maturity on day 13.

[0176] For mouse organoids, cells were mixed with Matrigel (Corning) (culture medium to Matrigel ratio of 1:1). In each well of a 96-well plate (μClear, Greiner, catalog number 655090), 5 μl droplets of different densities were inoculated. After solidification at 37°C for 20 min, 100 μl culture medium was covered.

[0177] Fixed sample preparation and imaging

[0178] To allow imaging of all organoids in a similar z-range, each well plate was centrifuged at 1000 rcf for 10 min in a pre-cooled centrifuge at 10°C prior to fixation. Organoids were fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 30 min at room temperature at indicated time points.

[0179] For image-based screening assays, organoids were permeabilized and blocked for 3 h (human) or 1 h (mouse) with 0.5% Triton X-100 (Sigma-Aldrich), 3% donkey serum (Sigma-Aldrich), 100 mM NH4Cl in PBS at RT. The primary and secondary antibodies were diluted in antibody buffer (0.1% Triton X-100, 3% donkey serum in PBS) and applied 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 mixture. Fixation, blocking / permeabilization and washing steps were performed using EL406Combination Washer Dispenser (BioTek Instruments).

[0180] High-throughput imaging was performed using one of two automated spinning disk microscopes (Molecular Devices ImageXpress Micro Confocal or Yokogawa CellVoyager 7000S). The Molecular Devices ImageXpress Micro Confocal was combined with a Nikon 20x Plan Apo 0.75NA objective for imaging. For each site, a z-plane spanning the size of the organoid was acquired. All experiments used 5 μm z-steps unless otherwise stated.

[0181] Alternatively, CellVoyager 7000S is also used for imaging in combination with Olympus 20x UPLSAPO 20x 0.75NA objective lens. Here, a search priority imaging method (SearchFirst (search priority) module of Wako Software Suite (software suite)) is used. For this reason, a visual field is obtained in each hole with 4 times resolution to cover the entire hole area. Then, the custom ImageJ macro that summarizes the coordinates of the output organoid position is used to dynamically segment the organoid. These coordinates are used to generate a position map (20x, NA=0.75) for high-resolution re-imaging. For each site, a z plane across the size of the organoid is obtained. Unless otherwise stated, all experiments use 5 μm z-steps.

[0182] Image analysis

[0183] The individually acquired fields of view are first combined to generate a well overview for each well. Next, a maximum intensity projection is generated for all overviews and channels. The trained RDCNet Network is then used4 Individual organoids were segmented using the DAPI channel. After segmentation, the regions of individual organoids were extracted from the overview based on the segmentation. All excised regions were subjected to a filtering step to remove incorrectly segmented organoids as well as fragments based on size, DAPI brightness or axis ratio. Additionally, organoids cut by the imaging boundaries were excluded from further analysis. The features of the remaining organoids were mainly calculated using the regionprops function in the python package scikit-image. Furthermore, outliers were removed by a quantile filter with lower and upper limits of 0.01 and 0.99, respectively.

[0184] For a given experiment, pseudo-time series were computed using the shape feature space. Briefly, a subset of shape features were hand-picked to simulate organoid maturation over time. 5 This subset was used to calculate pseudo-timing for all organoids. To do this, random organoids were picked from the earliest and latest time points to build trajectories between those using 500 intermediate random waypoints. This process was repeated five times and the average pseudo-timing was calculated for each organoid.

[0185] Single-cell RNA sequencing (scRNAseq) and raw data processing

[0186] Single cells were isolated from organoids at the indicated time points, passed through a cell strainer with a pore size of 40 μm, and used for FACS sorting to discard debris and dead cells. The cell suspension was loaded onto a 10x Genomics Chromium SingleCell instrument to generate single-cell GEMs. Single-cell RNA-Seq libraries were prepared using GemCode Single Cell 3'Gel Bead and Library Kit according to CG00052_SingleCell3'ReagentKitv2UserGuide_RevB. GEMRT was performed in a Bio-Rad PTC-200 Thermal cycler with a semi-skirted 96-well plate (Eppendorf P / N0030 128.605): 53°C for 45 minutes, 85°C for 5 minutes; hold at 4°C. After RT, GEMs were broken and used Single-stranded cDNA was cleaned up with MyOneTM Silane Beads (Life Technologies P / N 37002D). 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 min; cycle 12x: 98°C for 15 s, 67°C for 20 s, and 72°C for 1 min; 72°C for 1 min; hold at 4°C. Amplified cDNA products were cleaned up with the SPRIselect Reagent Kit (0.6X SPRI). Indexed sequencing libraries were constructed using reagents in the Chromium Single Cell 3'library kit V2 (10x Genomics P / N 120237), and the steps are as follows: 1) fragmentation, end repair, and A-tailing; 2) post-fragmentation, end repair, and A-tailing double-sided size selection using the SPRIselect10 Reagent Kit (0.6X SPRI and 0.8X SPRI); 3) adapter ligation; 4) post-ligation cleanup using SPRIselect (0.8X SPRI); 5) sample indexing PCR using the Chromium Multiplex kit (10xGenomics P / N-120262); 6) sample indexing followed by double-sided size selection - using the SPRIselect Reagent Kit (0.6X SPRI and 0.8X SPRI). The barcoded sequencing libraries were quantified using Qubit 2.0 with Qubit™ dsDNA HS Assay Kit (Invitrogen P / N Q32854), and the quality of the libraries was checked on a 2100 Bioanalyzer from Agilent using the Agilent High Sensitivity DNA Kit (Agilent P / N 5067-4626). The sequencing libraries were loaded at 10 pM onto an Illumina HiSeq 2500 with a 2×50 paired end kit, using the following read lengths: 26 cycle read 1, 8 cycle read i7 index, and 98 cycle read 2.The clustered gene expression matrix was generated using the CellRanger suite (1.3.0) from the BCL files generated by the sequencer based on the mm10 Cell Ranger human genome annotation file.

[0187] scRNA sequencing analysis

[0188] To exclude low-quality cells, the inventors removed cells with fewer than 4,500 reads (4,000 for the small intestine dataset) or cells with more than 26% of transcripts from mitochondrial genes. To exclude low-quality features, they removed transcripts detected in fewer than 27 cells. After removing unwanted cells and features from the dataset, they normalized the data based on total expression, multiplied it by the mean of the library size distribution, and log-transformed the results. The inventors further excluded feature types that contributed less to the total UMI counts (including non-coding RNAs, pseudogenes, ribosomal proteins, snoRNAs, and snRNAs) from the dataset. After preprocessing, our dataset counted 18,469 transcripts in 53,640 cells sampled at nine time points for colon organoids and 17,195 transcripts in 26,453 cells sampled at five time points for small intestine organoids.

[0189] The inventors used the modelGeneVar and getTopHVGs functions in the scran package in Bioconductor (an open source bioinformatics tool library) to identify variable genes, blocking time points and using a threshold of 0.05 as the false discovery rate. The inventors used sketchR (Hie et al. 2019 6 R implementation of the proposed geometry sketch) to perform density-equalized principal component analysis.

[0190] To visualize the data, they further reduced the dimensionality of the dataset using T-distributed stochastic neighbor embedding (t-SNE) based on the first 50 principal components to project the cells into 2D space. The inventors then used the Seurat R package for graph-based clustering. To visualize the clustered expression of known cell type markers, they used the DotPlot function in the Seurat package. They assigned clusters to known cell types based on relative gene expression. *https: / / www.stemcell.com / products / intesticult-organoid-differentiation-med ium-human.html#section-product-documents

[0191] Supplementary references

[0192] 1. Beumer, J. et al. High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells. Cell 181, 1291 - 1306.e19 (2020).

[0193] 2. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

[0194] 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).

[0195] 4. Ortiz, R., De Medeiros, G., Peters, A.H.F.M., Liberali, P. & Rempfler, M. RDCNet: Instance segmentation with a minimalist recurrent residual network.

[0196] 5. Setty, M. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat. Biotechnol. 37, 451–460 (2019).

[0197] 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).

[0198] Table 1

[0199]

[0200]

[0201]

[0202] Table 2

[0203]

[0204]

Claims

1. A culture medium for culturing intestinal organoids, characterized in that The culture medium contains neuregulin 1 (NRG1) and all-trans retinoic acid (atRA).

2. The culture medium of claim 1, wherein the intestinal organoid is a small intestine organoid, a colon organoid, a colorectal cancer organoid, a Crohn's disease organoid, or an ulcerative colitis organoid.

3. The culture medium of claim 1 or 2, wherein the concentration of NRG1 is between 1 ng / ml and 100 ng / ml, and wherein 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 in that The culture medium does not contain 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 of any one of claims 1 to 5, further comprising Wnt NGS.

7. A composition comprising the culture medium as claimed in any one of the preceding claims, and an extracellular matrix or a 3D matrix that simulates the extracellular matrix through the interaction of the extracellular matrix with cell membrane proteins.

8. The composition of claim 7, wherein the 3D matrix extracellular matrix is ​​a synthetic hydrogel or Matrigel TM .

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

10. A method for generating intestinal organoids, the method comprising the following steps: A suspension of single cells or fragments of intestinal tissue is provided, and the suspension of single cells or fragments of intestinal tissue is incubated with a first culture medium for at least one day, and then the first culture medium is replaced with a culture medium as described in claims 1-6.

11. The method for generating intestinal organoids as claimed in claim 10, wherein the single cell or fragment of the intestinal tissue is obtained from the small intestine, colon, colorectal cancer, Crohn's disease tissue, ulcerative colitis tissue or definitive endoderm cells.

12. The method of claim 10 or 11, wherein the first culture 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 A 83-01.

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

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

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